PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T1	0-76	Sentence	denotes	Flattening the COVID-19 Curve With Natural Killer Cell Based Immunotherapies
T2	78-86	Sentence	denotes	Abstract
T3	87-192	Sentence	denotes	Natural Killer (NK) cells are innate immune responders critical for viral clearance and immunomodulation.
T4	193-329	Sentence	denotes	Despite their vital role in viral infection, the contribution of NK cells in fighting SARS-CoV-2 has not yet been directly investigated.
T5	330-502	Sentence	denotes	Insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing NK cell phenotype and function during SARS, MERS, and COVID-19.
T6	503-686	Sentence	denotes	These studies suggest a reduction in circulating NK cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of NK cell responses by coronaviruses.
T7	687-810	Sentence	denotes	Reduced circulating NK cell levels and exhaustion may be directly responsible for the progression and severity of COVID-19.
T8	811-970	Sentence	denotes	Conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine NK cell potential in mediating immunopathology.
T9	971-1221	Sentence	denotes	A common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which NK cells are an important component.
T10	1222-1405	Sentence	denotes	In this review, we summarize the current understanding of how NK cells respond in both early and late coronavirus infections, and the implication for ongoing COVID-19 clinical trials.
T11	1406-1588	Sentence	denotes	Using this immunological lens, we outline recommendations for therapeutic strategies against COVID-19 in clearing the virus while preventing the harm of immunopathological responses.
T12	1590-1602	Sentence	denotes	Introduction
T13	1603-1769	Sentence	denotes	Natural Killer (NK) cells are a key component of the innate immune system and are critical in the response to many viral infections in humans and animal models (1–3).
T14	1770-1980	Sentence	denotes	In addition to their beneficial antiviral role, NK cells have also been associated with immunopathology in infections such as respiratory syncytial virus (RSV) (4), influenza A virus (5–8), and hepatitis B (9).
T15	1981-2197	Sentence	denotes	Additionally, in the context of non-respiratory viral infections by HIV and HCV, NK cells appear to act as a rheostat by eliminating activated CD4+ and CD8+ T cells, thus preventing T cell-mediated autoimmunity (10).
T16	2198-2396	Sentence	denotes	The etiologic agent of the 2019 outbreak of pneumonia in Wuhan, China, was identified as belonging to the Coronaviridae family and named Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).
T17	2397-2553	Sentence	denotes	This virus causes the coronavirus Disease 2019 (COVID-19) which was declared a pandemic by the World Health Organization (WHO) on March 11th, 2020 (11, 12).
T18	2554-2714	Sentence	denotes	With the paucity of information currently available, there is a lack of consensus on the role played by NK cells in the response to coronavirus (CoV) infection.
T19	2715-2842	Sentence	denotes	In this review, we will explore evidence for both the protective and pathological role that NK cells may play in CoV infection.
T20	2843-2979	Sentence	denotes	Based on this knowledge we will comment on immune modulating treatment options that are being developed for the current COVID-19 crisis.
T21	2981-3015	Sentence	denotes	Coronaviruses and Recent Outbreaks
T22	3016-3146	Sentence	denotes	First discovered in the 1960s, CoVs are part of the Coronaviridae family of enveloped positive single-strand RNA viruses (13, 14).
T23	3147-3281	Sentence	denotes	The subfamily Orthocoronaviridae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (15).
T24	3282-3450	Sentence	denotes	Alpha- and betacoronaviruses circulate in mammals, including bats, gammacoronaviruses infect mostly avian species, and deltacoronaviruses infect birds and mammals (15).
T25	3451-3612	Sentence	denotes	Low pathogenic human CoVs (hCoVs), such as HCoV-299E (16), infect upper airways and etiological studies suggest they account for 15–30% of common colds (17, 18).
T26	3613-3726	Sentence	denotes	On the other hand, highly pathogenic CoVs infect the lower respiratory tract and can cause severe pneumonia (19).
T27	3727-3982	Sentence	denotes	These highly pathogenic CoVs include SARS-CoV-1, the virus responsible for the 2002–2004 Severe Acute Respiratory Syndrome (SARS) epidemic, and MERS-CoV, the virus responsible for the outbreak of Middle Eastern Respiratory Syndrome (MERS) in 2015 (19–21).
T28	3983-4211	Sentence	denotes	While highly pathogenic CoVs have become a relatively recent issue for humans; feline, canine, and bovine CoVs have long been recognized as significant pathogens with implications in veterinary medicine and agriculture (22, 23).
T29	4212-4320	Sentence	denotes	All CoVs have a roughly 30 kb genome packed into an enveloped helical capsid ranging from 80 to 120 nm (24).
T30	4321-4513	Sentence	denotes	At minimum, Coronaviridae members encode 4 structural and 16 non-structural proteins (14) with the family owing its name to the crown-like appearance produced by their spike (S) proteins (25).
T31	4514-4734	Sentence	denotes	Mutations in the S protein have allowed SARS-CoV1/2 to co-opt ACE2 or MERS-CoV to co-opt dipeptidyl peptidase 4 (DPP4) receptor/CD26 as viral entry receptors, thus facilitating the zoonosis of non-human CoVs (15, 26–28).
T32	4735-5035	Sentence	denotes	In addition, another mechanism that may have allowed these viruses to adapt to human hosts is through S protein cleavage by host cell proteases to expose the S2 domain fusion peptide, which induces viral and cellular membrane fusion and results in the release of viral genome into the cytoplasm (15).
T33	5036-5189	Sentence	denotes	Genetic sequencing revealed SARS-CoV-2 to be a betacoronavirus that shares 79.0% nucleotide identity with SARS-CoV-1 and 51.8% identity to MERS-CoV (29).
T34	5190-5340	Sentence	denotes	The epidemic of SARS in 2002–2004 caused by SARS-CoV-1 illustrated the devastating potential of coronaviruses to cause serious disease in humans (24).
T35	5341-5445	Sentence	denotes	SARS ultimately reached 29 countries and 5 continents causing over 8,000 infections and over 900 deaths.
T36	5446-5581	Sentence	denotes	The basic reproductive rate (R0) or the number of expected cases arising from one infected individual, ranges from 2 to 4 (20, 30, 31).
T37	5582-5694	Sentence	denotes	With its reservoir in bats, SARS-CoV-1 is a zoonosis that was transmitted to humans by palm civets (24, 32, 33).
T38	5695-5828	Sentence	denotes	SARS-CoV-1 infects lung pneumocytes (34) and enterocytes in the digestive tract (35) most often producing flu-like symptoms (36, 37).
T39	5829-6048	Sentence	denotes	More severe presentations including pneumonia, pronounced lymphopenia, liver abnormalities, and acute respiratory distress syndrome (ARDS) were also reported, with most fatalities due to respiratory failure (19, 36–39).
T40	6049-6178	Sentence	denotes	The subsequent MERS-CoV outbreak in 2015 also originated in bats, with dromedary camels being the intermediary host (14, 40, 41).
T41	6179-6231	Sentence	denotes	The R0 for MERS-CoV is estimated to be under 1 (21).
T42	6232-6430	Sentence	denotes	The extent of MERS-CoV transmission was more limited than SARS-CoV-1, but its case fatality rate was greater with 2,494 cases over 27 countries and 858 deaths being reported at the end of 2019 (21).
T43	6431-6603	Sentence	denotes	Common presentations for MERS-CoV include fever, dyspnea, muscle pain, and digestive tract symptoms and disease progression is more likely in those with comorbidities (42).
T44	6604-6725	Sentence	denotes	Like SARS-CoV-1 and MERS-CoV, SARS-CoV-2 is thought to have originated in bats through an unknown intermediary host (43).
T45	6726-6877	Sentence	denotes	At the time of writing, the number of global infections is estimated to be over 5,000,000 with over 340,000 deaths (44) and the R0 is roughly 2.2 (45).
T46	6878-7076	Sentence	denotes	Like other diseases caused by infectious CoVs, most patients present with flu-like symptoms including fever, cough, and lethargy, with the development of pneumonia and ARDS often proving fatal (46).
T47	7077-7238	Sentence	denotes	Furthermore, patients with underlying conditions are at risk for further complications if infected with COVID-19, such as those with cardiovascular disease (47).
T48	7239-7359	Sentence	denotes	SARS-CoV-2 has been posthumously detected in not only the lungs, but the pharynx, heart, liver, brain, and kidneys (48).
T49	7360-7603	Sentence	denotes	Transmission of SARS-CoV-2 is thought to mainly occur through direct contact/inhalation of respiratory droplets and aerosols from infected carriers, but indirect transmission by fomites has also been reported, although less efficient (49, 50).
T50	7604-7756	Sentence	denotes	SARS-CoV-2 viral entrance is thought to be mediated by binding of the S protein to the ACE2 receptor (51, 52), although this is still under debate (53).
T51	7757-7970	Sentence	denotes	While direct cytopathic effects are thought to play a major role in CoV pathology, studies have suggested that a dysregulated immune response resulting in pathological inflammation is also partly responsible (19).
T52	7971-8150	Sentence	denotes	With the current pandemic already surpassing the previous CoV outbreaks (54), rapid deployment of novel approaches to understanding and treating coronavirus infections are needed.
T53	8152-8184	Sentence	denotes	NK Cells as Innate Viral Killers
T54	8186-8205	Sentence	denotes	Sensing RNA Viruses
T55	8206-8277	Sentence	denotes	Innate immunity is essential in disease prevention and viral clearance.
T56	8278-8558	Sentence	denotes	Among the first responders to viral infections, tissue-resident macrophages and dendritic cells (DCs) (55) recognize evolutionarily conserved microbial structures termed pathogen-associated molecular patterns (PAMPs) via germline-encoded pattern recognition receptors (PRRs) (56).
T57	8559-8731	Sentence	denotes	In the context of respiratory RNA viruses, airway epithelial cells, that also express some PRRs (57), are often infected and have a major role in the first line of defense.
T58	8732-8909	Sentence	denotes	TLR3, TLR7, TLR8, MDA-5, and RIG-I are PRR expressed by immune and non-immune cells that are especially relevant in fighting respiratory RNA viruses, such as Coronaviruses (57).
T59	8910-9113	Sentence	denotes	Sensing through PRRs results in the transcription of genes involved in the inflammatory response, with type I interferons (IFNs) (IFN-α/β) production being a critical part of the antiviral response (58).
T60	9114-9343	Sentence	denotes	Type I IFNs are produced by many immune and non-immune cells (55, 57, 59) and in addition to eliciting intrinsic antiviral responses (60), they are also essential to prime innate and adaptive lymphocytes, including NK cells (61).
T61	9345-9373	Sentence	denotes	NK Cells as Viral Responders
T62	9374-9558	Sentence	denotes	NK cells are cytotoxic lymphocytes that directly target infected, stressed, or transformed cells and play a critical role in bridging the innate and the adaptive immune responses (62).
T63	9559-9711	Sentence	denotes	In humans, mature NK cells comprise 10–15% of total peripheral blood leukocytes and are described phenotypically as CD3− CD14− CD19− CD56+ CD16+/− (63).
T64	9712-9850	Sentence	denotes	NK cells do not undergo clonal selection but instead express several germline-encoded receptors that regulate their activity (62, 64, 65).
T65	9851-10610	Sentence	denotes	Upon viral infection, host cells become more susceptible to NK cell killing through: (i) upregulation of self-encoded molecules induced by infection/cellular stress (66, 67) that bind activating NK cell receptors such as Natural Cytotoxicity Receptors (NCRs) (NKp30, NKp44, and NKp46) (68), C-type lectin-like receptors NKG2D and NKp80 (69), and co-activating receptors such as DNAM-1 (70); (ii) downregulation of ligands for inhibitory receptors such as Killer Immunoglobulin-like Receptors (KIRs) (71–73) and the C-type lectin-like receptor CD94-NKG2A (74, 75) which suppress NK cell activation, and; (iii) direct recognition of viral moieties, via engagement of PAMPS (76) or transmembrane activating receptors such as mouse Ly49H (77) or human NKG2C (78).
T66	10611-10818	Sentence	denotes	Moreover, NK cells can eliminate virus-infected cells via CD16-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), which has been shown to be particularly important for herpesvirus clearance (79).
T67	10819-11072	Sentence	denotes	Finally, NK cell activity is modulated by cytokines, including, but not limited to, the activating cytokines interleukin (IL)-2/12/15/18 (80) and type I IFN, which can be produced by virally infected cells or activated antigen presenting cells (81, 82).
T68	11073-11215	Sentence	denotes	IL-2/12/15/18, alone or in combination, promotes NK cell survival, proliferation, cytotoxicity, and cytokine production, including IFN-γ (80).
T69	11216-11307	Sentence	denotes	Therefore, NK cells are uniquely equipped to sense and quickly respond to viral infections.
T70	11309-11346	Sentence	denotes	NK Cell Effector Functions and Memory
T71	11347-11516	Sentence	denotes	NK cells are found in circulation and in peripheral tissues (63) and can be quickly recruited to sites of infection where they facilitate and accelerate viral clearance.
T72	11517-11669	Sentence	denotes	In fact, NK cells are not thought to have permanent tissue residency but instead move dynamically between the blood and tissues, such as the lungs (83).
T73	11670-11770	Sentence	denotes	NK recruitment is regulated by chemokine gradients that are sensed via chemokine receptors (84, 85).
T74	11771-12010	Sentence	denotes	Activated NK cells induce the apoptosis of target cells through the engagement of death receptors, such as TRAIL and Fas (86) or via direct cytotoxicity through Ca2+-dependent exocytosis of cytolytic granules (perforin and granzymes) (87).
T75	12011-12108	Sentence	denotes	Moreover, NK cells secrete cytokines, including IFN-γ, which have key anti-viral properties (88).
T76	12109-12291	Sentence	denotes	In addition to being essential first responders to viral infection, NK cells can elicit a stronger secondary response resembling the memory features of adaptive lymphocytes (89, 90).
T77	12292-12475	Sentence	denotes	NK cell memory has been initially described in mice infected by MCMV, where Ly49H+ NK cells quickly expand and have stronger responses after a secondary encounter with the virus (91).
T78	12476-12634	Sentence	denotes	Interestingly, a similar NK cell subset has been identified in humans, where NK cells expressing NKG2C are expanded and persist in CMV infected patients (92).
T79	12635-12791	Sentence	denotes	Both Ly49H and NKG2C bind viral determinants, highlighting how NK cell memory is linked with the ability of NK cells to directly recognize viruses (93, 94).
T80	12792-12965	Sentence	denotes	In addition to direct recognition of viral molecules, long-lasting changes in NK cells are induced by the cytokine milieu (89, 95), which can be elicited by viral infection.
T81	12967-13032	Sentence	denotes	NK Cell Dysfunction Is Linked With Increased Viral Susceptibility
T82	13033-13215	Sentence	denotes	The relevance of NK cells in fighting viral infections has been highlighted by several studies where NK cells, in mice and humans, were not present or had compromised functions (96).
T83	13216-13425	Sentence	denotes	For example, individuals with NK cell deficiencies (NKD), a subset of primary immunodeficiency diseases, are highly susceptible to viral infection, particularly by herpesvirus and papillomavirus families (96).
T84	13426-13682	Sentence	denotes	The seminal 1989 case of NKD in an adolescent female with severe herpesvirus infections (varicella pneumonia, disseminated CMV, and disseminated HSV) revealed how functional NK cell deficiencies have clinical consequences in terms of viral infections (97).
T85	13683-13883	Sentence	denotes	Cancer patients are also at risk of viral infections (98), which may be explained, at least in part, by an impairment of NK cell responses often observed in humans and in murine tumor models (99–104).
T86	13884-13984	Sentence	denotes	Unsurprisingly, cancer patients are at a significantly increased risk of severe COVID-19 (105, 106).
T87	13985-14054	Sentence	denotes	Elderly patients are also more susceptible to viral infections (107).
T88	14055-14218	Sentence	denotes	Mouse studies highlighted how a decreased number of circulating mature NK cells in aged animals paralleled with increased susceptibility to viral infections (108).
T89	14219-14360	Sentence	denotes	Studies in humans suggest that although NK cell numbers can actually increase with aging, NK cell activity declines significantly (109, 110).
T90	14361-14680	Sentence	denotes	Przemska-Kosicka et al. investigated NK cell function in response to seasonal influenza vaccination in young and old populations and observed quantitative and qualitative changes associated with impaired responses in the NK cell population and this was associated with poor seroconversion in the older population (111).
T91	14681-14921	Sentence	denotes	Additionally, obesity, which has been shown to cause systemic NK cell dysfunction (112, 113), has also been linked to increased COVID-19 severity and could be the reason behind the high prevalence of severe COVID-19 in younger people (113).
T92	14922-15034	Sentence	denotes	In short, NKD and individuals with reduced NK cell numbers or function are more susceptible to viral infections.
T93	15035-15224	Sentence	denotes	Unsurprisingly, the CDC has already highlighted a higher risk of infection and severity of COVID-19 in older individuals and individuals with comorbidities such as obesity and cancer (114).
T94	15225-15588	Sentence	denotes	However, this point is still controversial as a systematic review showed that primary immunodeficiencies are not linked with increased COVID-19 severity (115), but these data have to be interpreted keeping in mind that a large part of COVID-19 pathology is caused by excessive immune activation, which is arguably harder to reach in immunocompromised individuals.
T95	15589-15799	Sentence	denotes	Given the paradoxical role of the immune response in COVID-19 patients, it would be extremely useful to be able to rely on immunological functional biomarkers that could predict the outcome of disease severity.
T96	15800-15993	Sentence	denotes	Such assays are readily available for determining NK cell activity, e.g., NKVue™, and there is therefore an opportunity to conduct studies that would link NK cell functions to disease severity.
T97	15995-16031	Sentence	denotes	NK Cells and Coronavirus Infections:
T98	16032-16042	Sentence	denotes	Dual Roles
T99	16044-16096	Sentence	denotes	Coronaviruses Potently Suppress Type I IFN Responses
T100	16097-16185	Sentence	denotes	Evasion of host immune responses is necessary for the successful propagation of a virus.
T101	16186-16345	Sentence	denotes	Mechanisms employed by CoVs to evade the immune response could provide insights into how the immune system, and NK cells in particular, responds to SARS-CoV-2.
T102	16346-16523	Sentence	denotes	CoVs have been shown to target components of the innate IFN response, employing non-structural proteins (nsps), structural proteins, and accessory proteins to achieve this goal.
T103	16524-16634	Sentence	denotes	Nsp16 methylates viral RNA therefore preventing recognition by MDA5 and dampening type I IFN production (116).
T104	16635-16868	Sentence	denotes	Nsps also suppress type I IFN responses via the inhibition of the transcription factor STAT1 mRNA transcription (nsp1) and deubiquitination of transcription factors like Interferon Regulatory Transcription Factor (IRF)3 (nsp3) (116).
T105	16869-17023	Sentence	denotes	Moreover, viral-encoded accessory proteins from SARS-CoV-1 open reading frame (ORF)3b and MERS-CoV ORF4a/4b also block IFN production and signaling (116).
T106	17024-17128	Sentence	denotes	In addition, the MERS-CoV ORF6-encoded protein blocks p-STAT1 import, thus blocking IFN signaling (116).
T107	17129-17416	Sentence	denotes	Finally, the structural M protein of MERS-CoV (27) physically sequesters kinase proteins RIG-I, TBK1, IKKe, and TRAF3 and the SARS-CoV-1 N protein inhibits Activator Protein (AP)-1 signaling, protein kinase R function, and NFκB activation, all of which act to impede IFN responses (117).
T108	17417-17574	Sentence	denotes	In vivo murine studies report young mice rapidly clear SARS-CoV-1 infection, while old mice do not and that this discrepancy is due to a delay in type I IFN.
T109	17575-17693	Sentence	denotes	Furthermore, early administration of IFN-β induces a stronger immune response and reduces mortality in old mice (118).
T110	17694-17872	Sentence	denotes	Since type I IFNs are critical for NK cell activation and effector functions, it is possible that NK cell-mediated clearance of SARS-CoV-2 is being subverted by these mechanisms.
T111	17873-18029	Sentence	denotes	Further research into the role of NK cells in CoV clearance and potential immune evasion mechanisms are necessary to inform therapeutic development and use.
T112	18031-18084	Sentence	denotes	NK Cell Role in Clearing Acute Coronavirus Infections
T113	18085-18230	Sentence	denotes	There is currently a paucity of studies into the role of NK cells not only in COVID-19 pathophysiology, but also in other coronavirus infections.
T114	18231-18448	Sentence	denotes	An in vivo study reported that beige mice on a B6 background cleared SARS-CoV-1 normally, indicating that functional lymphocytes, including NK cells, may not be required to eliminate SARS-CoV-1 in murine models (119).
T115	18449-18726	Sentence	denotes	However, in a more recent study characterizing the cellular immune response to SARS-CoV-1 in 12–14-month old BALB/c mice, T cell depletion did not prevent control of SARS-CoV-1 replication (120), suggesting a role for the innate immune system, and NK cells, in viral clearance.
T116	18727-18984	Sentence	denotes	Importantly, in this study CD4-depletion resulted in enhanced lung immunopathology and delayed viral clearance, while CD8-depletion did not affect viral replication or clearance, thus highlighting an important role for CD4+ T cells in coronavirus infection.
T117	18985-19071	Sentence	denotes	These conflicting results may be due to the inherent limitations of CoV murine models.
T118	19072-19208	Sentence	denotes	In 4–8 week-old mice, SARS-CoV-1 is associated only with mild pneumonitis and cytokines are not detectable in the lungs (119, 121, 122).
T119	19209-19399	Sentence	denotes	A SARS-CoV-1 isolate (MA-15) replicates to a high titer and is associated with viremia and mortality, however the model lacks significant inflammatory cell infiltration into the lungs (123).
T120	19400-19553	Sentence	denotes	Thus, mouse models developed for the study of SARS fell short in terms of reproducing the clinical and histopathological signs of disease (119, 121–123).
T121	19554-19697	Sentence	denotes	It is therefore necessary to develop a usable animal model that is capable of reproducing the clinical and histopathological signs on COVID-19.
T122	19698-19910	Sentence	denotes	Israelow et al. recently described a SARS-CoV-2 murine model based on adeno associated virus (AAV)9-mediated expression of human (h)ACE2, which replicated the pathologic findings found in COVID-19 patients (124).
T123	19911-20088	Sentence	denotes	This model, which overcame the inability of murine (m)ACE2 to support SARS-CoV-2 infection, was used to show the inability of Type I IFN to control SARS-CoV-2 replication (124).
T124	20089-20387	Sentence	denotes	In a similar attempt to overcome the lack of infectability through mACE2, Dinnon et al. recently described a recombinant virus (SARS-CoV-2 MA) with a remodeled S protein mACE2 interface, which replicated in upper and lower airways in young and aged mice with disease being more severe in aged mice.
T125	20388-20528	Sentence	denotes	The authors used this model to screen therapeutics from vaccine challenge studies and assessed pegylated IFN-λ-1 as a promising therapeutic.
T126	20529-20704	Sentence	denotes	The authors suggested that this model has greater ease of use, cost, and utility over transgenic hACE2 models (125) to evaluate vaccine and therapeutic efficacy in mice (126).
T127	20705-20849	Sentence	denotes	A preliminary analysis of NK cell function and phenotype has been performed by Zheng et al. using peripheral blood from COVID-19 patients (127).
T128	20850-20946	Sentence	denotes	On admission, NK cell levels in the peripheral blood inversely correlated with disease severity.
T129	20947-21100	Sentence	denotes	Furthermore, COVID-19 patients with severe disease had significantly lower numbers of circulating NK cells, as compared to mild disease (p < 0.05) (127).
T130	21101-21379	Sentence	denotes	Additionally, circulating NK cells in severe disease displayed increased expression of the inhibitory receptor NKG2A and had an hyporesponsive phenotype with lower levels of IFN-γ, tumor necrosis factor (TNF)-α, IL-2, and granzyme B, although degranulation was maintained (127).
T131	21380-21532	Sentence	denotes	Finally, as compared to patients with active disease, patients recovering from COVID-19 had higher numbers of NK cells and lower NKG2A expression (127).
T132	21533-21838	Sentence	denotes	Liao et al. performed single-cell RNAseq on the cells obtained from bronchoalveolar lavage fluid of severe and mild COVID-19 patients and found that COVID-19 patients had significantly more NK cell infiltrates into the lungs, however patients with severe disease had reduced proportions of NK cells (128).
T133	21839-21923	Sentence	denotes	In addition, KLRC1 (NKG2A) and KLRD1 (CD94) were highly expressed by NK cells (128).
T134	21924-22358	Sentence	denotes	Carvelli et al. analyzed myeloid and lymphoid populations by immunophenotyping from blood and bronchoalveolar lavage fluid (BALF) in 10 healthy controls, 10 paucisymptomatic COVID-19 patients, 34 pneumonia patients, and 28 patients with ARDS due to SARS-CoV-2 and found that absolute numbers of peripheral blood lymphocytes, including NK cells, were significantly reduced in the pneumonia and ARDS groups compared to healthy controls.
T135	22359-22498	Sentence	denotes	Furthermore, the proportion of mature NK cells was reduced in patients with ARDS and NK cells showed increased NKG2A, PD-1, and CD39 (129).
T136	22499-22760	Sentence	denotes	Finally, Wilk et al. performed single-cell RNA-sequencing on 7 COVID-19 patients and 6 healthy controls and found that the CD56bright population was depleted in all COVID-19 patients but the CD56dim population was depleted only in patients with severe COVID-19.
T137	22761-22856	Sentence	denotes	Furthermore, NK cells had increased expression of the exhaustion markers LAG3 and HAVCR2 (130).
T138	22857-22956	Sentence	denotes	NK cell cytopenia seems to be a consistent characteristic among SARS-CoV-2 infected patients (131).
T139	22957-23191	Sentence	denotes	Altogether, these data indicate alterations in the NK cell phenotype and functional profile that are consistent with the hypothesis that to establish a productive and lasting infection, SARS-CoV-2 needs to dampen the NK cell response.
T140	23192-23276	Sentence	denotes	NK cell dysfunctions were also observed in patients from the previous CoV outbreaks.
T141	23277-23419	Sentence	denotes	Wang et al. assessed NK cell number and phenotype using peripheral blood from 221 SARS patients admitted to hospitals in Beijing, China (132).
T142	23420-23602	Sentence	denotes	NK cell proportion and absolute number were significantly reduced in SARS patients as compared to healthy donors and patients infected with the bacterium Mycoplasma pneumoniae (131).
T143	23603-23763	Sentence	denotes	NK cell number correlated inversely with disease severity and patients with anti-SARS CoV-specific IgG or IgM antibodies had significantly fewer NK cells (132).
T144	23764-23913	Sentence	denotes	The patients assessed had varied disease duration from 4 to 72 days (mean 31.7 days) and this allowed for patient stratification by disease duration.
T145	23914-24105	Sentence	denotes	Within the first 10 days of SARS-CoV-1 infection, NK cell numbers remained high but this period was followed by the development of lymphopenia with levels recovering only around day 40 (132).
T146	24106-24266	Sentence	denotes	Dong et al. also observed a reduction of NK cell numbers in SARS patients, and these levels were lower in patients with severe, as compared to mild, SARS (133).
T147	24267-24360	Sentence	denotes	In addition, MERS infection is strongly associated with leuko- and lymphopenia (42, 134–136).
T148	24361-24474	Sentence	denotes	The mechanisms underlying the reduction of circulating NK cells in patients infected with CoVs are still unclear.
T149	24475-24672	Sentence	denotes	As most studies have focused on peripheral blood NK cells, it is possible that the reduced number of circulating NK cells is due to redistribution of blood NK cells into the infected tissues (137).
T150	24673-24896	Sentence	denotes	While it is hard to assess NK cell migration to infected tissues in COVID-19 patients, this hypothesis was corroborated by mouse studies, where NK cells have been shown to migrate to the lungs in CoV infected animals (120).
T151	24897-25042	Sentence	denotes	An abundance of inhibitory factors, such as TGF-β, may be partially responsible for the NK cell hyporesponsiveness observed in COVID-19 patients.
T152	25043-25230	Sentence	denotes	In support of this hypothesis, Huang et al. found significantly higher TGF-β levels in SARS patients compared to healthy controls and this positively correlated with length of stay (138).
T153	25231-25455	Sentence	denotes	Given the importance of TGF-β in suppressing NK cell functions, it is possible that the higher levels of TGF-β (as well as other inhibitory cytokines) in CoV patients leads to suppression of NK cell antiviral activity (138).
T154	25456-25622	Sentence	denotes	Early studies of COVID-19 patients report secondary (super-) infections, including nosocomial pneumonia or bacteremia as a complication of SARS-CoV-2 infection (138).
T155	25623-25956	Sentence	denotes	Since NK cells are critical first responders that play a role in preventing and clearing infections (139), a poor NK cell count or exhausted phenotype, in addition to negatively influencing COVID-19 patient outcomes, could facilitate the development of secondary infections and have a significant negative impact on patient outcomes.
T156	25957-26258	Sentence	denotes	One of the main barriers in studying the role of NK cell activation in the early clearance of CoV infection in asymptomatic or mildly symptomatic patients is the fact that these individuals are rarely diagnosed in the clinic and therefore an opportunity to collect samples for research does not exist.
T157	26259-26475	Sentence	denotes	Thus, while there is currently no direct evidence to support a role for NK cells in the clearance of SARS-CoV-2, evidence showing that viral infection has a negative effect on the NK cell compartment is accumulating.
T158	26476-27003	Sentence	denotes	Given the importance of NK cell activity in early viral clearance and late immunopathology, having a rapid and reliable test to predict NK cell function, such as NKVue™ (ATGen Canada/NKMax), whereby whole blood is stimulated by an NK cell-specific activating cytokine mix and activity is measured via IFN-γ production, might allow researchers to predict who will mount an adequate response with asymptomatic or minimally symptomatic viral clearance and who will need ICU admission, as has been shown with cancer patients (140).
T159	27004-27153	Sentence	denotes	Further research will be required into the innate immune response to CoV infection to more fully understand NK cell contributions to viral clearance.
T160	27155-27198	Sentence	denotes	NK Cell Role in Coronavirus Immunopathology
T161	27199-27366	Sentence	denotes	In the context of CoVs, the significant morbidity and mortality associated with severe disease is due to acute lung injury (ALI) and the development of ARDS (19, 141).
T162	27367-27631	Sentence	denotes	Pathological analysis of tissues obtained from SARS and MERS patients showed edematous lungs with areas of consolidation, bronchial epithelial denudation, loss of cilia, squamous metaplasia, pneumocyte hyperplasia, and bronchial submucosal gland necrosis (19, 29).
T163	27632-27736	Sentence	denotes	Histological features include diffuse alveolar damage and acute fibrinous and organizing pneumonia (29).
T164	27737-27868	Sentence	denotes	A heightened inflammatory response in the lungs resulting in tissue damage has been hypothesized to explain the development of ALI.
T165	27869-27978	Sentence	denotes	There are several key factors that may be responsible for the induction of this dangerous inflammation (138).
T166	27979-28185	Sentence	denotes	Both SARS-CoV-1 and MERS-CoV replicate to high titers early in infection, which could lead to enhanced cytopathic effects and increased production of pro-inflammatory cytokines/chemokines by infected cells.
T167	28186-28431	Sentence	denotes	Chen et al. developed a pneumonia model where pulmonary replication of SARS-CoV-1 was associated with histopathological evidence of disease, including bronchiolitis, interstitial pneumonitis, diffuse alveolar damage, and fibrotic scarring (120).
T168	28432-28895	Sentence	denotes	They identified a biphasic cellular immune response in which cytokines (TNF-α and IL-6) and chemokines [interferon gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, RANTES] were produced early, likely by infected airway epithelial cells, alveolar macrophages, and recruited inflammatory monocyte-macrophages and neutrophils, which have been shown to replace resident alveolar macrophages (19, 142).
T169	28896-29134	Sentence	denotes	SARS-CoV-1 and MERS-CoV encode structural and non-structural proteins that antagonize the interferon response, which may initially delay the innate immune response but eventually potentiate inflammatory monocyte-macrophage responses (19).
T170	29135-29285	Sentence	denotes	In COVID-19 patients, Liao et al. reported increased lung infiltration by macrophages identified via RNA-seq analysis of bronchoalveolar lavage fluid.
T171	29286-29527	Sentence	denotes	Patients with mild cases exhibited infiltration by alveolar macrophages [Fatty Acid Binding Protein (FABP)4+] while patients with severe ARDS exhibited infiltration by highly inflammatory [Ficolin (FCN1)+] monocyte-derived macrophages (128).
T172	29528-29728	Sentence	denotes	In the SARS-CoV-1 pneumonia model, the first wave of cytokines and chemokines induced an accumulation of NK cells, as well as plasmacytoid (p)DCs, macrophages, CD4+ T cells and NKT cells in the lungs.
T173	29729-30015	Sentence	denotes	A second wave of inflammatory mediators was detected later on day 7 post-infection [cytokines TNF-α, IL-6, IFN-γ, IL-2, IL-5, and chemokines MCP-1, MIP-1a, RANTES, monokine induced by gamma interferon (MIG), IP-10] and correlated with lung infiltration of T cells and neutrophils (120).
T174	30016-30307	Sentence	denotes	These findings are consistent with studies that have shown increased levels of activating and inhibitory cytokines and chemokines in the blood and lungs of SARS patients, as well as histological studies of SARS and MERS-infected lungs which show extensive cell infiltrates (19, 29, 143–145).
T175	30308-30490	Sentence	denotes	When Huang et al. investigated the cytokine/chemokine profile in the acute phase of SARS infection in a cohort of Taiwanese patients, they observed an IFN-γ-led cytokine storm (138).
T176	30491-30730	Sentence	denotes	They assessed sera from hospitalized patients prior to the administration of immunomodulators and found significantly increased levels of IFN-γ, IL-18, IP-10, MCP-1, MIG, and IL-8 (138), which returned to basal levels in convalescent sera.
T177	30731-30829	Sentence	denotes	IP-10, MIG, MCP-1, and IL-18 levels were all significantly increased in death vs. survival groups.
T178	30830-31072	Sentence	denotes	Interestingly, they found an inverse relationship between IFN-γ levels and lymphocyte numbers and suggested this could either be due to IFN-γ-induced lymphocyte apoptosis or sequestration of chemokine-recruited lymphocytes in the lungs (138).
T179	31073-31167	Sentence	denotes	Indeed, this hyper-cytokinemia has been consistently observed in SARS-infected patients (146).
T180	31168-31431	Sentence	denotes	However, a recent study found that levels of six pro-inflammatory cytokines (IL-1b, IL-1Ra, IL-6, IL-8, IL-18, and TNF-α) implicated in the cytokine storm in COVID-19 patients did not differ significantly from levels in cytokine storms caused by other conditions.
T181	31432-31724	Sentence	denotes	They suggest that it is therefore possible that increased levels of pro-inflammatory cytokines in the context of severe COVID-19 may simply reflect an increased viral burden rather than an exuberant immune response and suggest that immunotherapies should therefore be used with caution (147).
T182	31725-32051	Sentence	denotes	Altogether these studies show that during acute CoV infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce cytokines and chemokines that induce the activation and migration of lymphocytes, including NK cells, to the lungs, where they could be one of the main producers of IFN-γ (148).
T183	32052-32187	Sentence	denotes	Under normal conditions, human lung NK cells are typically hyporesponsive but dynamically migrate in and out of pulmonary tissues (83).
T184	32188-32362	Sentence	denotes	This supports the hypothesis that during infectious respiratory diseases, an increased recruitment of hyperresponsive NK cells would worsen the festering immunopathology (8).
T185	32363-32607	Sentence	denotes	In fact, through Viral-Track scanning of unmapped single-cell RNA-sequencing data, Bost et al. showed that patients with severe COVID-19 exhibited a hyperinflammatory response with an enriched and highly proliferative NK cell compartment (142).
T186	32608-32886	Sentence	denotes	High levels of IFN-γ leads to epithelial and endothelial cell apoptosis and vascular leakage, suboptimal T cell response, accumulation of alternatively activated macrophages and altered tissue homeostasis, and ARDS (19), all of which may contribute to COVID-19 disease severity.
T187	32887-33152	Sentence	denotes	In summary, the evidence is consistent with the hypothesis that NK cells are involved in the cytokine storm associated with CoV infection and that this hyper-cytokinemia contributes significantly to disease severity via inflammation-mediated lung damage (Figure 1).
T188	33153-33478	Sentence	denotes	Figure 1 Hypothesized dual role of NK cells during coronavirus pathogenesis. (A) Healthy Natural Killer (NK) cells in low-risk individuals recognize SARS-CoV-2 infected cells via recognition of viral proteins on the surface of infected cells and through sensing of cytokines and chemokines produced in response to infection.
T189	33479-33806	Sentence	denotes	These cells are hypothesized to be able to directly induce apoptosis through death receptor ligation, antibody-dependent cell-mediated cytotoxicity (ADCC), and through the release of cytotoxic granules, in addition to indirectly targeting virally infected cells via modulation of the immune response through cytokine secretion.
T190	33807-34099	Sentence	denotes	An effective innate immune response may be able to clear SARS-CoV-2 infection and leave the patient's lungs undamaged. (B) High risk individuals may have dysfunctional NK cells which may not recognize and respond to SARS-CoV-2 infection due to immune evasion strategies employed by the virus.
T191	34100-34335	Sentence	denotes	It is hypothesized that an accumulation of infected epithelial cells and innate immune cells, monocyte-macrophages and neutrophils, release cytokines, and chemokines which further recruit immune cells, including NK cells, to the lungs.
T192	34336-34403	Sentence	denotes	This may result in the induction of a cytokine storm, led by IFN-γ.
T193	34404-34631	Sentence	denotes	This inflammatory state could act as the catalyst for the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), contributing to the significant morbidity, and mortality associated with COVID-19.
T194	34632-34790	Sentence	denotes	SARS-CoV-2 infection is associated with reduced NK cell levels and an exhausted phenotype which may impede viral clearance, in addition to severe lung damage.
T195	34791-34896	Sentence	denotes	Interestingly, this duality of NK cell roles mirrors what is seen in critically ill patients with sepsis.
T196	34897-35262	Sentence	denotes	Studies suggest that while early NK cell stimulation and IFN-γ production is beneficial to combat infections, excessive and prolonged stimulation of NK cells leads to reduced NK cell numbers and an exhausted phenotype and was associated with increased systemic inflammation in systemic inflammatory response syndrome (SIRS)/sepsis and increased mortality (149–152).
T197	35263-35385	Sentence	denotes	This review of the literature suggests that NK cells may play an important role in both CoV clearance and immunopathology.
T198	35386-35546	Sentence	denotes	The continued probing of NK cell involvement is essential for a more complete understanding of CoV pathophysiology and for the deployment of immunotherapeutics.
T199	35547-35891	Sentence	denotes	Depending on the patient, the stage of disease, and other still poorly understood factors, it may be necessary to either boost NK cell activity to ensure viral clearance, e.g., at exposure or during early infection, or to finely tune NK cell effector functions in late stage infections to prevent hyper-cytokinemia and inflammatory lung damage.
T200	35892-36045	Sentence	denotes	Indeed, all CoVs that infect humans are zoonoses and there is an extensive reservoir of CoVs that could serve as a source for future pandemics (14, 153).
T201	36046-36277	Sentence	denotes	Therefore, a broader understanding of the immune response to coronaviruses and insights into therapeutic implications will be of significant value not only for the current COVID-19 pandemic, but also for potential future pandemics.
T202	36279-36325	Sentence	denotes	Flattening the Curve With Natural Killer Cells
T203	36326-36474	Sentence	denotes	The race to vaccinate and find a cure for COVID-19 has resulted in a spectacular effort from researchers and medical practitioners around the world.
T204	36475-36668	Sentence	denotes	Early attempts at creating targeted therapeutics have mostly relied on historical evidence from related, but not identical, coronaviruses and on the paucity of studies investigating SARS-CoV-2.
T205	36669-36884	Sentence	denotes	These strategies have attempted to combat the virus by targeting various stages of its life cycle starting with neutralizing SARS-CoV-2 virions using monoclonal antibodies or plasma from convalescent patients (154).
T206	36885-37028	Sentence	denotes	The entry mechanism of CoVs has been shown to rely on binding the ACE2 receptor and using proteases such as TMPRSS2 for S protein priming (52).
T207	37029-37212	Sentence	denotes	Thus, preventing ACE2 receptor binding through blocking antibodies or competitive binding with soluble ACE2 and TMPRSS2 protease inhibitors (Camostat mesylate) are being tested (155).
T208	37213-37423	Sentence	denotes	Upon viral entry, the viral proteolysis or replication cycle can be targeted with protease inhibitors (Lopinovir and Ritonavir) (156) or RNA-dependent RNA polymerase inhibitors (Remdesivir and Ribavirin) (157).
T209	37424-37631	Sentence	denotes	At the time of writing this review, the results of these trials have not been released or are still preliminary and will require further evaluation to assess their clinical efficacy in larger cohort studies.
T210	37632-37907	Sentence	denotes	As NK cell activity is critical for viral clearance and may be involved in disease immunopathology, a rapid and reliable predictor of NK cell function may allow for the prediction of clinical progression and the stratification of patients to receive therapeutic intervention.
T211	37908-38076	Sentence	denotes	The remainder of this review will discuss the various ways immunotherapies are being deployed to tackle COVID-19, with a focus on therapies that use NK cells (Table 1).
T212	38077-38335	Sentence	denotes	Lastly, while NK cells play an important role in combating viral infections, we also need to be fully cognizant of the potential damage immunotherapies could have in severe cases of COVID-19, and how these adverse effects may need to be attenuated (Table 2).
T213	38336-38411	Sentence	denotes	Table 1 List of COVID-19 clinical trials using immunomodulatory therapies.
T214	38412-38545	Sentence	denotes	Category Therapeutic(s) Trial identifier Phase Location (centers) Patients (n) Study eligibility Mechanism of action(s)
T215	38546-38568	Sentence	denotes	NK cell-based products
T216	38569-38682	Sentence	denotes	Adoptive NK cells NK cells NCT04280224 I Henan, China 30 Pneumatic COVID19+ Adoptive NK cell therapy
T217	38683-38801	Sentence	denotes	CYNK-001 NCT04365101 I/II New Jersey, USA 86 Mild COVID19+ From human placental CD34+ cells and culture-expanded
T218	38802-38955	Sentence	denotes	NK cells isolated from healthy donor PBMCs NCT04344548 I/II Bagota, Colombia 10 NEWS of >4 Isolated NK cells ex vivo stimulated with IL-2 and IL-15
T219	38956-39284	Sentence	denotes	CAR-NK Cells NKG2D-ACE2 CAR-NK cell therapy from umbilical cord blood NCT04324996 I/II Chongqing, China 90 Within <14 dpi IL-15 prolongs NK cell lifespan; GM-CSF neutralizing scFV reduces recruitment of inflammatory cells; NKG2D-ACE2 CAR-NK cells can target virally infected cells; ACE2 CAR-NK can act as decoy cell
T220	39285-39309	Sentence	denotes	NK cell immunostimulants
T221	39310-39335	Sentence	denotes	Direct NK cell activation
T222	39336-39569	Sentence	denotes	IFN-α Therapy Recombinant human IFN-α ± Thymosin alpha 1 NCT04320238 III Hubei, China 2,944 Uninfected HCW IFN-alpha boosts immune system; thymosin alpha 1 activates TLRs in myeloid and pDCs leading to NK cell activation
T223	39570-39685	Sentence	denotes	Recombinant human IFN-α2β NCT04293887 I Hubei, China 328 Within <7 dpi IFN-alpha activates interferon pathway
T224	39686-39795	Sentence	denotes	Abidol Hydrochloride ± IFN atomization (Peg-IFN-α-2b) NCT04254874 IV Hubei, China 100 Pneumatic COVID19+
T225	39796-39895	Sentence	denotes	Rintatolimod ± Recombinant IFN-α2β NCT04379518 I/II New York, USA 80 Mild to moderate COVID19+
T226	39896-40040	Sentence	denotes	IFN-β Therapy Lopinavir/ritonavir ± IFN-β-1a NCT04315948 III France (3) 3,100 Moderate/Severe IFN-beta activates interferon pathway
T227	40041-40173	Sentence	denotes	Base therapy ± IFN-β-1a NCT04350671 IV Tehran, Iran 40 Within <10 dpi Base therapy = Hydropinchloroquine + Lopinavir/Ritonavir
T228	40174-40292	Sentence	denotes	Base therapy ± IFN-β-1b NCT04276688 II Hong Kong, HK 127 NEWS of ≥1 Base therapy = Lopinavir/ritonavir/Ribavirin
T229	40293-40440	Sentence	denotes	IFN-β-1a vs. IFN-β-1b (+Base therapy) NCT04343768 IV Tehran, Iran 60 Moderate/Severe Base therapy = Hydropinchloroquine + Lopinavir/Ritonavir
T230	40441-40468	Sentence	denotes	Indirect NK cell activation
T231	40469-40660	Sentence	denotes	IFN-λ Therapy Peg-IFN-Lambda-1a NCT04331899 II California, USA 120 Early COVID19+ IFN-lambda to boost NK cells indirectly through Monocytes, Macrophages, and pDCs IL-12 secretion
T232	40661-40728	Sentence	denotes	Peg-IFN-Lambda-1a NCT04343976 II Boston, USA 40 Early COVID19+
T233	40729-40814	Sentence	denotes	Peg-IFN-Lambda-1a NCT04354259 II Toronto, Canada 140 Ambulatory and hospitalized
T234	40815-40886	Sentence	denotes	Peg-IFN-Lambda-1a NCT04388709 II New York, USA 66 Non-ICU COVID19+
T235	40887-40964	Sentence	denotes	Peg-IFN-Lambda-1a NCT04344600 II Maryland, USA 164 Asymptomatic COVID19+
T236	40965-40991	Sentence	denotes	Immunoregulatory therapies
T237	40992-41219	Sentence	denotes	Immune checkpoint blockade Anti-PD-1 (vs. Thymosin) NCT04268537 II Nanjing, China 120 Respiratory failure within <48h of ICU Prevent T-cell regulation by blocking PD-1; in COVID19+ advanced or metastatic cancer patients;
T238	41220-41342	Sentence	denotes	Anti-PD-1 (pembrolizumab) + tocilizumab NCT04335305 II Nanjing, China 24 Pneumatic COVID19+ Tocilizumab = anti-IL-6R
T239	41343-41454	Sentence	denotes	Anti-PD-1 (nivolumab) vs. GNS651 NCT04333914 II France (4) 273 Early COVID19+ GNS651 = Chloroquine analog
T240	41455-41493	Sentence	denotes	Non-specific innate immune stimulation
T241	41494-41670	Sentence	denotes	Heterologous vaccines BCG NCT04328441 III Netherlands (9) 1,500 Uninfected HCW Trains and primes innate immunity against subsequent non-specific pathogen infection
T242	41671-41746	Sentence	denotes	BCG (Danish strain) NCT04327206 III Australia (8) 4,170 Uninfected HCW
T243	41747-41812	Sentence	denotes	BCG (Danish strain) NCT04350931 III Egypt 900 Uninfected HCW
T244	41813-41874	Sentence	denotes	BCG (Danish strain) NCT04379336 III South Africa 500 HCW
T245	41875-41939	Sentence	denotes	BCG (Tice strain) NCT04348370 IV USA (5) 700 Uninfected HCW
T246	41940-42002	Sentence	denotes	BCG NCT04369794 IV São Paulo, Brazil 1,000 Early COVID19+
T247	42003-42155	Sentence	denotes	TLR agonists PUL-042 (CpG ODN) NCT04313023 II Not listed 200 Unifected/asymptomatic Activates TLR2/6/9 leading to innate immune stimulation
T248	42156-42222	Sentence	denotes	PUL-042 (CpG ODN) NCT04312997 II Texas, USA 100 Within <7 dpi
T249	42223-42272	Sentence	denotes	Natural health products and alternative medicines
T250	42273-42434	Sentence	denotes	Vitamins Vitamin C NCT04323514 N/A Palermo, Italy 500 Pneumatic COVID19+ General immune boosting properties of vitamin C and natural health products
T251	42435-42494	Sentence	denotes	Vitamin C NCT04264533 II Hubei, China 140 ICU COVID19+
T252	42495-42557	Sentence	denotes	Vitamin C NCT03680274 III Quebec, Canada 800 ICU COVID19+
T253	42558-42618	Sentence	denotes	Vitamin C NCT04344184 II Virginia, USA 200 ICU COVID19+
T254	42619-42690	Sentence	denotes	Vitamin C + Zinc NCT04342728 N/A Ohio, USA 520 Outpatient COVID19+
T255	42691-42806	Sentence	denotes	Hydroxychloroquine; Azithromycin; Vitamin C, D; and Zinc NCT04334512 II California, USA 600 Low risk COVID-19+
T256	42807-42904	Sentence	denotes	Hydroxychloroquine; vitamin C, D; and Zinc NCT04335084 II California, USA 600 Uninfected HCW
T257	42905-43039	Sentence	denotes	Vitamin D NCT04334005 N/A Spain (2) 200 Non-severe COVID19+ Vitamin D is immunomodulatory and prevents nutritional deficiencies.
T258	43040-43210	Sentence	denotes	Zinc + Vitamin D (cholecalciferol) NCT04351490 N/A Lille, France 3,140 Asymptomatic COVID19+ To treat zinc and vitamin D deficiency and reduce inflammation and ARDS
T259	43211-43287	Sentence	denotes	High dose Vitamin D (4X) NCT04344041 III France (9) 260 Severe COVID19+
T260	43288-43410	Sentence	denotes	NEWS, National Early Warning Score; HCW, Healthcare Workers; Peg, pegylated; dpi, days post-infection; As of June 1, 2020.
T261	43411-43514	Sentence	denotes	Table 2 List of COVID-19 clinical trials investigating immunotherapies for mitigating immunopathology.
T262	43515-43648	Sentence	denotes	Category Therapeutic(s) Trial identifier Phase Location (centers) Patients (n) Study eligibility Mechanism of action(s)
T263	43649-43670	Sentence	denotes	Anti-cytokine therapy
T264	43671-43841	Sentence	denotes	Anti IL-6 Tocilizumab NCT04315480 II Italy 38 Pneumatic COVID19+ Anti-IL6R mAB to prevent virus-related cytokine storm and reduce symptoms of severe COVID-19
T265	43842-43907	Sentence	denotes	Tocilizumab NCT04317092 II Italy (27) 400 Pneumatic COVID19+
T266	43908-43974	Sentence	denotes	Tocilizumab NCT04320615 III Not listed 330 Pneumatic COVID19+
T267	43975-44035	Sentence	denotes	Tocilizumab NCT04332913 N/A Italy 30 Pneumatic COVID19+
T268	44036-44106	Sentence	denotes	Tocilizumab NCT04335071 II Switzerland (3) 100 Pneumatic COVID19+
T269	44107-44213	Sentence	denotes	Tocilizumab NCT04322773 II Denmark (2) 200 Pneumatic COVID19+ I.V. vs. S.C. routes of administration
T270	44214-44359	Sentence	denotes	Tocilizumab NCT04331795 II Illinois, USA 50 COVID19+, not on ventilator Low (80mg) vs. standard dose (200mg) in non-critically ill patients
T271	44360-44499	Sentence	denotes	Tocilizumab NCT04346355 II Italy (24) 398 Pneumatic COVID19+, non-ICU Early administration of tocilizumab on reduced ventilation time
T272	44500-44580	Sentence	denotes	Tocilizumab NCT04331808 II Paris, France 240 Group 1: non-ICU; Group 2: ICU
T273	44581-44705	Sentence	denotes	Tocilizumab (vs. CRRT) NCT04306705 N/A Hubei, China 120 Pneumatic COVID19+ CRRT = continuous renal replacement therapy
T274	44706-44823	Sentence	denotes	Tocilizumab (vs. Anakinra) NCT04339712 II Greece (17) 20 Pneumatic COVID19+, non-ICU Anakinra - IL1r antagonist
T275	44824-44992	Sentence	denotes	Tocilizumab (vs. GNS651) NCT04333914 II France (4) 273 Pneumatic COVID19+ Efficacy in COVID19+ advanced or metastatic cancer patients; GNS651 = chloroquine analog
T276	44993-45090	Sentence	denotes	Sarilumab NCT04315298 II/III USA (57) 400 Pneumatic COVID19+ Low vs. high dose of sarilumab
T277	45091-45170	Sentence	denotes	Sarilumab NCT04324073 II/III France (4) 239 Group 1: non-ICU; Group 2: ICU
T278	45171-45245	Sentence	denotes	Sarilumab NCT04327388 II/III Canada+France (8) 300 Pneumatic COVID19+
T279	45246-45368	Sentence	denotes	Hydroxychloroquine + Axithromycin ± Tocilizumab NCT04332094 II Barcelona, Spain 276 Early COVID19+, not on ventilator
T280	45369-45439	Sentence	denotes	Tocilizumab + Favipiravir NCT04310228 N/A China (11) 150 COVID19+
T281	45440-45592	Sentence	denotes	Tocilizumab + Anakinra + Siltuximab NCT04330638 III Belgium (9) 342 Pneumatic COVID19+ Anakinra - IL1r antagonist and Siltuximab - IL6r antagonist
T282	45593-45732	Sentence	denotes	Anti-IL-8 Anti-IL-8 (BMS-986253) NCT04347226 II New York, USA 138 Pneumatic COVID19+ Prevent recruitment of inflammatory cells
T283	45733-45892	Sentence	denotes	Anti IL-1R/Anti IFNγ Anakinra vs. Emapalumab NCT04324021 II/III Italy (4) 54 Pneumatic COVID19+ Anakinra (IL1r antagonist); Emapalumab (anti-IFNγ)
T284	45893-45965	Sentence	denotes	Anakinra ± Ruxolitinib NCT04366232 II France (3) 54 Severe COVID19+
T285	45966-46124	Sentence	denotes	Anti-GM-CSF Mavrilimumab NCT04337216 II Virginia, USA 10 Pneumatic COVID19+ GM-CSF is one of the main mediators of CRS in severe COVID19 patients
T286	46125-46273	Sentence	denotes	Jak Inhibitor Baricitinib NCT04399798 II Pavia, Italy 13 Pneumatic COVID19+ Inhibits JAK1-/JAK2-mediated cytokine release and TNF-alpha
T287	46274-46364	Sentence	denotes	Ritonavir ± Baricitinib NCT04320277 III Tuscany, Italy 60 Moderate/pneumonia COVID19+
T288	46365-46542	Sentence	denotes	Extracorporeal adsorption CytoSorb absorber NCT04324528 N/A Freiburg, Germany 30 Pneumatic COVID19+ “Absorbs” IL-6 in effort to reduce inflammation in ARDS patients
T289	46543-46562	Sentence	denotes	Anti-inflammatories
T290	46563-46737	Sentence	denotes	Corticosteroid Ciclesonide ± Hydroxychloroquine NCT04330586 II Seoul, Korea 141 Early COVID19+ (within 7 days) Ciclesonide is an anti-inflammatory corticosteroid
T291	46738-46855	Sentence	denotes	Prednisone NCT04344288 II Bron, France 304 Pneumatic COVID19+ Prednisone is an anti-inflammatory corticosteroid
T292	46856-46959	Sentence	denotes	Hydrocortisone NCT04348305 III Denmark 1000 Pneumatic COVID19+ Low-dose hydrocortisone for 7 days
T293	46960-47092	Sentence	denotes	Dexamethasone NCT04344730 N/A Paris, France 550 COVID19+ ICU within 48hrs Dexamethasone is an anti-inflammatory corticosteroid
T294	47093-47171	Sentence	denotes	Dexamethasone NCT04325061 IV Spain (24) 200 Severe COVID19+ on ventilator
T295	47172-47236	Sentence	denotes	Dexamethasone NCT04327401 III Brazil (21) 290 ARDS patients
T296	47237-47364	Sentence	denotes	Methylprednisolone NCT04343729 II Brazil 420 Pneumatic COVID19+ Methylprednisolone is an anti-inflammatory corticosteroid
T297	47365-47480	Sentence	denotes	Methylprednisolone (vs. Tocilizumab) NCT04345445 III Kuala Lumpur, Malaysia 310 COVID19+ with high risk of CRS
T298	47481-47576	Sentence	denotes	Methylprednisolone (vs. Siltuximab) NCT04329650 II Barcelona, Spain 200 Pneumatic COVID19+
T299	47577-47673	Sentence	denotes	NSAID Naproxen NCT04325633 III Paris, France 584 Pneumatic COVID19+ COX-2 inhibitor
T300	47674-47747	Sentence	denotes	Ibuprofen NCT04334629 IV Not listed 230 NEWS2 > 5 overall, Pneumatic
T301	47748-47828	Sentence	denotes	NEWS, National Early Warning Score; HCW, Healthcare Workers; As of June 1, 2020.
T302	47830-47852	Sentence	denotes	NK Cell-Based Products
T303	47853-48024	Sentence	denotes	In the absence of a clinically approved vaccine against SARS-CoV-2, scientists have begun developing therapeutics to halt the spread of COVID-19 by alternative strategies.
T304	48025-48243	Sentence	denotes	Studies have reported that patients infected with SARS-CoV-2 have lower levels of circulating NK cells and these express a greater level of inhibitory receptors (e.g., NKG2A) while producing less IFN-γ (127, 129, 130).
T305	48244-48344	Sentence	denotes	These findings provide a rationale for pursuing NK cell-based therapies as a tool to fight COVID-19.
T306	48345-48513	Sentence	denotes	Although NK cell-based therapies have mostly been developed for use against cancer, similar concepts and mechanisms could provide guidance in the fight against viruses.
T307	48514-48790	Sentence	denotes	Therapeutic NK cell products can be thought of as “living drugs” as they generally use either primary NK cells isolated from peripheral blood mononuclear cells (PBMCs) or are generated from stem cell precursors or genetically engineered immortalized human NK cell lines (158).
T308	48791-48946	Sentence	denotes	Primary NK cell products are often pre-treated and expanded in vitro with cytokines or via co-culture with target cells before being infused into patients.
T309	48947-49186	Sentence	denotes	Patients can also receive immune stimulants [e.g. recombinant IL-2 (159) or IL-15 (160)] with the goal of improving the in vivo activity and persistence of the NK cell products (161) as is being tested in this COVID-19 trial (NCT04344548).
T310	49187-49443	Sentence	denotes	The first cell-based investigational drug to be approved by the FDA for clinical testing in COVID-19 patients is an allogeneic, off-the-shelf, cryopreserved NK cell therapy made by Celularity (CYNK-001), originally developed for cancer immunotherapy (162).
T311	49444-49493	Sentence	denotes	The trial (NCT04365101) is split into two Phases.
T312	49494-49669	Sentence	denotes	Phase I will assess the frequency and severity of adverse events in mild, non-ICU COVID-19 patients (n = 14) following infusion of NK cells derived from placental CD34+ cells.
T313	49670-49793	Sentence	denotes	The subsequent Phase II trial will recruit up to 72 patients and include a standard of care comparator at a 1:1 allocation.
T314	49794-49882	Sentence	denotes	Genetically modified NK cells are also being investigated for efficacy against COVID-19.
T315	49883-50234	Sentence	denotes	Chimeric antigen receptor NK cells (CAR-NK cells) are engineered to express virtually any receptor(s) of interest and were originally designed to enhance the ability of NK cells to eliminate cancer cells via receptors targeting EGFR (163) or CD19 (164), which are present on many cancer types and B cell hematological malignancies, respectively (164).
T316	50235-50552	Sentence	denotes	Although the efficacy of CAR-NK cells to control viral infections has yet to be rigorously tested in large scale clinical trials, the promising safety profile of CAR-NK cells in cancer patients, who are often immunocompromised, suggests that CAR-NK therapy can be well-tolerated in early phase/mild COVID-19 patients.
T317	50553-50729	Sentence	denotes	Notably, CAR-NK cells are considered “safe” largely because they are less likely to lead to cytokine release syndrome (CRS), a severe adverse event of CAR-T cell therapy (165).
T318	50730-50867	Sentence	denotes	But as these are unchartered waters, it is critical that CAR-NK cells are used cautiously and not given to late/severe COVID-19 patients.
T319	50868-51118	Sentence	denotes	A Phase I/II study in early stage COVID-19 patients (within 14 days of illness) employing CAR-NK cell therapy is currently being tested using off-the-shelf NK cells derived from human umbilical cord blood expressing NKG2D and ACE2 CARs (NCT04324996).
T320	51119-51332	Sentence	denotes	This complex five-arm study will compare the efficacy of different CAR-NK constructs: (i) NK cells, (ii) NK cells secreting IL-15, (iii) NKG2D CAR-NK cells, (iv) ACE2 CAR-NK cells, and (v) NKG2D-ACE2 CAR-NK cells.
T321	51333-51595	Sentence	denotes	NKG2D CAR-NK cells have shown promising preclinical results in cancer studies (166), and although not proven for SARS-CoV-2, the rationale for expressing NKG2D derives from work showing that NKG2D-ligands (NKG2DL) are upregulated on virally infected cells (167).
T322	51596-51966	Sentence	denotes	Similarly, the investigators hypothesize that expressing ACE2 on NK cells will facilitate the elimination of SARS-CoV-2 virions and infected cells by binding the viral spike proteins–but it is unknown whether or not CAR-NK cells can eliminate virions or if infected cells display sufficient levels of spike protein to be recognized by ACE2-NK cells upon viral infection.
T323	51967-52168	Sentence	denotes	The investigators also suggest that expressing ACE2 on NK cells may also have a secondary benefit as a decoy cell that will be infected by the virus thereby indirectly protecting lung epithelial cells.
T324	52169-52375	Sentence	denotes	As described previously, it is unclear whether this strategy will work to stop viral spread to healthy epithelial cells or if it will serve to perpetuate viral spread if the virus can replicate in NK cells.
T325	52376-52554	Sentence	denotes	In arms ii-v of this trial, the CAR-NK cells have been engineered to secrete IL-15 based on studies showing improved in vivo persistence of CAR-NK cells in cancer patients (168).
T326	52555-52894	Sentence	denotes	However, the addition of the proinflammatory cytokine IL-15 to this treatment strategy should be monitored closely for life-threatening toxicities, as elevated IL-15 has been previously reported to accompany chronic pulmonary inflammatory diseases (169) and MERS-CoV infection (170) even if no correlation has been reported for SARS-CoV-2.
T327	52895-53132	Sentence	denotes	Interestingly, a study compared IL-15 levels from lung tissue homogenates following SARS-CoV infection in aged vs. juvenile monkeys and showed that IL-15 concentrations were only elevated in juvenile monkeys 10 days post-infection (171).
T328	53133-53310	Sentence	denotes	This study would suggest that IL-15 therapy may be tolerated and effective in older COVID-19 patients that may not be able to produce IL-15, however this has not been confirmed.
T329	53311-53613	Sentence	denotes	Lastly, all the CAR-NK cells in this trial secrete GM-CSF neutralizing scFv antibodies, since this cytokine has a known role in CRS in cancer patients treated with CAR-T cells (172), and has been shown to be correlated with COVID-19 disease severity in association with pathogenic CD4+ Th1 cells (173).
T330	53614-53836	Sentence	denotes	Although NK cell based therapies are versatile, have shown safety and efficacy in cancer patients, and can be utilized in immunocompromised individuals, their potential has yet to be fully realized as an antiviral therapy.
T331	53837-53958	Sentence	denotes	Furthermore, the logistics of manufacturing NK cell products (cost and time) may pose limitations and barriers to access.
T332	53959-54091	Sentence	denotes	For this reason, therapies focused on stimulating a patient's own NK cells offer many advantages over adoptive transfer of NK cells.
T333	54093-54124	Sentence	denotes	Interferon Therapy and NK Cells
T334	54125-54306	Sentence	denotes	The importance of the interferon pathway is underscored by the fact that many viruses actively interfere with host interferon responses, for which coronaviruses are a prime example.
T335	54307-54427	Sentence	denotes	As described above, CoVs utilize numerous tactics to avoid elimination by disrupting the host type I IFN response (174).
T336	54428-54602	Sentence	denotes	Therefore, since the majority of CoVs fail to induce any detectable type I IFN response, eliciting a type I IFN response is a very attractive therapeutic strategy (118, 175).
T337	54603-54809	Sentence	denotes	Given the robust immunomodulatory nature of type I IFNs, uninfected or early symptomatic patients would benefit the most from this therapy to prevent exacerbating immunopathology at later stages of disease.
T338	54810-54891	Sentence	denotes	Numerous clinical trials have been initiated investigating type I IFNs (Table 1).
T339	54892-55112	Sentence	denotes	A large study (NCT04320238) of ~3,000 medical staff allocated participants to two trial arms: (i) low-risk (non-isolated wards or laboratories) or (ii) high-risk (isolated wards in direct contact with COVID-19 patients).
T340	55113-55306	Sentence	denotes	In addition to the IFN-α-1b nasal drops, high-risk medical staff will also receive the immune-modulating TLR activator, thymosin α1, which indirectly activates NK cells through pDCs (176, 177).
T341	55307-55461	Sentence	denotes	Interestingly, reports in SARS-CoV-1 studies showed that IFN-β therapy had a 50-fold greater anti-viral activity in Vero cells than IFN-α treatment (178).
T342	55462-55942	Sentence	denotes	Promising results have been published from a Phase II study (NCT04276688) (179), showing that complementing lopinavir-ritonavir and ribavirin with subcutaneous IFN-β-1b in mild-to-moderate COVID-19 patients is safe with no serious adverse events reported in the triple combination therapy group, and highly effective, with significant and clinically meaningful reductions in time to complete alleviation of symptoms, hospital length of stay, and time to negative viral load (179).
T343	55943-56193	Sentence	denotes	Despite our best efforts in timing type I IFN therapy to mitigate immunopathology, these treatments still increase the risk of excessive activation of proinflammatory signals, which could damage host tissues and perpetuate immunopathology (180, 181).
T344	56194-56298	Sentence	denotes	For this reason, alternative therapeutic avenues to direct type I IFN administration are being explored.
T345	56299-56465	Sentence	denotes	Type III IFNs can be a valid alternative to type I IFNs, because they maintain antiviral functions yet are less toxic and less prone to mediate immunopathology (182).
T346	56466-56644	Sentence	denotes	The type III IFN, IFN-λ, activates NK cells indirectly (compared to type I IFNs which directly act on NK cells), resulting in a less potent and slower immune response (183, 184).
T347	56645-56767	Sentence	denotes	IFN-λ activates NK cells by stimulating macrophages to produce IL-12 which in turn induce NK cells to produce IFN-γ (185).
T348	56768-56902	Sentence	denotes	Pegylated IFN-λ is being tested in COVID-19 positive patients with mild symptoms in the absence of respiratory distress (NCT04331899).
T349	56903-57085	Sentence	denotes	While IFN-λ can lead to the eventual activation of NK cells, its primary utility is in preventing the tissue damaging potential of neutrophils at mucosal surfaces, such as the lungs.
T350	57086-57272	Sentence	denotes	However, IFN-λ also has been shown to reduce the rate of tissue repair, which in the context of COVID-19 which has a long disease course, could mean greater risk of secondary infections.
T351	57273-57517	Sentence	denotes	Since exogenous administration of any IFN therapy poses the risk of tipping the balance toward severe COVID-19 immunopathology, Broggi et al. assessed the levels of IFNs in upper and lower respiratory samples from healthy and COVID-19 patients.
T352	57518-57776	Sentence	denotes	In this preprint, they report that while the upper airway swabs showed similar mRNA expression levels of type I and III IFN compared to healthy controls, the BALF samples of severe COVID-19 patients had significantly elevated type I and III IFN levels (186).
T353	57777-57940	Sentence	denotes	Therefore, as with all of the therapies discussed in this review, careful consideration about safe and effective timing should guide our design of clinical trials.
T354	57942-57974	Sentence	denotes	Interleukin Therapy and NK Cells
T355	57975-58094	Sentence	denotes	In addition to IFN cytokine therapy, interleukin cytokine therapy can enhance the effector functions of NK cells (158).
T356	58095-58229	Sentence	denotes	The use of whole, unmodified recombinant cytokines as a monotherapy has resulted in minimal success in humans in cancer immunotherapy.
T357	58230-58380	Sentence	denotes	The earliest cytokine therapies to gain FDA-approval were IFN-α and recombinant IL-2, approved for renal cell carcinoma and metastatic melanoma (187).
T358	58381-58502	Sentence	denotes	Although approved, they were limited by their in vivo half-life, marginal anti-tumor activity, and associated toxicities.
T359	58503-58789	Sentence	denotes	The next generation of cytokine therapies were created to address these issues by first improving their biological stability through pegylation and fusion to chaperone molecules and secondly improving their specificity by fusing cytokines with antibodies or intratumoral administration.
T360	58790-58909	Sentence	denotes	These advances in the field have allowed for the reassessment of the therapeutic potential of specific cytokines (187).
T361	58910-59142	Sentence	denotes	Given the importance of IL-15 signaling and NK cell function, researchers have developed IL-15 “superagonists” which are IL-15:IL-15R heterodimers that have better in vivo stability and bioactivity compared to monomeric IL-15 (168).
T362	59143-59532	Sentence	denotes	Although at the time of writing IL-15 superagonists are not being studied for their efficacy in COVID-19 patients, IL-15 superagonists, such as ALT-803, are safe in humans (188) and have been used in conjunction with many of the therapies being discussed in this review including: CAR-NK cell therapy, adoptive NK cell transfers, checkpoint inhibitors, and the BCG vaccine in cancer (189).
T363	59533-59755	Sentence	denotes	It should be noted that although the therapeutic potential of cytokine therapy to specifically stimulate NK cells is enticing, exogenous cytokine therapy has a high risk for exacerbating CRS if given at the incorrect time.
T364	59757-59796	Sentence	denotes	Checkpoint Immunotherapies and NK Cells
T365	59797-59967	Sentence	denotes	Some viruses are known to induce a state of functional hyporesponsiveness in T cells that is essential for the productive establishment of chronic viral infections (190).
T366	59968-60108	Sentence	denotes	A vast body of literature has identified inhibitory checkpoint receptors, including CTLA4 and PD-1, as key regulators of this process (191).
T367	60109-60316	Sentence	denotes	Interestingly, cancer exploits similar mechanisms to escape the immune response, which provided the rationale for the introduction of antibodies targeting checkpoint receptors for cancer immunotherapy (192).
T368	60317-60560	Sentence	denotes	CTLA4 and PD-1/PD-L1 blockade have revolutionized cancer immunotherapy, and their success provides a strong rationale for the use of these drugs in COVID-19 patients, where emerging evidence suggests that the immune response is also subverted.
T369	60561-60729	Sentence	denotes	A clinical trial (NCT04268537) is currently assessing the efficacy of PD-1 blocking antibodies in severe COVID-19 patients within 48 h of reported respiratory distress.
T370	60730-60918	Sentence	denotes	PD-1 has also been shown to play a role in regulating NK cell responses, in addition to modulating T cell functions (193–197), and has been reportedly increased in COVID-19 patients (129).
T371	60919-61035	Sentence	denotes	Inhibitory receptors on the surface of NK cells regulate NK cell activation and can be targeted by antibody therapy.
T372	61036-61142	Sentence	denotes	One of the most promising is certainly the inhibitory receptor NKG2A, which binds to HLA-E (74, 198, 199).
T373	61143-61337	Sentence	denotes	NKG2A expression is increased in circulating (127) and BALF NK cells from COVID-19 patients, in contrast to NKG2C, an activating receptor closely related to NKG2A, which remains unchanged (129).
T374	61338-61553	Sentence	denotes	However, it is unclear whether the observed increase in NKG2A+ NK cells is due selective proliferation of NKG2A+ cells or if it is the result of NKG2A negative cells migrating out of circulation to infected tissues.
T375	61554-61778	Sentence	denotes	Circulating NK cells from patients with active hepatitis B disease had higher levels of NKG2A compared to patients without active disease, however antiviral administration was associated with a reduction in NKG2A expression.
T376	61779-61888	Sentence	denotes	Additionally, blocking NKG2A in vitro with NKG2A monoclonal antibodies led to improved NK cytotoxicity (200).
T377	61889-62175	Sentence	denotes	Given the association between NKG2A expression in patients with severe COVID-19 (127, 201), a promising avenue of investigation would be anti-NKG2A therapy, even in light of results showing that NKG2A+ NK cells are tuned to present a higher level of responsiveness to stimulation (202).
T378	62177-62238	Sentence	denotes	Indirect NK Cell Activation Through Innate Immune Stimulation
T379	62239-62487	Sentence	denotes	While NK cells can be stimulated directly by cytokines such as interferons and interleukins, their activity can also be enhanced through a by-stander effect following stimulation of other innate immune cells, such as macrophages and pDCs (Table 1).
T380	62488-62615	Sentence	denotes	This type of coordinated innate immune response may be more effective at CoV viral clearance and mitigation of severe COVID-19.
T381	62617-62659	Sentence	denotes	Trained Immunity and Heterologous Vaccines
T382	62660-62893	Sentence	denotes	Trained immunity has been recently described as an epigenetic re-wiring occurring in myeloid cells and progenitors upon stimulation that primes for a stronger response to subsequent stimuli, even of a different nature (90, 203, 204).
T383	62894-63101	Sentence	denotes	Whereas, the consensus is that myeloid cells are primarily responsible for trained immunity (205), it is likely that the resulting alteration in the cytokine milieu also has an effect on NK cells (204, 206).
T384	63102-63248	Sentence	denotes	This is the case for the BCG vaccine, which has been shown to provide non-specific protection against yellow fever viral infection (90, 207, 208).
T385	63249-63419	Sentence	denotes	The BCG vaccine is composed of a live attenuated strain of Mycobacterium bovis originally given to young children to protect against tuberculosis (M. tuberculosis) (209).
T386	63420-63627	Sentence	denotes	This vaccine provides an initial boost to innate immunity, but more importantly, results in the secretion of IL-1β from monocytes/macrophages, which feeds back to further stimulate the innate response (204).
T387	63628-63858	Sentence	denotes	The use of a heterologous vaccine to provide enhanced protection against non-specific/new pathogens makes this a compelling strategy against COVID-19 that warrants thorough investigation in randomized controlled trials (209, 210).
T388	63859-64123	Sentence	denotes	The BCG vaccine is undergoing clinical trials in healthcare workers in the Netherlands (NCT04328441), Australia (NCT04327206), Egypt (NCT04350931), and the USA (NCT04348370) to enhance overall innate immunity and provide heterologous protection against SARS-CoV-2.
T389	64124-64290	Sentence	denotes	Interestingly, an association was found that linked lower COVID-19-attributable mortality rates in countries using BCG in their national immunization schedules (211).
T390	64291-64467	Sentence	denotes	On the contrary, a study that assessed the association of childhood BCG vaccination in adults living in Israel did not show a beneficial difference in COVID-19 infection rates.
T391	64468-64723	Sentence	denotes	The discrepancy between these two reports likely stem from the fact that the latter study only included adults who were previously vaccinated during childhood, supporting the fact that heterologous vaccination may not result in long-term protection (212).
T392	64724-65013	Sentence	denotes	Childhood BCG immunization has a limited window of opportunity to protect younger individuals from infection (213), but it is hypothesized that reducing the number of infected children can have a meaningful impact on curbing the spread of COVID-19 to the rest of the population (206, 211).
T393	65014-65135	Sentence	denotes	Another heterologous vaccine in the process of clinical trial development for COVID-19 studies is IMM-101 (CCTG ID# IC8).
T394	65136-65299	Sentence	denotes	Created by Immodulon Therapeutics LTD, IMM-101 is composed of heat-killed Mycobacterium obuense and may have an improved safety profile over the BCG vaccine (214).
T395	65300-65483	Sentence	denotes	IMM-101 has been studied in multiple clinical trials for its non-specific immune stimulating properties as a cancer immunotherapy in pancreatic (215) and melanoma patients (216, 217).
T396	65485-65519	Sentence	denotes	Toll-like Receptor Agonist Therapy
T397	65520-65762	Sentence	denotes	Agonists of Toll-Like Receptors (TLRs) have been shown to broadly activate different immune populations and have had both preclinical and clinical success as adjuvants in vaccination and in the treatment of a variety of viral pathogens (218).
T398	65763-65939	Sentence	denotes	For example, CpG oligodeoxynucleotides (CpG ODNs) are short DNA sequences that contain unmethylated CpG dinucleotides which activate TLR9 particularly on DCs and B cells (219).
T399	65940-66125	Sentence	denotes	Bao et al. showed that their CpG ODN construct, BW001, had protective effects against SARS-CoV-1 in a mechanism that relied on NK cell activation likely through a DC intermediate (220).
T400	66126-66292	Sentence	denotes	Amidst the ongoing SARS-CoV-2 pandemic, two clinical trials (NCT04313023, NCT04312997) have opened using the TLR2/6/9 agonist, PUL-042, in order to prevent infection.
T401	66294-66333	Sentence	denotes	Immune-Boosting Natural Health Products
T402	66334-66479	Sentence	denotes	Ascorbic acid, more commonly known as vitamin C, has been shown to exhibit potent immunomodulatory, antioxidant, and antimicrobial effects (221).
T403	66480-66668	Sentence	denotes	Vitamin C has been shown to restore NK cell cytotoxicity in individuals exposed to toxic chemicals through protein kinase C expression, a critical component in lymphocyte metabolism (222).
T404	66669-66911	Sentence	denotes	Additional reports have shown that vitamin C also enhances the expression of NKp46, CD69, CD25 and IFN-γ production by NK cells (223) and can increase the expression of IRF3 in lung tissues of influenza infected, pneumonia-induced mice (224).
T405	66912-67049	Sentence	denotes	Vitamin C also harbors potent antioxidant attributes which can scavenge reactive oxygen species (ROS) and prevent lung injury (225, 226).
T406	67050-67234	Sentence	denotes	Although ROS production is an important component in the host defense response to viruses, they can be harmful to cells and lead to the pathogenesis of viral-induced host injury (227).
T407	67235-67517	Sentence	denotes	The underlying rationale to investigate the therapeutic potential of vitamin C has been based on two key observations: (i) critically ill patients have lower levels of vitamin C (228–230) and (ii) vitamin C has pleiotropic immunomodulatory, antioxidant, and antiviral effects (221).
T408	67518-67651	Sentence	denotes	It is important to underscore that reports on the clinical outcomes of vitamin C treatment in humans are mixed and context dependent.
T409	67652-67772	Sentence	denotes	A thorough meta-analysis on vitamin C supplementation for the common cold has been reported by Hemilä and Chalker (231).
T410	67773-67952	Sentence	denotes	Briefly, they concluded that while the incidence of colds was not reduced, the duration and severity of colds was reduced when assessing studies of regular vitamin C intake (231).
T411	67953-68127	Sentence	denotes	Interestingly, a separate meta-analysis on vitamin C and cardiac surgery showed a reduction in the length of ICU stay and shortened the need for mechanical ventilation (232).
T412	68128-68305	Sentence	denotes	This is an important correlation as clinical trials are currently investigating the efficacy of vitamin C to reduce mortality and hospital burden in COVID-19 patients (Table 1).
T413	68306-68453	Sentence	denotes	A Phase II clinical trial (NCT04264533) was initiated in Wuhan where COVID-19 patients will be given a high dose intravenous infusion of vitamin C.
T414	68454-68782	Sentence	denotes	Lastly, whether oral dosing of vitamin C can achieve therapeutically relevant concentrations, as described in the above studies, is currently unknown, thus caution should be taken as exceeding the recommended dietary allowance of 100–200 mg/day may lead to mild toxicities including abdominal discomfort and diarrhea (231, 233).
T415	68784-68838	Sentence	denotes	Mitigating Immunopathology in Severe COVID-19 Patients
T416	68839-68999	Sentence	denotes	The main cause of death for COVID-19 patients has been pulmonary complications and respiratory failure often as a result of an unregulated cytokine storm (234).
T417	69000-69195	Sentence	denotes	It is unclear whether the hyperinflammation seen in severe cases of COVID-19 is the result of the viral replication within pulmonary epithelial cells or an overactive/avalanching immune response.
T418	69196-69376	Sentence	denotes	However, studies in SARS-CoV-1 reported hyperinflammation in later stages of disease progression, despite reduced viral titers, suggesting that the damage was immune-mediated (19).
T419	69377-69497	Sentence	denotes	The most appropriate course of therapy can only be determined by elucidating the pathophysiology of disease progression.
T420	69498-69731	Sentence	denotes	Scientists and physicians, however, have had to respond quickly to the growing number of severe COVID-19 cases and this has resulted in therapy mainly through a combination of anti-inflammatory and anti-viral interventions (Table 2).
T421	69732-69859	Sentence	denotes	As described above, there is a potential for NK cells to contribute to the cytokine storm and therefore the development of ALI.
T422	69860-70091	Sentence	denotes	A possible explanation for the observed lymphopenia in COVID-19 patients is that NK cells and other lymphocytes migrate out of the circulation and into pulmonary tissues to aid in the elimination of infected epithelial cells (235).
T423	70092-70212	Sentence	denotes	This could be the premise for the large, unintended, amount of tissue damage that worsen the respiratory distress (148).
T424	70213-70353	Sentence	denotes	For this reason, therapeutics that dampen the immune response have been effective in mitigating immunopathology in severe COVID-19 patients.
T425	70354-70556	Sentence	denotes	The following review papers have thoroughly discussed many of these immunotherapies already (236–241), therefore, this section will focus on immunotherapies and their potential implications on NK cells.
T426	70558-70579	Sentence	denotes	Anti-cytokine Therapy
T427	70580-70772	Sentence	denotes	The main cytokines responsible for the life threatening respiratory distress seen in reported cases of severe COVID-19 are IL-2, IL-6, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP1A, and TNF-α (234).
T428	70773-70980	Sentence	denotes	Many clinical trials have focused on targeting IL-6 signaling with anti-IL-6R monoclonal antibodies (e.g., tocilizumab, sarilumab, siltuximab) because of the important role IL-6 has in propagating CRS (242).
T429	70981-71172	Sentence	denotes	Tocilizumab, in particular, is being used as the primary therapy in the majority of these trials, likely owing to its FDA approved status as a therapeutic for CRS in CAR-T cell therapy (243).
T430	71173-71394	Sentence	denotes	A case report demonstrated the potential for tocilizumab therapy in treating severe COVID-19 illness, where a single dose on day 24 of symptoms led to progressive reduction in IL-6 levels and resolution of symptoms (244).
T431	71395-71661	Sentence	denotes	A Phase III study (NCT04320615) led by Hoffman-La Roche is recruiting patients to study the safety and efficacy of tocilizumab therapy in a randomized, double-blind, placebo-controlled, multicenter study in over 300 patients with severe COVID-19 pneumonia (Table 2).
T432	71662-71845	Sentence	denotes	Targeting the IL-6 axis in severe COVID-19 patients may also serve to improve NK cell functions as Cifaldi et al. showed that increased IL-6 negatively impacts NK cell function (245).
T433	71846-71931	Sentence	denotes	They also showed that tocilizumab treatment improved NK cell function in vitro (245).
T434	71932-72072	Sentence	denotes	Mazzoni et al. recently reported that serum IL-6 levels were inversely correlated (p = 0.01) with NK cell function in COVID-19 ICU patients.
T435	72073-72249	Sentence	denotes	Additionally, in a small subset of COVID-19 ICU patients (n = 5), NK cells displayed improved markers of activation (granzyme A and perforin) after tocilizumab treatment (246).
T436	72250-72404	Sentence	denotes	Similar therapies have emerged in the fight against COVID-19 including an IL-1R antagonist (Anakinra; NCT04330638) (247) and Cytosorb (NCT04324528) (248).
T437	72405-72562	Sentence	denotes	High dose anakinra therapy has shown promising safety and efficacy in a small retrospective study, as part of the COVID-19 Biobank study (NCT04318366) (247).
T438	72563-72835	Sentence	denotes	Cytosorb therapy is used in conjunction with conventional dialysis through a whole blood cartridge-based filtration system designed to remove middle molecular weight molecules (which include inflammatory cytokines <75 kDa) through extracorporeal cytokine adsorption (248).
T439	72836-72981	Sentence	denotes	It is reported to be effective at removing Ferritin and IL-6 in a case study of a 14-year-old with severe CRS following CAR-T cell therapy (249).
T440	72982-73115	Sentence	denotes	Jak1/2 inhibitors (JAKi) are also undergoing clinical trials in moderate-severe COVID-19 patients, such as baricitinib (NCT04320277).
T441	73116-73305	Sentence	denotes	In addition to their ability to impede the production of IL-6, thus curb the excessive inflammation, they may also block clathrin mediated endocytosis–indicating a dual role for JAKi (241).
T442	73306-73497	Sentence	denotes	However, JAKi can also lead to the transient increase in NK cells as shown in baricitinib treated Rheumatoid Arthritis patients (250), which could be detrimental for severe COVID-19 patients.
T443	73499-73514	Sentence	denotes	Corticosteroids
T444	73515-73629	Sentence	denotes	Corticosteroids have played a key role in the treatment of auto-immune diseases over the past 70 years (251, 252).
T445	73630-73819	Sentence	denotes	Whether endogenous or exogenous, corticosteroids decrease the number of circulating monocytes and lymphocytes and decrease synthesis of pro-inflammatory cytokines (IL-2, IL-6, TNF-α) (251).
T446	73820-73997	Sentence	denotes	Their strong anti-inflammatory and immunosuppressive effects make them good candidates for rapidly suppressing inflammation during early auto-immune disease or viral infections.
T447	73998-74084	Sentence	denotes	Corticosteroids have been shown to inhibit NK cells in ex vivo experiments (253, 254).
T448	74085-74308	Sentence	denotes	While corticosteroids may delay clearance of infections, their major benefit lies in suppressing excessive innate immune responses, thus preventing lung damage and ARDS commonly present in severe viral infections (255–257).
T449	74309-74431	Sentence	denotes	In fact, this was the main rationale for the widespread use of corticosteroids during MERS and SARS infections (255, 256).
T450	74432-74631	Sentence	denotes	Specific to COVID-19, some groups have advocated for the use of low-dose corticosteroids in a specific subset of critically-ill patients with refractory ARDS, sepsis, or septic shock (Table 2) (257).
T451	74632-74794	Sentence	denotes	There is one known ongoing randomized clinical trial examining the effect of the corticosteroid ciclesonide in adults with mild COVID-19 infections (NCT04330586).
T452	74795-74908	Sentence	denotes	This trial is based on preclinical studies showing in vitro antiviral activity of ciclesonide against SARS-CoV-2.
T453	74909-75175	Sentence	denotes	While there may be a benefit to using corticosteroids in a subset of critically-ill patients with refractory ARDS or sepsis (257), their routine use in COVID-19 is not recommended outside of clinical trials, based on expert opinion and WHO recommendations (258–260).
T454	75176-75317	Sentence	denotes	Corticosteroids also cause a multitude of side effects, most notably diabetes mellitus, osteoporosis, and increased risk of infections (251).
T455	75318-75509	Sentence	denotes	Controversially, a 2019 systematic review of over 6,500 influenza patients showed that corticosteroids actually led to increased mortality, length of ICU stay, and secondary infections (261).
T456	75510-75768	Sentence	denotes	Additionally, one retrospective observational study examined the use of corticosteroids in 31 COVID-19 patients, and reported no significant association between corticosteroids and viral clearance time, hospital length of stay, or duration of symptoms (262).
T457	75769-75851	Sentence	denotes	These studies highlight the need to be vigilant in our attempts to fight COVID-19.
T458	75853-75899	Sentence	denotes	Non-steroidal Anti-inflammatory Drugs (NSAIDs)
T459	75900-76039	Sentence	denotes	Non-steroidal anti-inflammatory drugs, or NSAIDs, are one of the most commonly prescribed drugs for treating fever, pain, and inflammation.
T460	76040-76129	Sentence	denotes	NSAIDs include over-the-counter household names such as ibuprofen, naproxen, and aspirin.
T461	76130-76308	Sentence	denotes	Given the widespread use of these medications it is appropriate that researchers have investigated the potential benefits and harms of NSAIDs in patients diagnosed with COVID-19.
T462	76309-76473	Sentence	denotes	Thus far, the evidence for using NSAIDs in the context of CoVs are mixed and might not be generalizable to all NSAIDs as reports tended to focus on specific NSAIDs.
T463	76474-76629	Sentence	denotes	These studies also focused on the potential for NSAIDs to act as an antiviral, with a potential added benefit of being able to treat inflammatory symptoms.
T464	76630-76777	Sentence	denotes	One report showed that the NSAID indomethacin could directly inhibit SARS-CoV replication in Vero cell monolayers in a dose-dependent manner (263).
T465	76778-77046	Sentence	denotes	The antiviral properties of naproxen have been described in the context of influenza virus (264, 265) and has prompted the initiation of a clinical trial investigating the efficacy of naproxen as a treatment for critically ill COVID-19 infected patients (NCT04325633).
T466	77047-77243	Sentence	denotes	NSAID therapy should be used with caution as they have been shown to interfere with immune responses and ability to produce antibodies, with ibuprofen having the greatest suppressive effect (266).
T467	77244-77385	Sentence	denotes	Furthermore, ibuprofen has been reported to increase the expression of the ACE2 receptor (267) which could facilitate SARS-CoV-2 viral entry.
T468	77386-77516	Sentence	denotes	This finding should be considered for any current (NCT04334629) and potential COVID-19 clinical trial assessing ibuprofen therapy.
T469	77517-77682	Sentence	denotes	NSAIDs also have been shown to have a direct suppressive effect on NK cell IFN-γ and TNF-α production (268) which may be beneficial for late stage COVID-19 patients.
T470	77684-77713	Sentence	denotes	Conclusions and Further Study
T471	77714-77894	Sentence	denotes	The relevance of NK cells as antiviral first responders is highlighted in patients with NKD and immunocompromised individuals who show increased susceptibility to viral infections.
T472	77895-78050	Sentence	denotes	While there is currently little direct evidence to support a role for NK cells in the clearance of SARS-CoV-2 there is a paucity of research in this field.
T473	78051-78226	Sentence	denotes	However, studies in admitted COVID-19 patients with mild and severe disease reported a reduction in circulating NK cell levels and function as compared to healthy individuals.
T474	78227-78383	Sentence	denotes	Furthermore, reduced NK cell levels and function were inversely correlated with disease severity, suggesting that NK cells may be involved in some capacity.
T475	78384-78516	Sentence	denotes	One of the potential mechanisms by which NK cells may become hyporesponsive is via SARS-CoV-2 interference with type I IFN pathways.
T476	78517-78803	Sentence	denotes	In investigating the pathogenesis of other CoV infections, namely SARS and MERS, studies suggest that during acute CoV infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce chemokines and cytokines that induce NK cell migration and activation.
T477	78804-79033	Sentence	denotes	As NK cells are one of the main producers of IFN-γ, they may be involved in the IFN-γ-led cytokine storm that is responsible for the induction of inflammation-mediated ALI, ARDS, and subsequent mortality associated with COVID-19.
T478	79034-79110	Sentence	denotes	Inarguably, more research into the role of NK cells in COVID-19 is required.
T479	79111-79295	Sentence	denotes	Despite the knowledge gaps in COVID-19 pathophysiology, there has been a surge of clinical trials as the FDA continues to fast-track the approval of investigational therapeutics (269).
T480	79296-79616	Sentence	denotes	Here we have outlined potential therapeutics with a focus on mediating NK cell activity, including prophylactic treatments that could boost innate immunity in addition to therapeutics that could mitigate the immunopathological consequences of COVID-19, thereby relieving the burden on our health care systems (Figure 2).
T481	79617-79768	Sentence	denotes	Rigorous preclinical testing and thoughtfully designed clinical trials will be necessary for the development of robust therapeutics against SARS-CoV-2.
T482	79769-79885	Sentence	denotes	Importantly, we must be aware of the potential dangers immunotherapies may have in potentiating CoV immunopathology.
T483	79886-79932	Sentence	denotes	The fight against COVID-19 is not an easy one.
T484	79933-80049	Sentence	denotes	As with any novel disease, we will have to rely on incomplete pictures to guide reasoning for appropriate treatment.
T485	80050-80280	Sentence	denotes	We believe that immunotherapeutics targeting the innate immune response, and specifically NK cells, have the potential to flatten the curve and will be important instruments in our armamentarium against this pandemic and the next.
T486	80281-80934	Sentence	denotes	Figure 2 Short-list of immune modulating therapies undergoing clinical trial in COVID-19 patients and recommendations on who should receive therapy. (A) Healthy, uninfected individuals, who are at a high risk of becoming infected (through situational circumstances such as healthcare workers) would be most fit and suitable to receive investigational prophylactic therapies such as exogenous IFNs and heterologous vaccines. (B) Individuals who have tested positive for COVID-19 that are asymptomatic or have mild to moderate disease progression may benefit from receiving investigational immune stimulating therapies, including NK cell-based therapies.
T487	80935-81271	Sentence	denotes	It is critical that investigators must be vigilant to assess the safety profile and potential immunopathologies associated with these immunotherapies. (C) In severe COVID-19 patients, the most appropriate therapies to investigate would be those that mitigate immunopathologies, such as anti-inflammatory and immunosuppressive therapies.
T488	81272-81494	Sentence	denotes	Given the relatively low chance of toxicity and the wide range of beneficial immune effects, natural health products such as vitamin C and vitamin D can be suitable for investigation at all categories of COVID-19 patients.
T489	81496-81516	Sentence	denotes	Author Contributions
T490	81517-81586	Sentence	denotes	MM, LA, AM, DB, OO, GT, DMB, and JN contributed to literature review.
T491	81587-81637	Sentence	denotes	MM, LA, AM, DB, OO, and GT contributed to writing.
T492	81638-81686	Sentence	denotes	MM, LA, MA, and RA were responsible for editing.
T493	81687-81729	Sentence	denotes	MM and LA were responsible for formatting.
T494	81730-81805	Sentence	denotes	MA and RA oversaw the writing process and provided mentorship and guidance.
T495	81806-81880	Sentence	denotes	All authors contributed to the article and approved the submitted version.
T496	81882-81902	Sentence	denotes	Conflict of Interest
T497	81903-82075	Sentence	denotes	The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
T498	82077-82190	Sentence	denotes	The authors would like to thank Dr. Doug Gray for his thorough editing, proofreading, and thoughtful suggestions.
T499	82191-82199	Sentence	denotes	Funding.
T500	82200-82431	Sentence	denotes	The authors have received funding from the Canadian Institutes of Health Research, the Cancer Research Society, and the Ontario Student Assistance Program through a Queen Elizabeth II Graduate Scholarship in Science and Technology.

T1

0-76

Sentence

denotes

Flattening the COVID-19 Curve With Natural Killer Cell Based Immunotherapies

T2

78-86

Sentence

denotes

Abstract

T3

87-192

Sentence

denotes

Natural Killer (NK) cells are innate immune responders critical for viral clearance and immunomodulation.

T4

193-329

Sentence

denotes

Despite their vital role in viral infection, the contribution of NK cells in fighting SARS-CoV-2 has not yet been directly investigated.

T5

330-502

Sentence

denotes

Insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing NK cell phenotype and function during SARS, MERS, and COVID-19.

T6

503-686

Sentence

denotes

These studies suggest a reduction in circulating NK cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of NK cell responses by coronaviruses.

T7

687-810

Sentence

denotes

Reduced circulating NK cell levels and exhaustion may be directly responsible for the progression and severity of COVID-19.

T8

811-970

Sentence

denotes

Conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine NK cell potential in mediating immunopathology.

T9

971-1221

Sentence

denotes

A common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which NK cells are an important component.

T10

1222-1405

Sentence

denotes

In this review, we summarize the current understanding of how NK cells respond in both early and late coronavirus infections, and the implication for ongoing COVID-19 clinical trials.

T11

1406-1588

Sentence

denotes

Using this immunological lens, we outline recommendations for therapeutic strategies against COVID-19 in clearing the virus while preventing the harm of immunopathological responses.

T12

1590-1602

Sentence

denotes

Introduction

T13

1603-1769

Sentence

denotes

Natural Killer (NK) cells are a key component of the innate immune system and are critical in the response to many viral infections in humans and animal models (1–3).

T14

1770-1980

Sentence

denotes

In addition to their beneficial antiviral role, NK cells have also been associated with immunopathology in infections such as respiratory syncytial virus (RSV) (4), influenza A virus (5–8), and hepatitis B (9).

T15

1981-2197

Sentence

denotes

Additionally, in the context of non-respiratory viral infections by HIV and HCV, NK cells appear to act as a rheostat by eliminating activated CD4+ and CD8+ T cells, thus preventing T cell-mediated autoimmunity (10).

T16

2198-2396

Sentence

denotes

The etiologic agent of the 2019 outbreak of pneumonia in Wuhan, China, was identified as belonging to the Coronaviridae family and named Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).

T17

2397-2553

Sentence

denotes

This virus causes the coronavirus Disease 2019 (COVID-19) which was declared a pandemic by the World Health Organization (WHO) on March 11th, 2020 (11, 12).

T18

2554-2714

Sentence

denotes

With the paucity of information currently available, there is a lack of consensus on the role played by NK cells in the response to coronavirus (CoV) infection.

T19

2715-2842

Sentence

denotes

In this review, we will explore evidence for both the protective and pathological role that NK cells may play in CoV infection.

T20

2843-2979

Sentence

denotes

Based on this knowledge we will comment on immune modulating treatment options that are being developed for the current COVID-19 crisis.

T21

2981-3015

Sentence

denotes

Coronaviruses and Recent Outbreaks

T22

3016-3146

Sentence

denotes

First discovered in the 1960s, CoVs are part of the Coronaviridae family of enveloped positive single-strand RNA viruses (13, 14).

T23

3147-3281

Sentence

denotes

The subfamily Orthocoronaviridae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (15).

T24

3282-3450

Sentence

denotes

Alpha- and betacoronaviruses circulate in mammals, including bats, gammacoronaviruses infect mostly avian species, and deltacoronaviruses infect birds and mammals (15).

T25

3451-3612

Sentence

denotes

Low pathogenic human CoVs (hCoVs), such as HCoV-299E (16), infect upper airways and etiological studies suggest they account for 15–30% of common colds (17, 18).

T26

3613-3726

Sentence

denotes

On the other hand, highly pathogenic CoVs infect the lower respiratory tract and can cause severe pneumonia (19).

T27

3727-3982

Sentence

denotes

These highly pathogenic CoVs include SARS-CoV-1, the virus responsible for the 2002–2004 Severe Acute Respiratory Syndrome (SARS) epidemic, and MERS-CoV, the virus responsible for the outbreak of Middle Eastern Respiratory Syndrome (MERS) in 2015 (19–21).

T28

3983-4211

Sentence

denotes

While highly pathogenic CoVs have become a relatively recent issue for humans; feline, canine, and bovine CoVs have long been recognized as significant pathogens with implications in veterinary medicine and agriculture (22, 23).

T29

4212-4320

Sentence

denotes

All CoVs have a roughly 30 kb genome packed into an enveloped helical capsid ranging from 80 to 120 nm (24).

T30

4321-4513

Sentence

denotes

At minimum, Coronaviridae members encode 4 structural and 16 non-structural proteins (14) with the family owing its name to the crown-like appearance produced by their spike (S) proteins (25).

T31

4514-4734

Sentence

denotes

Mutations in the S protein have allowed SARS-CoV1/2 to co-opt ACE2 or MERS-CoV to co-opt dipeptidyl peptidase 4 (DPP4) receptor/CD26 as viral entry receptors, thus facilitating the zoonosis of non-human CoVs (15, 26–28).

T32

4735-5035

Sentence

denotes

In addition, another mechanism that may have allowed these viruses to adapt to human hosts is through S protein cleavage by host cell proteases to expose the S2 domain fusion peptide, which induces viral and cellular membrane fusion and results in the release of viral genome into the cytoplasm (15).

T33

5036-5189

Sentence

denotes

Genetic sequencing revealed SARS-CoV-2 to be a betacoronavirus that shares 79.0% nucleotide identity with SARS-CoV-1 and 51.8% identity to MERS-CoV (29).

T34

5190-5340

Sentence

denotes

The epidemic of SARS in 2002–2004 caused by SARS-CoV-1 illustrated the devastating potential of coronaviruses to cause serious disease in humans (24).

T35

5341-5445

Sentence

denotes

SARS ultimately reached 29 countries and 5 continents causing over 8,000 infections and over 900 deaths.

T36

5446-5581

Sentence

denotes

The basic reproductive rate (R0) or the number of expected cases arising from one infected individual, ranges from 2 to 4 (20, 30, 31).

T37

5582-5694

Sentence

denotes

With its reservoir in bats, SARS-CoV-1 is a zoonosis that was transmitted to humans by palm civets (24, 32, 33).

T38

5695-5828

Sentence

denotes

SARS-CoV-1 infects lung pneumocytes (34) and enterocytes in the digestive tract (35) most often producing flu-like symptoms (36, 37).

T39

5829-6048

Sentence

denotes

More severe presentations including pneumonia, pronounced lymphopenia, liver abnormalities, and acute respiratory distress syndrome (ARDS) were also reported, with most fatalities due to respiratory failure (19, 36–39).

T40

6049-6178

Sentence

denotes

The subsequent MERS-CoV outbreak in 2015 also originated in bats, with dromedary camels being the intermediary host (14, 40, 41).

T41

6179-6231

Sentence

denotes

The R0 for MERS-CoV is estimated to be under 1 (21).

T42

6232-6430

Sentence

denotes

The extent of MERS-CoV transmission was more limited than SARS-CoV-1, but its case fatality rate was greater with 2,494 cases over 27 countries and 858 deaths being reported at the end of 2019 (21).

T43

6431-6603

Sentence

denotes

Common presentations for MERS-CoV include fever, dyspnea, muscle pain, and digestive tract symptoms and disease progression is more likely in those with comorbidities (42).

T44

6604-6725

Sentence

denotes

Like SARS-CoV-1 and MERS-CoV, SARS-CoV-2 is thought to have originated in bats through an unknown intermediary host (43).

T45

6726-6877

Sentence

denotes

At the time of writing, the number of global infections is estimated to be over 5,000,000 with over 340,000 deaths (44) and the R0 is roughly 2.2 (45).

T46

6878-7076

Sentence

denotes

Like other diseases caused by infectious CoVs, most patients present with flu-like symptoms including fever, cough, and lethargy, with the development of pneumonia and ARDS often proving fatal (46).

T47

7077-7238

Sentence

denotes

Furthermore, patients with underlying conditions are at risk for further complications if infected with COVID-19, such as those with cardiovascular disease (47).

T48

7239-7359

Sentence

denotes

SARS-CoV-2 has been posthumously detected in not only the lungs, but the pharynx, heart, liver, brain, and kidneys (48).

T49

7360-7603

Sentence

denotes

Transmission of SARS-CoV-2 is thought to mainly occur through direct contact/inhalation of respiratory droplets and aerosols from infected carriers, but indirect transmission by fomites has also been reported, although less efficient (49, 50).

T50

7604-7756

Sentence

denotes

SARS-CoV-2 viral entrance is thought to be mediated by binding of the S protein to the ACE2 receptor (51, 52), although this is still under debate (53).

T51

7757-7970

Sentence

denotes

While direct cytopathic effects are thought to play a major role in CoV pathology, studies have suggested that a dysregulated immune response resulting in pathological inflammation is also partly responsible (19).

T52

7971-8150

Sentence

denotes

With the current pandemic already surpassing the previous CoV outbreaks (54), rapid deployment of novel approaches to understanding and treating coronavirus infections are needed.

T53

8152-8184

Sentence

denotes

NK Cells as Innate Viral Killers

T54

8186-8205

Sentence

denotes

Sensing RNA Viruses

T55

8206-8277

Sentence

denotes

Innate immunity is essential in disease prevention and viral clearance.

T56

8278-8558

Sentence

denotes

Among the first responders to viral infections, tissue-resident macrophages and dendritic cells (DCs) (55) recognize evolutionarily conserved microbial structures termed pathogen-associated molecular patterns (PAMPs) via germline-encoded pattern recognition receptors (PRRs) (56).

T57

8559-8731

Sentence

denotes

In the context of respiratory RNA viruses, airway epithelial cells, that also express some PRRs (57), are often infected and have a major role in the first line of defense.

T58

8732-8909

Sentence

denotes

TLR3, TLR7, TLR8, MDA-5, and RIG-I are PRR expressed by immune and non-immune cells that are especially relevant in fighting respiratory RNA viruses, such as Coronaviruses (57).

T59

8910-9113

Sentence

denotes

Sensing through PRRs results in the transcription of genes involved in the inflammatory response, with type I interferons (IFNs) (IFN-α/β) production being a critical part of the antiviral response (58).

T60

9114-9343

Sentence

denotes

Type I IFNs are produced by many immune and non-immune cells (55, 57, 59) and in addition to eliciting intrinsic antiviral responses (60), they are also essential to prime innate and adaptive lymphocytes, including NK cells (61).

T61

9345-9373

Sentence

denotes

NK Cells as Viral Responders

T62

9374-9558

Sentence

denotes

NK cells are cytotoxic lymphocytes that directly target infected, stressed, or transformed cells and play a critical role in bridging the innate and the adaptive immune responses (62).

T63

9559-9711

Sentence

denotes

In humans, mature NK cells comprise 10–15% of total peripheral blood leukocytes and are described phenotypically as CD3− CD14− CD19− CD56+ CD16+/− (63).

T64

9712-9850

Sentence

denotes

NK cells do not undergo clonal selection but instead express several germline-encoded receptors that regulate their activity (62, 64, 65).

T65

9851-10610

Sentence

denotes

Upon viral infection, host cells become more susceptible to NK cell killing through: (i) upregulation of self-encoded molecules induced by infection/cellular stress (66, 67) that bind activating NK cell receptors such as Natural Cytotoxicity Receptors (NCRs) (NKp30, NKp44, and NKp46) (68), C-type lectin-like receptors NKG2D and NKp80 (69), and co-activating receptors such as DNAM-1 (70); (ii) downregulation of ligands for inhibitory receptors such as Killer Immunoglobulin-like Receptors (KIRs) (71–73) and the C-type lectin-like receptor CD94-NKG2A (74, 75) which suppress NK cell activation, and; (iii) direct recognition of viral moieties, via engagement of PAMPS (76) or transmembrane activating receptors such as mouse Ly49H (77) or human NKG2C (78).

T66

10611-10818

Sentence

denotes

Moreover, NK cells can eliminate virus-infected cells via CD16-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), which has been shown to be particularly important for herpesvirus clearance (79).

T67

10819-11072

Sentence

denotes

Finally, NK cell activity is modulated by cytokines, including, but not limited to, the activating cytokines interleukin (IL)-2/12/15/18 (80) and type I IFN, which can be produced by virally infected cells or activated antigen presenting cells (81, 82).

T68

11073-11215

Sentence

denotes

IL-2/12/15/18, alone or in combination, promotes NK cell survival, proliferation, cytotoxicity, and cytokine production, including IFN-γ (80).

T69

11216-11307

Sentence

denotes

Therefore, NK cells are uniquely equipped to sense and quickly respond to viral infections.

T70

11309-11346

Sentence

denotes

NK Cell Effector Functions and Memory

T71

11347-11516

Sentence

denotes

NK cells are found in circulation and in peripheral tissues (63) and can be quickly recruited to sites of infection where they facilitate and accelerate viral clearance.

T72

11517-11669

Sentence

denotes

In fact, NK cells are not thought to have permanent tissue residency but instead move dynamically between the blood and tissues, such as the lungs (83).

T73

11670-11770

Sentence

denotes

NK recruitment is regulated by chemokine gradients that are sensed via chemokine receptors (84, 85).

T74

11771-12010

Sentence

denotes

Activated NK cells induce the apoptosis of target cells through the engagement of death receptors, such as TRAIL and Fas (86) or via direct cytotoxicity through Ca2+-dependent exocytosis of cytolytic granules (perforin and granzymes) (87).

T75

12011-12108

Sentence

denotes

Moreover, NK cells secrete cytokines, including IFN-γ, which have key anti-viral properties (88).

T76

12109-12291

Sentence

denotes

In addition to being essential first responders to viral infection, NK cells can elicit a stronger secondary response resembling the memory features of adaptive lymphocytes (89, 90).

T77

12292-12475

Sentence

denotes

NK cell memory has been initially described in mice infected by MCMV, where Ly49H+ NK cells quickly expand and have stronger responses after a secondary encounter with the virus (91).

T78

12476-12634

Sentence

denotes

Interestingly, a similar NK cell subset has been identified in humans, where NK cells expressing NKG2C are expanded and persist in CMV infected patients (92).

T79

12635-12791

Sentence

denotes

Both Ly49H and NKG2C bind viral determinants, highlighting how NK cell memory is linked with the ability of NK cells to directly recognize viruses (93, 94).

T80

12792-12965

Sentence

denotes

In addition to direct recognition of viral molecules, long-lasting changes in NK cells are induced by the cytokine milieu (89, 95), which can be elicited by viral infection.

T81

12967-13032

Sentence

denotes

NK Cell Dysfunction Is Linked With Increased Viral Susceptibility

T82

13033-13215

Sentence

denotes

The relevance of NK cells in fighting viral infections has been highlighted by several studies where NK cells, in mice and humans, were not present or had compromised functions (96).

T83

13216-13425

Sentence

denotes

For example, individuals with NK cell deficiencies (NKD), a subset of primary immunodeficiency diseases, are highly susceptible to viral infection, particularly by herpesvirus and papillomavirus families (96).

T84

13426-13682

Sentence

denotes

The seminal 1989 case of NKD in an adolescent female with severe herpesvirus infections (varicella pneumonia, disseminated CMV, and disseminated HSV) revealed how functional NK cell deficiencies have clinical consequences in terms of viral infections (97).

T85

13683-13883

Sentence

denotes

Cancer patients are also at risk of viral infections (98), which may be explained, at least in part, by an impairment of NK cell responses often observed in humans and in murine tumor models (99–104).

T86

13884-13984

Sentence

denotes

Unsurprisingly, cancer patients are at a significantly increased risk of severe COVID-19 (105, 106).

T87

13985-14054

Sentence

denotes

Elderly patients are also more susceptible to viral infections (107).

T88

14055-14218

Sentence

denotes

Mouse studies highlighted how a decreased number of circulating mature NK cells in aged animals paralleled with increased susceptibility to viral infections (108).

T89

14219-14360

Sentence

denotes

Studies in humans suggest that although NK cell numbers can actually increase with aging, NK cell activity declines significantly (109, 110).

T90

14361-14680

Sentence

denotes

Przemska-Kosicka et al. investigated NK cell function in response to seasonal influenza vaccination in young and old populations and observed quantitative and qualitative changes associated with impaired responses in the NK cell population and this was associated with poor seroconversion in the older population (111).

T91

14681-14921

Sentence

denotes

Additionally, obesity, which has been shown to cause systemic NK cell dysfunction (112, 113), has also been linked to increased COVID-19 severity and could be the reason behind the high prevalence of severe COVID-19 in younger people (113).

T92

14922-15034

Sentence

denotes

In short, NKD and individuals with reduced NK cell numbers or function are more susceptible to viral infections.

T93

15035-15224

Sentence

denotes

Unsurprisingly, the CDC has already highlighted a higher risk of infection and severity of COVID-19 in older individuals and individuals with comorbidities such as obesity and cancer (114).

T94

15225-15588

Sentence

denotes

However, this point is still controversial as a systematic review showed that primary immunodeficiencies are not linked with increased COVID-19 severity (115), but these data have to be interpreted keeping in mind that a large part of COVID-19 pathology is caused by excessive immune activation, which is arguably harder to reach in immunocompromised individuals.

T95

15589-15799

Sentence

denotes

Given the paradoxical role of the immune response in COVID-19 patients, it would be extremely useful to be able to rely on immunological functional biomarkers that could predict the outcome of disease severity.

T96

15800-15993

Sentence

denotes

Such assays are readily available for determining NK cell activity, e.g., NKVue™, and there is therefore an opportunity to conduct studies that would link NK cell functions to disease severity.

T97

15995-16031

Sentence

denotes

NK Cells and Coronavirus Infections:

T98

16032-16042

Sentence

denotes

Dual Roles

T99

16044-16096

Sentence

denotes

Coronaviruses Potently Suppress Type I IFN Responses

T100

16097-16185

Sentence

denotes

Evasion of host immune responses is necessary for the successful propagation of a virus.

T101

16186-16345

Sentence

denotes

Mechanisms employed by CoVs to evade the immune response could provide insights into how the immune system, and NK cells in particular, responds to SARS-CoV-2.

T102

16346-16523

Sentence

denotes

CoVs have been shown to target components of the innate IFN response, employing non-structural proteins (nsps), structural proteins, and accessory proteins to achieve this goal.

T103

16524-16634

Sentence

denotes

Nsp16 methylates viral RNA therefore preventing recognition by MDA5 and dampening type I IFN production (116).

T104

16635-16868

Sentence

denotes

Nsps also suppress type I IFN responses via the inhibition of the transcription factor STAT1 mRNA transcription (nsp1) and deubiquitination of transcription factors like Interferon Regulatory Transcription Factor (IRF)3 (nsp3) (116).

T105

16869-17023

Sentence

denotes

Moreover, viral-encoded accessory proteins from SARS-CoV-1 open reading frame (ORF)3b and MERS-CoV ORF4a/4b also block IFN production and signaling (116).

T106

17024-17128

Sentence

denotes

In addition, the MERS-CoV ORF6-encoded protein blocks p-STAT1 import, thus blocking IFN signaling (116).

T107

17129-17416

Sentence

denotes

Finally, the structural M protein of MERS-CoV (27) physically sequesters kinase proteins RIG-I, TBK1, IKKe, and TRAF3 and the SARS-CoV-1 N protein inhibits Activator Protein (AP)-1 signaling, protein kinase R function, and NFκB activation, all of which act to impede IFN responses (117).

T108

17417-17574

Sentence

denotes

In vivo murine studies report young mice rapidly clear SARS-CoV-1 infection, while old mice do not and that this discrepancy is due to a delay in type I IFN.

T109

17575-17693

Sentence

denotes

Furthermore, early administration of IFN-β induces a stronger immune response and reduces mortality in old mice (118).

T110

17694-17872

Sentence

denotes

Since type I IFNs are critical for NK cell activation and effector functions, it is possible that NK cell-mediated clearance of SARS-CoV-2 is being subverted by these mechanisms.

T111

17873-18029

Sentence

denotes

Further research into the role of NK cells in CoV clearance and potential immune evasion mechanisms are necessary to inform therapeutic development and use.

T112

18031-18084

Sentence

denotes

NK Cell Role in Clearing Acute Coronavirus Infections

T113

18085-18230

Sentence

denotes

There is currently a paucity of studies into the role of NK cells not only in COVID-19 pathophysiology, but also in other coronavirus infections.

T114

18231-18448

Sentence

denotes

An in vivo study reported that beige mice on a B6 background cleared SARS-CoV-1 normally, indicating that functional lymphocytes, including NK cells, may not be required to eliminate SARS-CoV-1 in murine models (119).

T115

18449-18726

Sentence

denotes

However, in a more recent study characterizing the cellular immune response to SARS-CoV-1 in 12–14-month old BALB/c mice, T cell depletion did not prevent control of SARS-CoV-1 replication (120), suggesting a role for the innate immune system, and NK cells, in viral clearance.

T116

18727-18984

Sentence

denotes

Importantly, in this study CD4-depletion resulted in enhanced lung immunopathology and delayed viral clearance, while CD8-depletion did not affect viral replication or clearance, thus highlighting an important role for CD4+ T cells in coronavirus infection.

T117

18985-19071

Sentence

denotes

These conflicting results may be due to the inherent limitations of CoV murine models.

T118

19072-19208

Sentence

denotes

In 4–8 week-old mice, SARS-CoV-1 is associated only with mild pneumonitis and cytokines are not detectable in the lungs (119, 121, 122).

T119

19209-19399

Sentence

denotes

A SARS-CoV-1 isolate (MA-15) replicates to a high titer and is associated with viremia and mortality, however the model lacks significant inflammatory cell infiltration into the lungs (123).

T120

19400-19553

Sentence

denotes

Thus, mouse models developed for the study of SARS fell short in terms of reproducing the clinical and histopathological signs of disease (119, 121–123).

T121

19554-19697

Sentence

denotes

It is therefore necessary to develop a usable animal model that is capable of reproducing the clinical and histopathological signs on COVID-19.

T122

19698-19910

Sentence

denotes

Israelow et al. recently described a SARS-CoV-2 murine model based on adeno associated virus (AAV)9-mediated expression of human (h)ACE2, which replicated the pathologic findings found in COVID-19 patients (124).

T123

19911-20088

Sentence

denotes

This model, which overcame the inability of murine (m)ACE2 to support SARS-CoV-2 infection, was used to show the inability of Type I IFN to control SARS-CoV-2 replication (124).

T124

20089-20387

Sentence

denotes

In a similar attempt to overcome the lack of infectability through mACE2, Dinnon et al. recently described a recombinant virus (SARS-CoV-2 MA) with a remodeled S protein mACE2 interface, which replicated in upper and lower airways in young and aged mice with disease being more severe in aged mice.

T125

20388-20528

Sentence

denotes

The authors used this model to screen therapeutics from vaccine challenge studies and assessed pegylated IFN-λ-1 as a promising therapeutic.

T126

20529-20704

Sentence

denotes

The authors suggested that this model has greater ease of use, cost, and utility over transgenic hACE2 models (125) to evaluate vaccine and therapeutic efficacy in mice (126).

T127

20705-20849

Sentence

denotes

A preliminary analysis of NK cell function and phenotype has been performed by Zheng et al. using peripheral blood from COVID-19 patients (127).

T128

20850-20946

Sentence

denotes

On admission, NK cell levels in the peripheral blood inversely correlated with disease severity.

T129

20947-21100

Sentence

denotes

Furthermore, COVID-19 patients with severe disease had significantly lower numbers of circulating NK cells, as compared to mild disease (p < 0.05) (127).

T130

21101-21379

Sentence

denotes

Additionally, circulating NK cells in severe disease displayed increased expression of the inhibitory receptor NKG2A and had an hyporesponsive phenotype with lower levels of IFN-γ, tumor necrosis factor (TNF)-α, IL-2, and granzyme B, although degranulation was maintained (127).

T131

21380-21532

Sentence

denotes

Finally, as compared to patients with active disease, patients recovering from COVID-19 had higher numbers of NK cells and lower NKG2A expression (127).

T132

21533-21838

Sentence

denotes

Liao et al. performed single-cell RNAseq on the cells obtained from bronchoalveolar lavage fluid of severe and mild COVID-19 patients and found that COVID-19 patients had significantly more NK cell infiltrates into the lungs, however patients with severe disease had reduced proportions of NK cells (128).

T133

21839-21923

Sentence

denotes

In addition, KLRC1 (NKG2A) and KLRD1 (CD94) were highly expressed by NK cells (128).

T134

21924-22358

Sentence

denotes

Carvelli et al. analyzed myeloid and lymphoid populations by immunophenotyping from blood and bronchoalveolar lavage fluid (BALF) in 10 healthy controls, 10 paucisymptomatic COVID-19 patients, 34 pneumonia patients, and 28 patients with ARDS due to SARS-CoV-2 and found that absolute numbers of peripheral blood lymphocytes, including NK cells, were significantly reduced in the pneumonia and ARDS groups compared to healthy controls.

T135

22359-22498

Sentence

denotes

Furthermore, the proportion of mature NK cells was reduced in patients with ARDS and NK cells showed increased NKG2A, PD-1, and CD39 (129).

T136

22499-22760

Sentence

denotes

Finally, Wilk et al. performed single-cell RNA-sequencing on 7 COVID-19 patients and 6 healthy controls and found that the CD56bright population was depleted in all COVID-19 patients but the CD56dim population was depleted only in patients with severe COVID-19.

T137

22761-22856

Sentence

denotes

Furthermore, NK cells had increased expression of the exhaustion markers LAG3 and HAVCR2 (130).

T138

22857-22956

Sentence

denotes

NK cell cytopenia seems to be a consistent characteristic among SARS-CoV-2 infected patients (131).

T139

22957-23191

Sentence

denotes

Altogether, these data indicate alterations in the NK cell phenotype and functional profile that are consistent with the hypothesis that to establish a productive and lasting infection, SARS-CoV-2 needs to dampen the NK cell response.

T140

23192-23276

Sentence

denotes

NK cell dysfunctions were also observed in patients from the previous CoV outbreaks.

T141

23277-23419

Sentence

denotes

Wang et al. assessed NK cell number and phenotype using peripheral blood from 221 SARS patients admitted to hospitals in Beijing, China (132).

T142

23420-23602

Sentence

denotes

NK cell proportion and absolute number were significantly reduced in SARS patients as compared to healthy donors and patients infected with the bacterium Mycoplasma pneumoniae (131).

T143

23603-23763

Sentence

denotes

NK cell number correlated inversely with disease severity and patients with anti-SARS CoV-specific IgG or IgM antibodies had significantly fewer NK cells (132).

T144

23764-23913

Sentence

denotes

The patients assessed had varied disease duration from 4 to 72 days (mean 31.7 days) and this allowed for patient stratification by disease duration.

T145

23914-24105

Sentence

denotes

Within the first 10 days of SARS-CoV-1 infection, NK cell numbers remained high but this period was followed by the development of lymphopenia with levels recovering only around day 40 (132).

T146

24106-24266

Sentence

denotes

Dong et al. also observed a reduction of NK cell numbers in SARS patients, and these levels were lower in patients with severe, as compared to mild, SARS (133).

T147

24267-24360

Sentence

denotes

In addition, MERS infection is strongly associated with leuko- and lymphopenia (42, 134–136).

T148

24361-24474

Sentence

denotes

The mechanisms underlying the reduction of circulating NK cells in patients infected with CoVs are still unclear.

T149

24475-24672

Sentence

denotes

As most studies have focused on peripheral blood NK cells, it is possible that the reduced number of circulating NK cells is due to redistribution of blood NK cells into the infected tissues (137).

T150

24673-24896

Sentence

denotes

While it is hard to assess NK cell migration to infected tissues in COVID-19 patients, this hypothesis was corroborated by mouse studies, where NK cells have been shown to migrate to the lungs in CoV infected animals (120).

T151

24897-25042

Sentence

denotes

An abundance of inhibitory factors, such as TGF-β, may be partially responsible for the NK cell hyporesponsiveness observed in COVID-19 patients.

T152

25043-25230

Sentence

denotes

In support of this hypothesis, Huang et al. found significantly higher TGF-β levels in SARS patients compared to healthy controls and this positively correlated with length of stay (138).

T153

25231-25455

Sentence

denotes

Given the importance of TGF-β in suppressing NK cell functions, it is possible that the higher levels of TGF-β (as well as other inhibitory cytokines) in CoV patients leads to suppression of NK cell antiviral activity (138).

T154

25456-25622

Sentence

denotes

Early studies of COVID-19 patients report secondary (super-) infections, including nosocomial pneumonia or bacteremia as a complication of SARS-CoV-2 infection (138).

T155

25623-25956

Sentence

denotes

Since NK cells are critical first responders that play a role in preventing and clearing infections (139), a poor NK cell count or exhausted phenotype, in addition to negatively influencing COVID-19 patient outcomes, could facilitate the development of secondary infections and have a significant negative impact on patient outcomes.

T156

25957-26258

Sentence

denotes

One of the main barriers in studying the role of NK cell activation in the early clearance of CoV infection in asymptomatic or mildly symptomatic patients is the fact that these individuals are rarely diagnosed in the clinic and therefore an opportunity to collect samples for research does not exist.

T157

26259-26475

Sentence

denotes

Thus, while there is currently no direct evidence to support a role for NK cells in the clearance of SARS-CoV-2, evidence showing that viral infection has a negative effect on the NK cell compartment is accumulating.

T158

26476-27003

Sentence

denotes

Given the importance of NK cell activity in early viral clearance and late immunopathology, having a rapid and reliable test to predict NK cell function, such as NKVue™ (ATGen Canada/NKMax), whereby whole blood is stimulated by an NK cell-specific activating cytokine mix and activity is measured via IFN-γ production, might allow researchers to predict who will mount an adequate response with asymptomatic or minimally symptomatic viral clearance and who will need ICU admission, as has been shown with cancer patients (140).

T159

27004-27153

Sentence

denotes

Further research will be required into the innate immune response to CoV infection to more fully understand NK cell contributions to viral clearance.

T160

27155-27198

Sentence

denotes

NK Cell Role in Coronavirus Immunopathology

T161

27199-27366

Sentence

denotes

In the context of CoVs, the significant morbidity and mortality associated with severe disease is due to acute lung injury (ALI) and the development of ARDS (19, 141).

T162

27367-27631

Sentence

denotes

Pathological analysis of tissues obtained from SARS and MERS patients showed edematous lungs with areas of consolidation, bronchial epithelial denudation, loss of cilia, squamous metaplasia, pneumocyte hyperplasia, and bronchial submucosal gland necrosis (19, 29).

T163

27632-27736

Sentence

denotes

Histological features include diffuse alveolar damage and acute fibrinous and organizing pneumonia (29).

T164

27737-27868

Sentence

denotes

A heightened inflammatory response in the lungs resulting in tissue damage has been hypothesized to explain the development of ALI.

T165

27869-27978

Sentence

denotes

There are several key factors that may be responsible for the induction of this dangerous inflammation (138).

T166

27979-28185

Sentence

denotes

Both SARS-CoV-1 and MERS-CoV replicate to high titers early in infection, which could lead to enhanced cytopathic effects and increased production of pro-inflammatory cytokines/chemokines by infected cells.

T167

28186-28431

Sentence

denotes

Chen et al. developed a pneumonia model where pulmonary replication of SARS-CoV-1 was associated with histopathological evidence of disease, including bronchiolitis, interstitial pneumonitis, diffuse alveolar damage, and fibrotic scarring (120).

T168

28432-28895

Sentence

denotes

They identified a biphasic cellular immune response in which cytokines (TNF-α and IL-6) and chemokines [interferon gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, RANTES] were produced early, likely by infected airway epithelial cells, alveolar macrophages, and recruited inflammatory monocyte-macrophages and neutrophils, which have been shown to replace resident alveolar macrophages (19, 142).

T169

28896-29134

Sentence

denotes

SARS-CoV-1 and MERS-CoV encode structural and non-structural proteins that antagonize the interferon response, which may initially delay the innate immune response but eventually potentiate inflammatory monocyte-macrophage responses (19).

T170

29135-29285

Sentence

denotes

In COVID-19 patients, Liao et al. reported increased lung infiltration by macrophages identified via RNA-seq analysis of bronchoalveolar lavage fluid.

T171

29286-29527

Sentence

denotes

Patients with mild cases exhibited infiltration by alveolar macrophages [Fatty Acid Binding Protein (FABP)4+] while patients with severe ARDS exhibited infiltration by highly inflammatory [Ficolin (FCN1)+] monocyte-derived macrophages (128).

T172

29528-29728

Sentence

denotes

In the SARS-CoV-1 pneumonia model, the first wave of cytokines and chemokines induced an accumulation of NK cells, as well as plasmacytoid (p)DCs, macrophages, CD4+ T cells and NKT cells in the lungs.

T173

29729-30015

Sentence

denotes

A second wave of inflammatory mediators was detected later on day 7 post-infection [cytokines TNF-α, IL-6, IFN-γ, IL-2, IL-5, and chemokines MCP-1, MIP-1a, RANTES, monokine induced by gamma interferon (MIG), IP-10] and correlated with lung infiltration of T cells and neutrophils (120).

T174

30016-30307

Sentence

denotes

These findings are consistent with studies that have shown increased levels of activating and inhibitory cytokines and chemokines in the blood and lungs of SARS patients, as well as histological studies of SARS and MERS-infected lungs which show extensive cell infiltrates (19, 29, 143–145).

T175

30308-30490

Sentence

denotes

When Huang et al. investigated the cytokine/chemokine profile in the acute phase of SARS infection in a cohort of Taiwanese patients, they observed an IFN-γ-led cytokine storm (138).

T176

30491-30730

Sentence

denotes

They assessed sera from hospitalized patients prior to the administration of immunomodulators and found significantly increased levels of IFN-γ, IL-18, IP-10, MCP-1, MIG, and IL-8 (138), which returned to basal levels in convalescent sera.

T177

30731-30829

Sentence

denotes

IP-10, MIG, MCP-1, and IL-18 levels were all significantly increased in death vs. survival groups.

T178

30830-31072

Sentence

denotes

Interestingly, they found an inverse relationship between IFN-γ levels and lymphocyte numbers and suggested this could either be due to IFN-γ-induced lymphocyte apoptosis or sequestration of chemokine-recruited lymphocytes in the lungs (138).

T179

31073-31167

Sentence

denotes

Indeed, this hyper-cytokinemia has been consistently observed in SARS-infected patients (146).

T180

31168-31431

Sentence

denotes

However, a recent study found that levels of six pro-inflammatory cytokines (IL-1b, IL-1Ra, IL-6, IL-8, IL-18, and TNF-α) implicated in the cytokine storm in COVID-19 patients did not differ significantly from levels in cytokine storms caused by other conditions.

T181

31432-31724

Sentence

denotes

They suggest that it is therefore possible that increased levels of pro-inflammatory cytokines in the context of severe COVID-19 may simply reflect an increased viral burden rather than an exuberant immune response and suggest that immunotherapies should therefore be used with caution (147).

T182

31725-32051

Sentence

denotes

Altogether these studies show that during acute CoV infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce cytokines and chemokines that induce the activation and migration of lymphocytes, including NK cells, to the lungs, where they could be one of the main producers of IFN-γ (148).

T183

32052-32187

Sentence

denotes

Under normal conditions, human lung NK cells are typically hyporesponsive but dynamically migrate in and out of pulmonary tissues (83).

T184

32188-32362

Sentence

denotes

This supports the hypothesis that during infectious respiratory diseases, an increased recruitment of hyperresponsive NK cells would worsen the festering immunopathology (8).

T185

32363-32607

Sentence

denotes

In fact, through Viral-Track scanning of unmapped single-cell RNA-sequencing data, Bost et al. showed that patients with severe COVID-19 exhibited a hyperinflammatory response with an enriched and highly proliferative NK cell compartment (142).

T186

32608-32886

Sentence

denotes

High levels of IFN-γ leads to epithelial and endothelial cell apoptosis and vascular leakage, suboptimal T cell response, accumulation of alternatively activated macrophages and altered tissue homeostasis, and ARDS (19), all of which may contribute to COVID-19 disease severity.

T187

32887-33152

Sentence

denotes

In summary, the evidence is consistent with the hypothesis that NK cells are involved in the cytokine storm associated with CoV infection and that this hyper-cytokinemia contributes significantly to disease severity via inflammation-mediated lung damage (Figure 1).

T188

33153-33478

Sentence

denotes

Figure 1 Hypothesized dual role of NK cells during coronavirus pathogenesis. (A) Healthy Natural Killer (NK) cells in low-risk individuals recognize SARS-CoV-2 infected cells via recognition of viral proteins on the surface of infected cells and through sensing of cytokines and chemokines produced in response to infection.

T189

33479-33806

Sentence

denotes

These cells are hypothesized to be able to directly induce apoptosis through death receptor ligation, antibody-dependent cell-mediated cytotoxicity (ADCC), and through the release of cytotoxic granules, in addition to indirectly targeting virally infected cells via modulation of the immune response through cytokine secretion.

T190

33807-34099

Sentence

denotes

An effective innate immune response may be able to clear SARS-CoV-2 infection and leave the patient's lungs undamaged. (B) High risk individuals may have dysfunctional NK cells which may not recognize and respond to SARS-CoV-2 infection due to immune evasion strategies employed by the virus.

T191

34100-34335

Sentence

denotes

It is hypothesized that an accumulation of infected epithelial cells and innate immune cells, monocyte-macrophages and neutrophils, release cytokines, and chemokines which further recruit immune cells, including NK cells, to the lungs.

T192

34336-34403

Sentence

denotes

This may result in the induction of a cytokine storm, led by IFN-γ.

T193

34404-34631

Sentence

denotes

This inflammatory state could act as the catalyst for the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), contributing to the significant morbidity, and mortality associated with COVID-19.

T194

34632-34790

Sentence

denotes

SARS-CoV-2 infection is associated with reduced NK cell levels and an exhausted phenotype which may impede viral clearance, in addition to severe lung damage.

T195

34791-34896

Sentence

denotes

Interestingly, this duality of NK cell roles mirrors what is seen in critically ill patients with sepsis.

T196

34897-35262

Sentence

denotes

Studies suggest that while early NK cell stimulation and IFN-γ production is beneficial to combat infections, excessive and prolonged stimulation of NK cells leads to reduced NK cell numbers and an exhausted phenotype and was associated with increased systemic inflammation in systemic inflammatory response syndrome (SIRS)/sepsis and increased mortality (149–152).

T197

35263-35385

Sentence

denotes

This review of the literature suggests that NK cells may play an important role in both CoV clearance and immunopathology.

T198

35386-35546

Sentence

denotes

The continued probing of NK cell involvement is essential for a more complete understanding of CoV pathophysiology and for the deployment of immunotherapeutics.

T199

35547-35891

Sentence

denotes

Depending on the patient, the stage of disease, and other still poorly understood factors, it may be necessary to either boost NK cell activity to ensure viral clearance, e.g., at exposure or during early infection, or to finely tune NK cell effector functions in late stage infections to prevent hyper-cytokinemia and inflammatory lung damage.

T200

35892-36045

Sentence

denotes

Indeed, all CoVs that infect humans are zoonoses and there is an extensive reservoir of CoVs that could serve as a source for future pandemics (14, 153).

T201

36046-36277

Sentence

denotes

Therefore, a broader understanding of the immune response to coronaviruses and insights into therapeutic implications will be of significant value not only for the current COVID-19 pandemic, but also for potential future pandemics.

T202

36279-36325

Sentence

denotes

Flattening the Curve With Natural Killer Cells

T203

36326-36474

Sentence

denotes

The race to vaccinate and find a cure for COVID-19 has resulted in a spectacular effort from researchers and medical practitioners around the world.

T204

36475-36668

Sentence

denotes

Early attempts at creating targeted therapeutics have mostly relied on historical evidence from related, but not identical, coronaviruses and on the paucity of studies investigating SARS-CoV-2.

T205

36669-36884

Sentence

denotes

These strategies have attempted to combat the virus by targeting various stages of its life cycle starting with neutralizing SARS-CoV-2 virions using monoclonal antibodies or plasma from convalescent patients (154).

T206

36885-37028

Sentence

denotes

The entry mechanism of CoVs has been shown to rely on binding the ACE2 receptor and using proteases such as TMPRSS2 for S protein priming (52).

T207

37029-37212

Sentence

denotes

Thus, preventing ACE2 receptor binding through blocking antibodies or competitive binding with soluble ACE2 and TMPRSS2 protease inhibitors (Camostat mesylate) are being tested (155).

T208

37213-37423

Sentence

denotes

Upon viral entry, the viral proteolysis or replication cycle can be targeted with protease inhibitors (Lopinovir and Ritonavir) (156) or RNA-dependent RNA polymerase inhibitors (Remdesivir and Ribavirin) (157).

T209

37424-37631

Sentence

denotes

At the time of writing this review, the results of these trials have not been released or are still preliminary and will require further evaluation to assess their clinical efficacy in larger cohort studies.

T210

37632-37907

Sentence

denotes

As NK cell activity is critical for viral clearance and may be involved in disease immunopathology, a rapid and reliable predictor of NK cell function may allow for the prediction of clinical progression and the stratification of patients to receive therapeutic intervention.

T211

37908-38076

Sentence

denotes

The remainder of this review will discuss the various ways immunotherapies are being deployed to tackle COVID-19, with a focus on therapies that use NK cells (Table 1).

T212

38077-38335

Sentence

denotes

Lastly, while NK cells play an important role in combating viral infections, we also need to be fully cognizant of the potential damage immunotherapies could have in severe cases of COVID-19, and how these adverse effects may need to be attenuated (Table 2).

T213

38336-38411

Sentence

denotes

Table 1 List of COVID-19 clinical trials using immunomodulatory therapies.

T214

38412-38545

Sentence

denotes

Category Therapeutic(s) Trial identifier Phase Location (centers) Patients (n) Study eligibility Mechanism of action(s)

T215

38546-38568

Sentence

denotes

NK cell-based products

T216

38569-38682

Sentence

denotes

Adoptive NK cells NK cells NCT04280224 I Henan, China 30 Pneumatic COVID19+ Adoptive NK cell therapy

T217

38683-38801

Sentence

denotes

CYNK-001 NCT04365101 I/II New Jersey, USA 86 Mild COVID19+ From human placental CD34+ cells and culture-expanded

T218

38802-38955

Sentence

denotes

NK cells isolated from healthy donor PBMCs NCT04344548 I/II Bagota, Colombia 10 NEWS of >4 Isolated NK cells ex vivo stimulated with IL-2 and IL-15

T219

38956-39284

Sentence

denotes

CAR-NK Cells NKG2D-ACE2 CAR-NK cell therapy from umbilical cord blood NCT04324996 I/II Chongqing, China 90 Within <14 dpi IL-15 prolongs NK cell lifespan; GM-CSF neutralizing scFV reduces recruitment of inflammatory cells; NKG2D-ACE2 CAR-NK cells can target virally infected cells; ACE2 CAR-NK can act as decoy cell

T220

39285-39309

Sentence

denotes

NK cell immunostimulants

T221

39310-39335

Sentence

denotes

Direct NK cell activation

T222

39336-39569

Sentence

denotes

IFN-α Therapy Recombinant human IFN-α ± Thymosin alpha 1 NCT04320238 III Hubei, China 2,944 Uninfected HCW IFN-alpha boosts immune system; thymosin alpha 1 activates TLRs in myeloid and pDCs leading to NK cell activation

T223

39570-39685

Sentence

denotes

Recombinant human IFN-α2β NCT04293887 I Hubei, China 328 Within <7 dpi IFN-alpha activates interferon pathway

T224

39686-39795

Sentence

denotes

Abidol Hydrochloride ± IFN atomization (Peg-IFN-α-2b) NCT04254874 IV Hubei, China 100 Pneumatic COVID19+

T225

39796-39895

Sentence

denotes

Rintatolimod ± Recombinant IFN-α2β NCT04379518 I/II New York, USA 80 Mild to moderate COVID19+

T226

39896-40040

Sentence

denotes

IFN-β Therapy Lopinavir/ritonavir ± IFN-β-1a NCT04315948 III France (3) 3,100 Moderate/Severe IFN-beta activates interferon pathway

T227

40041-40173

Sentence

denotes

Base therapy ± IFN-β-1a NCT04350671 IV Tehran, Iran 40 Within <10 dpi Base therapy = Hydropinchloroquine + Lopinavir/Ritonavir

T228

40174-40292

Sentence

denotes

Base therapy ± IFN-β-1b NCT04276688 II Hong Kong, HK 127 NEWS of ≥1 Base therapy = Lopinavir/ritonavir/Ribavirin

T229

40293-40440

Sentence

denotes

IFN-β-1a vs. IFN-β-1b (+Base therapy) NCT04343768 IV Tehran, Iran 60 Moderate/Severe Base therapy = Hydropinchloroquine + Lopinavir/Ritonavir

T230

40441-40468

Sentence

denotes

Indirect NK cell activation

T231

40469-40660

Sentence

denotes

IFN-λ Therapy Peg-IFN-Lambda-1a NCT04331899 II California, USA 120 Early COVID19+ IFN-lambda to boost NK cells indirectly through Monocytes, Macrophages, and pDCs IL-12 secretion

T232

40661-40728

Sentence

denotes

Peg-IFN-Lambda-1a NCT04343976 II Boston, USA 40 Early COVID19+

T233

40729-40814

Sentence

denotes

Peg-IFN-Lambda-1a NCT04354259 II Toronto, Canada 140 Ambulatory and hospitalized

T234

40815-40886

Sentence

denotes

Peg-IFN-Lambda-1a NCT04388709 II New York, USA 66 Non-ICU COVID19+

T235

40887-40964

Sentence

denotes

Peg-IFN-Lambda-1a NCT04344600 II Maryland, USA 164 Asymptomatic COVID19+

T236

40965-40991

Sentence

denotes

Immunoregulatory therapies

T237

40992-41219

Sentence

denotes

Immune checkpoint blockade Anti-PD-1 (vs. Thymosin) NCT04268537 II Nanjing, China 120 Respiratory failure within <48h of ICU Prevent T-cell regulation by blocking PD-1; in COVID19+ advanced or metastatic cancer patients;

T238

41220-41342

Sentence

denotes

Anti-PD-1 (pembrolizumab) + tocilizumab NCT04335305 II Nanjing, China 24 Pneumatic COVID19+ Tocilizumab = anti-IL-6R

T239

41343-41454

Sentence

denotes

Anti-PD-1 (nivolumab) vs. GNS651 NCT04333914 II France (4) 273 Early COVID19+ GNS651 = Chloroquine analog

T240

41455-41493

Sentence

denotes

Non-specific innate immune stimulation

T241

41494-41670

Sentence

denotes

Heterologous vaccines BCG NCT04328441 III Netherlands (9) 1,500 Uninfected HCW Trains and primes innate immunity against subsequent non-specific pathogen infection

T242

41671-41746

Sentence

denotes

BCG (Danish strain) NCT04327206 III Australia (8) 4,170 Uninfected HCW

T243

41747-41812

Sentence

denotes

BCG (Danish strain) NCT04350931 III Egypt 900 Uninfected HCW

T244

41813-41874

Sentence

denotes

BCG (Danish strain) NCT04379336 III South Africa 500 HCW

T245

41875-41939

Sentence

denotes

BCG (Tice strain) NCT04348370 IV USA (5) 700 Uninfected HCW

T246

41940-42002

Sentence

denotes

BCG NCT04369794 IV São Paulo, Brazil 1,000 Early COVID19+

T247

42003-42155

Sentence

denotes

TLR agonists PUL-042 (CpG ODN) NCT04313023 II Not listed 200 Unifected/asymptomatic Activates TLR2/6/9 leading to innate immune stimulation

T248

42156-42222

Sentence

denotes

PUL-042 (CpG ODN) NCT04312997 II Texas, USA 100 Within <7 dpi

T249

42223-42272

Sentence

denotes

Natural health products and alternative medicines

T250

42273-42434

Sentence

denotes

Vitamins Vitamin C NCT04323514 N/A Palermo, Italy 500 Pneumatic COVID19+ General immune boosting properties of vitamin C and natural health products

T251

42435-42494

Sentence

denotes

Vitamin C NCT04264533 II Hubei, China 140 ICU COVID19+

T252

42495-42557

Sentence

denotes

Vitamin C NCT03680274 III Quebec, Canada 800 ICU COVID19+

T253

42558-42618

Sentence

denotes

Vitamin C NCT04344184 II Virginia, USA 200 ICU COVID19+

T254

42619-42690

Sentence

denotes

Vitamin C + Zinc NCT04342728 N/A Ohio, USA 520 Outpatient COVID19+

T255

42691-42806

Sentence

denotes

Hydroxychloroquine; Azithromycin; Vitamin C, D; and Zinc NCT04334512 II California, USA 600 Low risk COVID-19+

T256

42807-42904

Sentence

denotes

Hydroxychloroquine; vitamin C, D; and Zinc NCT04335084 II California, USA 600 Uninfected HCW

T257

42905-43039

Sentence

denotes

Vitamin D NCT04334005 N/A Spain (2) 200 Non-severe COVID19+ Vitamin D is immunomodulatory and prevents nutritional deficiencies.

T258

43040-43210

Sentence

denotes

Zinc + Vitamin D (cholecalciferol) NCT04351490 N/A Lille, France 3,140 Asymptomatic COVID19+ To treat zinc and vitamin D deficiency and reduce inflammation and ARDS

T259

43211-43287

Sentence

denotes

High dose Vitamin D (4X) NCT04344041 III France (9) 260 Severe COVID19+

T260

43288-43410

Sentence

denotes

NEWS, National Early Warning Score; HCW, Healthcare Workers; Peg, pegylated; dpi, days post-infection; As of June 1, 2020.

T261

43411-43514

Sentence

denotes

Table 2 List of COVID-19 clinical trials investigating immunotherapies for mitigating immunopathology.

T262

43515-43648

Sentence

denotes

Category Therapeutic(s) Trial identifier Phase Location (centers) Patients (n) Study eligibility Mechanism of action(s)

T263

43649-43670

Sentence

denotes

Anti-cytokine therapy

T264

43671-43841

Sentence

denotes

Anti IL-6 Tocilizumab NCT04315480 II Italy 38 Pneumatic COVID19+ Anti-IL6R mAB to prevent virus-related cytokine storm and reduce symptoms of severe COVID-19

T265

43842-43907

Sentence

denotes

Tocilizumab NCT04317092 II Italy (27) 400 Pneumatic COVID19+

T266

43908-43974

Sentence

denotes

Tocilizumab NCT04320615 III Not listed 330 Pneumatic COVID19+

T267

43975-44035

Sentence

denotes

Tocilizumab NCT04332913 N/A Italy 30 Pneumatic COVID19+

T268

44036-44106

Sentence

denotes

Tocilizumab NCT04335071 II Switzerland (3) 100 Pneumatic COVID19+

T269

44107-44213

Sentence

denotes

Tocilizumab NCT04322773 II Denmark (2) 200 Pneumatic COVID19+ I.V. vs. S.C. routes of administration

T270

44214-44359

Sentence

denotes

Tocilizumab NCT04331795 II Illinois, USA 50 COVID19+, not on ventilator Low (80mg) vs. standard dose (200mg) in non-critically ill patients

T271

44360-44499

Sentence

denotes

Tocilizumab NCT04346355 II Italy (24) 398 Pneumatic COVID19+, non-ICU Early administration of tocilizumab on reduced ventilation time

T272

44500-44580

Sentence

denotes

Tocilizumab NCT04331808 II Paris, France 240 Group 1: non-ICU; Group 2: ICU

T273

44581-44705

Sentence

denotes

Tocilizumab (vs. CRRT) NCT04306705 N/A Hubei, China 120 Pneumatic COVID19+ CRRT = continuous renal replacement therapy

T274

44706-44823

Sentence

denotes

Tocilizumab (vs. Anakinra) NCT04339712 II Greece (17) 20 Pneumatic COVID19+, non-ICU Anakinra - IL1r antagonist

T275

44824-44992

Sentence

denotes

Tocilizumab (vs. GNS651) NCT04333914 II France (4) 273 Pneumatic COVID19+ Efficacy in COVID19+ advanced or metastatic cancer patients; GNS651 = chloroquine analog

T276

44993-45090

Sentence

denotes

Sarilumab NCT04315298 II/III USA (57) 400 Pneumatic COVID19+ Low vs. high dose of sarilumab

T277

45091-45170

Sentence

denotes

Sarilumab NCT04324073 II/III France (4) 239 Group 1: non-ICU; Group 2: ICU

T278

45171-45245

Sentence

denotes

Sarilumab NCT04327388 II/III Canada+France (8) 300 Pneumatic COVID19+

T279

45246-45368

Sentence

denotes

Hydroxychloroquine + Axithromycin ± Tocilizumab NCT04332094 II Barcelona, Spain 276 Early COVID19+, not on ventilator

T280

45369-45439

Sentence

denotes

Tocilizumab + Favipiravir NCT04310228 N/A China (11) 150 COVID19+

T281

45440-45592

Sentence

denotes

Tocilizumab + Anakinra + Siltuximab NCT04330638 III Belgium (9) 342 Pneumatic COVID19+ Anakinra - IL1r antagonist and Siltuximab - IL6r antagonist

T282

45593-45732

Sentence

denotes

Anti-IL-8 Anti-IL-8 (BMS-986253) NCT04347226 II New York, USA 138 Pneumatic COVID19+ Prevent recruitment of inflammatory cells

T283

45733-45892

Sentence

denotes

Anti IL-1R/Anti IFNγ Anakinra vs. Emapalumab NCT04324021 II/III Italy (4) 54 Pneumatic COVID19+ Anakinra (IL1r antagonist); Emapalumab (anti-IFNγ)

T284

45893-45965

Sentence

denotes

Anakinra ± Ruxolitinib NCT04366232 II France (3) 54 Severe COVID19+

T285

45966-46124

Sentence

denotes

Anti-GM-CSF Mavrilimumab NCT04337216 II Virginia, USA 10 Pneumatic COVID19+ GM-CSF is one of the main mediators of CRS in severe COVID19 patients

T286

46125-46273

Sentence

denotes

Jak Inhibitor Baricitinib NCT04399798 II Pavia, Italy 13 Pneumatic COVID19+ Inhibits JAK1-/JAK2-mediated cytokine release and TNF-alpha

T287

46274-46364

Sentence

denotes

Ritonavir ± Baricitinib NCT04320277 III Tuscany, Italy 60 Moderate/pneumonia COVID19+

T288

46365-46542

Sentence

denotes

Extracorporeal adsorption CytoSorb absorber NCT04324528 N/A Freiburg, Germany 30 Pneumatic COVID19+ “Absorbs” IL-6 in effort to reduce inflammation in ARDS patients

T289

46543-46562

Sentence

denotes

Anti-inflammatories

T290

46563-46737

Sentence

denotes

Corticosteroid Ciclesonide ± Hydroxychloroquine NCT04330586 II Seoul, Korea 141 Early COVID19+ (within 7 days) Ciclesonide is an anti-inflammatory corticosteroid

T291

46738-46855

Sentence

denotes

Prednisone NCT04344288 II Bron, France 304 Pneumatic COVID19+ Prednisone is an anti-inflammatory corticosteroid

T292

46856-46959

Sentence

denotes

Hydrocortisone NCT04348305 III Denmark 1000 Pneumatic COVID19+ Low-dose hydrocortisone for 7 days

T293

46960-47092

Sentence

denotes

Dexamethasone NCT04344730 N/A Paris, France 550 COVID19+ ICU within 48hrs Dexamethasone is an anti-inflammatory corticosteroid

T294

47093-47171

Sentence

denotes

Dexamethasone NCT04325061 IV Spain (24) 200 Severe COVID19+ on ventilator

T295

47172-47236

Sentence

denotes

Dexamethasone NCT04327401 III Brazil (21) 290 ARDS patients

T296

47237-47364

Sentence

denotes

Methylprednisolone NCT04343729 II Brazil 420 Pneumatic COVID19+ Methylprednisolone is an anti-inflammatory corticosteroid

T297

47365-47480

Sentence

denotes

Methylprednisolone (vs. Tocilizumab) NCT04345445 III Kuala Lumpur, Malaysia 310 COVID19+ with high risk of CRS

T298

47481-47576

Sentence

denotes

Methylprednisolone (vs. Siltuximab) NCT04329650 II Barcelona, Spain 200 Pneumatic COVID19+

T299

47577-47673

Sentence

denotes

NSAID Naproxen NCT04325633 III Paris, France 584 Pneumatic COVID19+ COX-2 inhibitor

T300

47674-47747

Sentence

denotes

Ibuprofen NCT04334629 IV Not listed 230 NEWS2 > 5 overall, Pneumatic

T301

47748-47828

Sentence

denotes

NEWS, National Early Warning Score; HCW, Healthcare Workers; As of June 1, 2020.

T302

47830-47852

Sentence

denotes

NK Cell-Based Products

T303

47853-48024

Sentence

denotes

In the absence of a clinically approved vaccine against SARS-CoV-2, scientists have begun developing therapeutics to halt the spread of COVID-19 by alternative strategies.

T304

48025-48243

Sentence

denotes

Studies have reported that patients infected with SARS-CoV-2 have lower levels of circulating NK cells and these express a greater level of inhibitory receptors (e.g., NKG2A) while producing less IFN-γ (127, 129, 130).

T305

48244-48344

Sentence

denotes

These findings provide a rationale for pursuing NK cell-based therapies as a tool to fight COVID-19.

T306

48345-48513

Sentence

denotes

Although NK cell-based therapies have mostly been developed for use against cancer, similar concepts and mechanisms could provide guidance in the fight against viruses.

T307

48514-48790

Sentence

denotes

Therapeutic NK cell products can be thought of as “living drugs” as they generally use either primary NK cells isolated from peripheral blood mononuclear cells (PBMCs) or are generated from stem cell precursors or genetically engineered immortalized human NK cell lines (158).

T308

48791-48946

Sentence

denotes

Primary NK cell products are often pre-treated and expanded in vitro with cytokines or via co-culture with target cells before being infused into patients.

T309

48947-49186

Sentence

denotes

Patients can also receive immune stimulants [e.g. recombinant IL-2 (159) or IL-15 (160)] with the goal of improving the in vivo activity and persistence of the NK cell products (161) as is being tested in this COVID-19 trial (NCT04344548).

T310

49187-49443

Sentence

denotes

The first cell-based investigational drug to be approved by the FDA for clinical testing in COVID-19 patients is an allogeneic, off-the-shelf, cryopreserved NK cell therapy made by Celularity (CYNK-001), originally developed for cancer immunotherapy (162).

T311

49444-49493

Sentence

denotes

The trial (NCT04365101) is split into two Phases.

T312

49494-49669

Sentence

denotes

Phase I will assess the frequency and severity of adverse events in mild, non-ICU COVID-19 patients (n = 14) following infusion of NK cells derived from placental CD34+ cells.

T313

49670-49793

Sentence

denotes

The subsequent Phase II trial will recruit up to 72 patients and include a standard of care comparator at a 1:1 allocation.

T314

49794-49882

Sentence

denotes

Genetically modified NK cells are also being investigated for efficacy against COVID-19.

T315

49883-50234

Sentence

denotes

Chimeric antigen receptor NK cells (CAR-NK cells) are engineered to express virtually any receptor(s) of interest and were originally designed to enhance the ability of NK cells to eliminate cancer cells via receptors targeting EGFR (163) or CD19 (164), which are present on many cancer types and B cell hematological malignancies, respectively (164).

T316

50235-50552

Sentence

denotes

Although the efficacy of CAR-NK cells to control viral infections has yet to be rigorously tested in large scale clinical trials, the promising safety profile of CAR-NK cells in cancer patients, who are often immunocompromised, suggests that CAR-NK therapy can be well-tolerated in early phase/mild COVID-19 patients.

T317

50553-50729

Sentence

denotes

Notably, CAR-NK cells are considered “safe” largely because they are less likely to lead to cytokine release syndrome (CRS), a severe adverse event of CAR-T cell therapy (165).

T318

50730-50867

Sentence

denotes

But as these are unchartered waters, it is critical that CAR-NK cells are used cautiously and not given to late/severe COVID-19 patients.

T319

50868-51118

Sentence

denotes

A Phase I/II study in early stage COVID-19 patients (within 14 days of illness) employing CAR-NK cell therapy is currently being tested using off-the-shelf NK cells derived from human umbilical cord blood expressing NKG2D and ACE2 CARs (NCT04324996).

T320

51119-51332

Sentence

denotes

This complex five-arm study will compare the efficacy of different CAR-NK constructs: (i) NK cells, (ii) NK cells secreting IL-15, (iii) NKG2D CAR-NK cells, (iv) ACE2 CAR-NK cells, and (v) NKG2D-ACE2 CAR-NK cells.

T321

51333-51595

Sentence

denotes

NKG2D CAR-NK cells have shown promising preclinical results in cancer studies (166), and although not proven for SARS-CoV-2, the rationale for expressing NKG2D derives from work showing that NKG2D-ligands (NKG2DL) are upregulated on virally infected cells (167).

T322

51596-51966

Sentence

denotes

Similarly, the investigators hypothesize that expressing ACE2 on NK cells will facilitate the elimination of SARS-CoV-2 virions and infected cells by binding the viral spike proteins–but it is unknown whether or not CAR-NK cells can eliminate virions or if infected cells display sufficient levels of spike protein to be recognized by ACE2-NK cells upon viral infection.

T323

51967-52168

Sentence

denotes

The investigators also suggest that expressing ACE2 on NK cells may also have a secondary benefit as a decoy cell that will be infected by the virus thereby indirectly protecting lung epithelial cells.

T324

52169-52375

Sentence

denotes

As described previously, it is unclear whether this strategy will work to stop viral spread to healthy epithelial cells or if it will serve to perpetuate viral spread if the virus can replicate in NK cells.

T325

52376-52554

Sentence

denotes

In arms ii-v of this trial, the CAR-NK cells have been engineered to secrete IL-15 based on studies showing improved in vivo persistence of CAR-NK cells in cancer patients (168).

T326

52555-52894

Sentence

denotes

However, the addition of the proinflammatory cytokine IL-15 to this treatment strategy should be monitored closely for life-threatening toxicities, as elevated IL-15 has been previously reported to accompany chronic pulmonary inflammatory diseases (169) and MERS-CoV infection (170) even if no correlation has been reported for SARS-CoV-2.

T327

52895-53132

Sentence

denotes

Interestingly, a study compared IL-15 levels from lung tissue homogenates following SARS-CoV infection in aged vs. juvenile monkeys and showed that IL-15 concentrations were only elevated in juvenile monkeys 10 days post-infection (171).

T328

53133-53310

Sentence

denotes

This study would suggest that IL-15 therapy may be tolerated and effective in older COVID-19 patients that may not be able to produce IL-15, however this has not been confirmed.

T329

53311-53613

Sentence

denotes

Lastly, all the CAR-NK cells in this trial secrete GM-CSF neutralizing scFv antibodies, since this cytokine has a known role in CRS in cancer patients treated with CAR-T cells (172), and has been shown to be correlated with COVID-19 disease severity in association with pathogenic CD4+ Th1 cells (173).

T330

53614-53836

Sentence

denotes

Although NK cell based therapies are versatile, have shown safety and efficacy in cancer patients, and can be utilized in immunocompromised individuals, their potential has yet to be fully realized as an antiviral therapy.

T331

53837-53958

Sentence

denotes

Furthermore, the logistics of manufacturing NK cell products (cost and time) may pose limitations and barriers to access.

T332

53959-54091

Sentence

denotes

For this reason, therapies focused on stimulating a patient's own NK cells offer many advantages over adoptive transfer of NK cells.

T333

54093-54124

Sentence

denotes

Interferon Therapy and NK Cells

T334

54125-54306

Sentence

denotes

The importance of the interferon pathway is underscored by the fact that many viruses actively interfere with host interferon responses, for which coronaviruses are a prime example.

T335

54307-54427

Sentence

denotes

As described above, CoVs utilize numerous tactics to avoid elimination by disrupting the host type I IFN response (174).

T336

54428-54602

Sentence

denotes

Therefore, since the majority of CoVs fail to induce any detectable type I IFN response, eliciting a type I IFN response is a very attractive therapeutic strategy (118, 175).

T337

54603-54809

Sentence

denotes

Given the robust immunomodulatory nature of type I IFNs, uninfected or early symptomatic patients would benefit the most from this therapy to prevent exacerbating immunopathology at later stages of disease.

T338

54810-54891

Sentence

denotes

Numerous clinical trials have been initiated investigating type I IFNs (Table 1).

T339

54892-55112

Sentence

denotes

A large study (NCT04320238) of ~3,000 medical staff allocated participants to two trial arms: (i) low-risk (non-isolated wards or laboratories) or (ii) high-risk (isolated wards in direct contact with COVID-19 patients).

T340

55113-55306

Sentence

denotes

In addition to the IFN-α-1b nasal drops, high-risk medical staff will also receive the immune-modulating TLR activator, thymosin α1, which indirectly activates NK cells through pDCs (176, 177).

T341

55307-55461

Sentence

denotes

Interestingly, reports in SARS-CoV-1 studies showed that IFN-β therapy had a 50-fold greater anti-viral activity in Vero cells than IFN-α treatment (178).

T342

55462-55942

Sentence

denotes

Promising results have been published from a Phase II study (NCT04276688) (179), showing that complementing lopinavir-ritonavir and ribavirin with subcutaneous IFN-β-1b in mild-to-moderate COVID-19 patients is safe with no serious adverse events reported in the triple combination therapy group, and highly effective, with significant and clinically meaningful reductions in time to complete alleviation of symptoms, hospital length of stay, and time to negative viral load (179).

T343

55943-56193

Sentence

denotes

Despite our best efforts in timing type I IFN therapy to mitigate immunopathology, these treatments still increase the risk of excessive activation of proinflammatory signals, which could damage host tissues and perpetuate immunopathology (180, 181).

T344

56194-56298

Sentence

denotes

For this reason, alternative therapeutic avenues to direct type I IFN administration are being explored.

T345

56299-56465

Sentence

denotes

Type III IFNs can be a valid alternative to type I IFNs, because they maintain antiviral functions yet are less toxic and less prone to mediate immunopathology (182).

T346

56466-56644

Sentence

denotes

The type III IFN, IFN-λ, activates NK cells indirectly (compared to type I IFNs which directly act on NK cells), resulting in a less potent and slower immune response (183, 184).

T347

56645-56767

Sentence

denotes

IFN-λ activates NK cells by stimulating macrophages to produce IL-12 which in turn induce NK cells to produce IFN-γ (185).

T348

56768-56902

Sentence

denotes

Pegylated IFN-λ is being tested in COVID-19 positive patients with mild symptoms in the absence of respiratory distress (NCT04331899).

T349

56903-57085

Sentence

denotes

While IFN-λ can lead to the eventual activation of NK cells, its primary utility is in preventing the tissue damaging potential of neutrophils at mucosal surfaces, such as the lungs.

T350

57086-57272

Sentence

denotes

However, IFN-λ also has been shown to reduce the rate of tissue repair, which in the context of COVID-19 which has a long disease course, could mean greater risk of secondary infections.

T351

57273-57517

Sentence

denotes

Since exogenous administration of any IFN therapy poses the risk of tipping the balance toward severe COVID-19 immunopathology, Broggi et al. assessed the levels of IFNs in upper and lower respiratory samples from healthy and COVID-19 patients.

T352

57518-57776

Sentence

denotes

In this preprint, they report that while the upper airway swabs showed similar mRNA expression levels of type I and III IFN compared to healthy controls, the BALF samples of severe COVID-19 patients had significantly elevated type I and III IFN levels (186).

T353

57777-57940

Sentence

denotes

Therefore, as with all of the therapies discussed in this review, careful consideration about safe and effective timing should guide our design of clinical trials.

T354

57942-57974

Sentence

denotes

Interleukin Therapy and NK Cells

T355

57975-58094

Sentence

denotes

In addition to IFN cytokine therapy, interleukin cytokine therapy can enhance the effector functions of NK cells (158).

T356

58095-58229

Sentence

denotes

The use of whole, unmodified recombinant cytokines as a monotherapy has resulted in minimal success in humans in cancer immunotherapy.

T357

58230-58380

Sentence

denotes

The earliest cytokine therapies to gain FDA-approval were IFN-α and recombinant IL-2, approved for renal cell carcinoma and metastatic melanoma (187).

T358

58381-58502

Sentence

denotes

Although approved, they were limited by their in vivo half-life, marginal anti-tumor activity, and associated toxicities.

T359

58503-58789

Sentence

denotes

The next generation of cytokine therapies were created to address these issues by first improving their biological stability through pegylation and fusion to chaperone molecules and secondly improving their specificity by fusing cytokines with antibodies or intratumoral administration.

T360

58790-58909

Sentence

denotes

These advances in the field have allowed for the reassessment of the therapeutic potential of specific cytokines (187).

T361

58910-59142

Sentence

denotes

Given the importance of IL-15 signaling and NK cell function, researchers have developed IL-15 “superagonists” which are IL-15:IL-15R heterodimers that have better in vivo stability and bioactivity compared to monomeric IL-15 (168).

T362

59143-59532

Sentence

denotes

Although at the time of writing IL-15 superagonists are not being studied for their efficacy in COVID-19 patients, IL-15 superagonists, such as ALT-803, are safe in humans (188) and have been used in conjunction with many of the therapies being discussed in this review including: CAR-NK cell therapy, adoptive NK cell transfers, checkpoint inhibitors, and the BCG vaccine in cancer (189).

T363

59533-59755

Sentence

denotes

It should be noted that although the therapeutic potential of cytokine therapy to specifically stimulate NK cells is enticing, exogenous cytokine therapy has a high risk for exacerbating CRS if given at the incorrect time.

T364

59757-59796

Sentence

denotes

Checkpoint Immunotherapies and NK Cells

T365

59797-59967

Sentence

denotes

Some viruses are known to induce a state of functional hyporesponsiveness in T cells that is essential for the productive establishment of chronic viral infections (190).

T366

59968-60108

Sentence

denotes

A vast body of literature has identified inhibitory checkpoint receptors, including CTLA4 and PD-1, as key regulators of this process (191).

T367

60109-60316

Sentence

denotes

Interestingly, cancer exploits similar mechanisms to escape the immune response, which provided the rationale for the introduction of antibodies targeting checkpoint receptors for cancer immunotherapy (192).

T368

60317-60560

Sentence

denotes

CTLA4 and PD-1/PD-L1 blockade have revolutionized cancer immunotherapy, and their success provides a strong rationale for the use of these drugs in COVID-19 patients, where emerging evidence suggests that the immune response is also subverted.

T369

60561-60729

Sentence

denotes

A clinical trial (NCT04268537) is currently assessing the efficacy of PD-1 blocking antibodies in severe COVID-19 patients within 48 h of reported respiratory distress.

T370

60730-60918

Sentence

denotes

PD-1 has also been shown to play a role in regulating NK cell responses, in addition to modulating T cell functions (193–197), and has been reportedly increased in COVID-19 patients (129).

T371

60919-61035

Sentence

denotes

Inhibitory receptors on the surface of NK cells regulate NK cell activation and can be targeted by antibody therapy.

T372

61036-61142

Sentence

denotes

One of the most promising is certainly the inhibitory receptor NKG2A, which binds to HLA-E (74, 198, 199).

T373

61143-61337

Sentence

denotes

NKG2A expression is increased in circulating (127) and BALF NK cells from COVID-19 patients, in contrast to NKG2C, an activating receptor closely related to NKG2A, which remains unchanged (129).

T374

61338-61553

Sentence

denotes

However, it is unclear whether the observed increase in NKG2A+ NK cells is due selective proliferation of NKG2A+ cells or if it is the result of NKG2A negative cells migrating out of circulation to infected tissues.

T375

61554-61778

Sentence

denotes

Circulating NK cells from patients with active hepatitis B disease had higher levels of NKG2A compared to patients without active disease, however antiviral administration was associated with a reduction in NKG2A expression.

T376

61779-61888

Sentence

denotes

Additionally, blocking NKG2A in vitro with NKG2A monoclonal antibodies led to improved NK cytotoxicity (200).

T377

61889-62175

Sentence

denotes

Given the association between NKG2A expression in patients with severe COVID-19 (127, 201), a promising avenue of investigation would be anti-NKG2A therapy, even in light of results showing that NKG2A+ NK cells are tuned to present a higher level of responsiveness to stimulation (202).

T378

62177-62238

Sentence

denotes

Indirect NK Cell Activation Through Innate Immune Stimulation

T379

62239-62487

Sentence

denotes

While NK cells can be stimulated directly by cytokines such as interferons and interleukins, their activity can also be enhanced through a by-stander effect following stimulation of other innate immune cells, such as macrophages and pDCs (Table 1).

T380

62488-62615

Sentence

denotes

This type of coordinated innate immune response may be more effective at CoV viral clearance and mitigation of severe COVID-19.

T381

62617-62659

Sentence

denotes

Trained Immunity and Heterologous Vaccines

T382

62660-62893

Sentence

denotes

Trained immunity has been recently described as an epigenetic re-wiring occurring in myeloid cells and progenitors upon stimulation that primes for a stronger response to subsequent stimuli, even of a different nature (90, 203, 204).

T383

62894-63101

Sentence

denotes

Whereas, the consensus is that myeloid cells are primarily responsible for trained immunity (205), it is likely that the resulting alteration in the cytokine milieu also has an effect on NK cells (204, 206).

T384

63102-63248

Sentence

denotes

This is the case for the BCG vaccine, which has been shown to provide non-specific protection against yellow fever viral infection (90, 207, 208).

T385

63249-63419

Sentence

denotes

The BCG vaccine is composed of a live attenuated strain of Mycobacterium bovis originally given to young children to protect against tuberculosis (M. tuberculosis) (209).

T386

63420-63627

Sentence

denotes

This vaccine provides an initial boost to innate immunity, but more importantly, results in the secretion of IL-1β from monocytes/macrophages, which feeds back to further stimulate the innate response (204).

T387

63628-63858

Sentence

denotes

The use of a heterologous vaccine to provide enhanced protection against non-specific/new pathogens makes this a compelling strategy against COVID-19 that warrants thorough investigation in randomized controlled trials (209, 210).

T388

63859-64123

Sentence

denotes

The BCG vaccine is undergoing clinical trials in healthcare workers in the Netherlands (NCT04328441), Australia (NCT04327206), Egypt (NCT04350931), and the USA (NCT04348370) to enhance overall innate immunity and provide heterologous protection against SARS-CoV-2.

T389

64124-64290

Sentence

denotes

Interestingly, an association was found that linked lower COVID-19-attributable mortality rates in countries using BCG in their national immunization schedules (211).

T390

64291-64467

Sentence

denotes

On the contrary, a study that assessed the association of childhood BCG vaccination in adults living in Israel did not show a beneficial difference in COVID-19 infection rates.

T391

64468-64723

Sentence

denotes

The discrepancy between these two reports likely stem from the fact that the latter study only included adults who were previously vaccinated during childhood, supporting the fact that heterologous vaccination may not result in long-term protection (212).

T392

64724-65013

Sentence

denotes

Childhood BCG immunization has a limited window of opportunity to protect younger individuals from infection (213), but it is hypothesized that reducing the number of infected children can have a meaningful impact on curbing the spread of COVID-19 to the rest of the population (206, 211).

T393

65014-65135

Sentence

denotes

Another heterologous vaccine in the process of clinical trial development for COVID-19 studies is IMM-101 (CCTG ID# IC8).

T394

65136-65299

Sentence

denotes

Created by Immodulon Therapeutics LTD, IMM-101 is composed of heat-killed Mycobacterium obuense and may have an improved safety profile over the BCG vaccine (214).

T395

65300-65483

Sentence

denotes

IMM-101 has been studied in multiple clinical trials for its non-specific immune stimulating properties as a cancer immunotherapy in pancreatic (215) and melanoma patients (216, 217).

T396

65485-65519

Sentence

denotes

Toll-like Receptor Agonist Therapy

T397

65520-65762

Sentence

denotes

Agonists of Toll-Like Receptors (TLRs) have been shown to broadly activate different immune populations and have had both preclinical and clinical success as adjuvants in vaccination and in the treatment of a variety of viral pathogens (218).

T398

65763-65939

Sentence

denotes

For example, CpG oligodeoxynucleotides (CpG ODNs) are short DNA sequences that contain unmethylated CpG dinucleotides which activate TLR9 particularly on DCs and B cells (219).

T399

65940-66125

Sentence

denotes

Bao et al. showed that their CpG ODN construct, BW001, had protective effects against SARS-CoV-1 in a mechanism that relied on NK cell activation likely through a DC intermediate (220).

T400

66126-66292

Sentence

denotes

Amidst the ongoing SARS-CoV-2 pandemic, two clinical trials (NCT04313023, NCT04312997) have opened using the TLR2/6/9 agonist, PUL-042, in order to prevent infection.

T401

66294-66333

Sentence

denotes

Immune-Boosting Natural Health Products

T402

66334-66479

Sentence

denotes

Ascorbic acid, more commonly known as vitamin C, has been shown to exhibit potent immunomodulatory, antioxidant, and antimicrobial effects (221).

T403

66480-66668

Sentence

denotes

Vitamin C has been shown to restore NK cell cytotoxicity in individuals exposed to toxic chemicals through protein kinase C expression, a critical component in lymphocyte metabolism (222).

T404

66669-66911

Sentence

denotes

Additional reports have shown that vitamin C also enhances the expression of NKp46, CD69, CD25 and IFN-γ production by NK cells (223) and can increase the expression of IRF3 in lung tissues of influenza infected, pneumonia-induced mice (224).

T405

66912-67049

Sentence

denotes

Vitamin C also harbors potent antioxidant attributes which can scavenge reactive oxygen species (ROS) and prevent lung injury (225, 226).

T406

67050-67234

Sentence

denotes

Although ROS production is an important component in the host defense response to viruses, they can be harmful to cells and lead to the pathogenesis of viral-induced host injury (227).

T407

67235-67517

Sentence

denotes

The underlying rationale to investigate the therapeutic potential of vitamin C has been based on two key observations: (i) critically ill patients have lower levels of vitamin C (228–230) and (ii) vitamin C has pleiotropic immunomodulatory, antioxidant, and antiviral effects (221).

T408

67518-67651

Sentence

denotes

It is important to underscore that reports on the clinical outcomes of vitamin C treatment in humans are mixed and context dependent.

T409

67652-67772

Sentence

denotes

A thorough meta-analysis on vitamin C supplementation for the common cold has been reported by Hemilä and Chalker (231).

T410

67773-67952

Sentence

denotes

Briefly, they concluded that while the incidence of colds was not reduced, the duration and severity of colds was reduced when assessing studies of regular vitamin C intake (231).

T411

67953-68127

Sentence

denotes

Interestingly, a separate meta-analysis on vitamin C and cardiac surgery showed a reduction in the length of ICU stay and shortened the need for mechanical ventilation (232).

T412

68128-68305

Sentence

denotes

This is an important correlation as clinical trials are currently investigating the efficacy of vitamin C to reduce mortality and hospital burden in COVID-19 patients (Table 1).

T413

68306-68453

Sentence

denotes

A Phase II clinical trial (NCT04264533) was initiated in Wuhan where COVID-19 patients will be given a high dose intravenous infusion of vitamin C.

T414

68454-68782

Sentence

denotes

Lastly, whether oral dosing of vitamin C can achieve therapeutically relevant concentrations, as described in the above studies, is currently unknown, thus caution should be taken as exceeding the recommended dietary allowance of 100–200 mg/day may lead to mild toxicities including abdominal discomfort and diarrhea (231, 233).

T415

68784-68838

Sentence

denotes

Mitigating Immunopathology in Severe COVID-19 Patients

T416

68839-68999

Sentence

denotes

The main cause of death for COVID-19 patients has been pulmonary complications and respiratory failure often as a result of an unregulated cytokine storm (234).

T417

69000-69195

Sentence

denotes

It is unclear whether the hyperinflammation seen in severe cases of COVID-19 is the result of the viral replication within pulmonary epithelial cells or an overactive/avalanching immune response.

T418

69196-69376

Sentence

denotes

However, studies in SARS-CoV-1 reported hyperinflammation in later stages of disease progression, despite reduced viral titers, suggesting that the damage was immune-mediated (19).

T419

69377-69497

Sentence

denotes

The most appropriate course of therapy can only be determined by elucidating the pathophysiology of disease progression.

T420

69498-69731

Sentence

denotes

Scientists and physicians, however, have had to respond quickly to the growing number of severe COVID-19 cases and this has resulted in therapy mainly through a combination of anti-inflammatory and anti-viral interventions (Table 2).

T421

69732-69859

Sentence

denotes

As described above, there is a potential for NK cells to contribute to the cytokine storm and therefore the development of ALI.

T422

69860-70091

Sentence

denotes

A possible explanation for the observed lymphopenia in COVID-19 patients is that NK cells and other lymphocytes migrate out of the circulation and into pulmonary tissues to aid in the elimination of infected epithelial cells (235).

T423

70092-70212

Sentence

denotes

This could be the premise for the large, unintended, amount of tissue damage that worsen the respiratory distress (148).

T424

70213-70353

Sentence

denotes

For this reason, therapeutics that dampen the immune response have been effective in mitigating immunopathology in severe COVID-19 patients.

T425

70354-70556

Sentence

denotes

The following review papers have thoroughly discussed many of these immunotherapies already (236–241), therefore, this section will focus on immunotherapies and their potential implications on NK cells.

T426

70558-70579

Sentence

denotes

Anti-cytokine Therapy

T427

70580-70772

Sentence

denotes

The main cytokines responsible for the life threatening respiratory distress seen in reported cases of severe COVID-19 are IL-2, IL-6, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP1A, and TNF-α (234).

T428

70773-70980

Sentence

denotes

Many clinical trials have focused on targeting IL-6 signaling with anti-IL-6R monoclonal antibodies (e.g., tocilizumab, sarilumab, siltuximab) because of the important role IL-6 has in propagating CRS (242).

T429

70981-71172

Sentence

denotes

Tocilizumab, in particular, is being used as the primary therapy in the majority of these trials, likely owing to its FDA approved status as a therapeutic for CRS in CAR-T cell therapy (243).

T430

71173-71394

Sentence

denotes

A case report demonstrated the potential for tocilizumab therapy in treating severe COVID-19 illness, where a single dose on day 24 of symptoms led to progressive reduction in IL-6 levels and resolution of symptoms (244).

T431

71395-71661

Sentence

denotes

A Phase III study (NCT04320615) led by Hoffman-La Roche is recruiting patients to study the safety and efficacy of tocilizumab therapy in a randomized, double-blind, placebo-controlled, multicenter study in over 300 patients with severe COVID-19 pneumonia (Table 2).

T432

71662-71845

Sentence

denotes

Targeting the IL-6 axis in severe COVID-19 patients may also serve to improve NK cell functions as Cifaldi et al. showed that increased IL-6 negatively impacts NK cell function (245).

T433

71846-71931

Sentence

denotes

They also showed that tocilizumab treatment improved NK cell function in vitro (245).

T434

71932-72072

Sentence

denotes

Mazzoni et al. recently reported that serum IL-6 levels were inversely correlated (p = 0.01) with NK cell function in COVID-19 ICU patients.

T435

72073-72249

Sentence

denotes

Additionally, in a small subset of COVID-19 ICU patients (n = 5), NK cells displayed improved markers of activation (granzyme A and perforin) after tocilizumab treatment (246).

T436

72250-72404

Sentence

denotes

Similar therapies have emerged in the fight against COVID-19 including an IL-1R antagonist (Anakinra; NCT04330638) (247) and Cytosorb (NCT04324528) (248).

T437

72405-72562

Sentence

denotes

High dose anakinra therapy has shown promising safety and efficacy in a small retrospective study, as part of the COVID-19 Biobank study (NCT04318366) (247).

T438

72563-72835

Sentence

denotes

Cytosorb therapy is used in conjunction with conventional dialysis through a whole blood cartridge-based filtration system designed to remove middle molecular weight molecules (which include inflammatory cytokines <75 kDa) through extracorporeal cytokine adsorption (248).

T439

72836-72981

Sentence

denotes

It is reported to be effective at removing Ferritin and IL-6 in a case study of a 14-year-old with severe CRS following CAR-T cell therapy (249).

T440

72982-73115

Sentence

denotes

Jak1/2 inhibitors (JAKi) are also undergoing clinical trials in moderate-severe COVID-19 patients, such as baricitinib (NCT04320277).

T441

73116-73305

Sentence

denotes

In addition to their ability to impede the production of IL-6, thus curb the excessive inflammation, they may also block clathrin mediated endocytosis–indicating a dual role for JAKi (241).

T442

73306-73497

Sentence

denotes

However, JAKi can also lead to the transient increase in NK cells as shown in baricitinib treated Rheumatoid Arthritis patients (250), which could be detrimental for severe COVID-19 patients.

T443

73499-73514

Sentence

denotes

Corticosteroids

T444

73515-73629

Sentence

denotes

Corticosteroids have played a key role in the treatment of auto-immune diseases over the past 70 years (251, 252).

T445

73630-73819

Sentence

denotes

Whether endogenous or exogenous, corticosteroids decrease the number of circulating monocytes and lymphocytes and decrease synthesis of pro-inflammatory cytokines (IL-2, IL-6, TNF-α) (251).

T446

73820-73997

Sentence

denotes

Their strong anti-inflammatory and immunosuppressive effects make them good candidates for rapidly suppressing inflammation during early auto-immune disease or viral infections.

T447

73998-74084

Sentence

denotes

Corticosteroids have been shown to inhibit NK cells in ex vivo experiments (253, 254).

T448

74085-74308

Sentence

denotes

While corticosteroids may delay clearance of infections, their major benefit lies in suppressing excessive innate immune responses, thus preventing lung damage and ARDS commonly present in severe viral infections (255–257).

T449

74309-74431

Sentence

denotes

In fact, this was the main rationale for the widespread use of corticosteroids during MERS and SARS infections (255, 256).

T450

74432-74631

Sentence

denotes

Specific to COVID-19, some groups have advocated for the use of low-dose corticosteroids in a specific subset of critically-ill patients with refractory ARDS, sepsis, or septic shock (Table 2) (257).

T451

74632-74794

Sentence

denotes

There is one known ongoing randomized clinical trial examining the effect of the corticosteroid ciclesonide in adults with mild COVID-19 infections (NCT04330586).

T452

74795-74908

Sentence

denotes

This trial is based on preclinical studies showing in vitro antiviral activity of ciclesonide against SARS-CoV-2.

T453

74909-75175

Sentence

denotes

While there may be a benefit to using corticosteroids in a subset of critically-ill patients with refractory ARDS or sepsis (257), their routine use in COVID-19 is not recommended outside of clinical trials, based on expert opinion and WHO recommendations (258–260).

T454

75176-75317

Sentence

denotes

Corticosteroids also cause a multitude of side effects, most notably diabetes mellitus, osteoporosis, and increased risk of infections (251).

T455

75318-75509

Sentence

denotes

Controversially, a 2019 systematic review of over 6,500 influenza patients showed that corticosteroids actually led to increased mortality, length of ICU stay, and secondary infections (261).

T456

75510-75768

Sentence

denotes

Additionally, one retrospective observational study examined the use of corticosteroids in 31 COVID-19 patients, and reported no significant association between corticosteroids and viral clearance time, hospital length of stay, or duration of symptoms (262).

T457

75769-75851

Sentence

denotes

These studies highlight the need to be vigilant in our attempts to fight COVID-19.

T458

75853-75899

Sentence

denotes

Non-steroidal Anti-inflammatory Drugs (NSAIDs)

T459

75900-76039

Sentence

denotes

Non-steroidal anti-inflammatory drugs, or NSAIDs, are one of the most commonly prescribed drugs for treating fever, pain, and inflammation.

T460

76040-76129

Sentence

denotes

NSAIDs include over-the-counter household names such as ibuprofen, naproxen, and aspirin.

T461

76130-76308

Sentence

denotes

Given the widespread use of these medications it is appropriate that researchers have investigated the potential benefits and harms of NSAIDs in patients diagnosed with COVID-19.

T462

76309-76473

Sentence

denotes

Thus far, the evidence for using NSAIDs in the context of CoVs are mixed and might not be generalizable to all NSAIDs as reports tended to focus on specific NSAIDs.

T463

76474-76629

Sentence

denotes

These studies also focused on the potential for NSAIDs to act as an antiviral, with a potential added benefit of being able to treat inflammatory symptoms.

T464

76630-76777

Sentence

denotes

One report showed that the NSAID indomethacin could directly inhibit SARS-CoV replication in Vero cell monolayers in a dose-dependent manner (263).

T465

76778-77046

Sentence

denotes

The antiviral properties of naproxen have been described in the context of influenza virus (264, 265) and has prompted the initiation of a clinical trial investigating the efficacy of naproxen as a treatment for critically ill COVID-19 infected patients (NCT04325633).

T466

77047-77243

Sentence

denotes

NSAID therapy should be used with caution as they have been shown to interfere with immune responses and ability to produce antibodies, with ibuprofen having the greatest suppressive effect (266).

T467

77244-77385

Sentence

denotes

Furthermore, ibuprofen has been reported to increase the expression of the ACE2 receptor (267) which could facilitate SARS-CoV-2 viral entry.

T468

77386-77516

Sentence

denotes

This finding should be considered for any current (NCT04334629) and potential COVID-19 clinical trial assessing ibuprofen therapy.

T469

77517-77682

Sentence

denotes

NSAIDs also have been shown to have a direct suppressive effect on NK cell IFN-γ and TNF-α production (268) which may be beneficial for late stage COVID-19 patients.

T470

77684-77713

Sentence

denotes

Conclusions and Further Study

T471

77714-77894

Sentence

denotes

The relevance of NK cells as antiviral first responders is highlighted in patients with NKD and immunocompromised individuals who show increased susceptibility to viral infections.

T472

77895-78050

Sentence

denotes

While there is currently little direct evidence to support a role for NK cells in the clearance of SARS-CoV-2 there is a paucity of research in this field.

T473

78051-78226

Sentence

denotes

However, studies in admitted COVID-19 patients with mild and severe disease reported a reduction in circulating NK cell levels and function as compared to healthy individuals.

T474

78227-78383

Sentence

denotes

Furthermore, reduced NK cell levels and function were inversely correlated with disease severity, suggesting that NK cells may be involved in some capacity.

T475

78384-78516

Sentence

denotes

One of the potential mechanisms by which NK cells may become hyporesponsive is via SARS-CoV-2 interference with type I IFN pathways.

T476

78517-78803

Sentence

denotes

In investigating the pathogenesis of other CoV infections, namely SARS and MERS, studies suggest that during acute CoV infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce chemokines and cytokines that induce NK cell migration and activation.

T477

78804-79033

Sentence

denotes

As NK cells are one of the main producers of IFN-γ, they may be involved in the IFN-γ-led cytokine storm that is responsible for the induction of inflammation-mediated ALI, ARDS, and subsequent mortality associated with COVID-19.

T478

79034-79110

Sentence

denotes

Inarguably, more research into the role of NK cells in COVID-19 is required.

T479

79111-79295

Sentence

denotes

Despite the knowledge gaps in COVID-19 pathophysiology, there has been a surge of clinical trials as the FDA continues to fast-track the approval of investigational therapeutics (269).

T480

79296-79616

Sentence

denotes

Here we have outlined potential therapeutics with a focus on mediating NK cell activity, including prophylactic treatments that could boost innate immunity in addition to therapeutics that could mitigate the immunopathological consequences of COVID-19, thereby relieving the burden on our health care systems (Figure 2).

T481

79617-79768

Sentence

denotes

Rigorous preclinical testing and thoughtfully designed clinical trials will be necessary for the development of robust therapeutics against SARS-CoV-2.

T482

79769-79885

Sentence

denotes

Importantly, we must be aware of the potential dangers immunotherapies may have in potentiating CoV immunopathology.

T483

79886-79932

Sentence

denotes

The fight against COVID-19 is not an easy one.

T484

79933-80049

Sentence

denotes

As with any novel disease, we will have to rely on incomplete pictures to guide reasoning for appropriate treatment.

T485

80050-80280

Sentence

denotes

We believe that immunotherapeutics targeting the innate immune response, and specifically NK cells, have the potential to flatten the curve and will be important instruments in our armamentarium against this pandemic and the next.

T486

80281-80934

Sentence

denotes

Figure 2 Short-list of immune modulating therapies undergoing clinical trial in COVID-19 patients and recommendations on who should receive therapy. (A) Healthy, uninfected individuals, who are at a high risk of becoming infected (through situational circumstances such as healthcare workers) would be most fit and suitable to receive investigational prophylactic therapies such as exogenous IFNs and heterologous vaccines. (B) Individuals who have tested positive for COVID-19 that are asymptomatic or have mild to moderate disease progression may benefit from receiving investigational immune stimulating therapies, including NK cell-based therapies.

T487

80935-81271

Sentence

denotes

It is critical that investigators must be vigilant to assess the safety profile and potential immunopathologies associated with these immunotherapies. (C) In severe COVID-19 patients, the most appropriate therapies to investigate would be those that mitigate immunopathologies, such as anti-inflammatory and immunosuppressive therapies.

T488

81272-81494

Sentence

denotes

Given the relatively low chance of toxicity and the wide range of beneficial immune effects, natural health products such as vitamin C and vitamin D can be suitable for investigation at all categories of COVID-19 patients.

T489

81496-81516

Sentence

denotes

Author Contributions

T490

81517-81586

Sentence

denotes

MM, LA, AM, DB, OO, GT, DMB, and JN contributed to literature review.

T491

81587-81637

Sentence

denotes

MM, LA, AM, DB, OO, and GT contributed to writing.

T492

81638-81686

Sentence

denotes

MM, LA, MA, and RA were responsible for editing.

T493

81687-81729

Sentence

denotes

MM and LA were responsible for formatting.

T494

81730-81805

Sentence

denotes

MA and RA oversaw the writing process and provided mentorship and guidance.

T495

81806-81880

Sentence

denotes

All authors contributed to the article and approved the submitted version.

T496

81882-81902

Sentence

denotes

Conflict of Interest

T497

81903-82075

Sentence

denotes

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

T498

82077-82190

Sentence

denotes

The authors would like to thank Dr. Doug Gray for his thorough editing, proofreading, and thoughtful suggestions.

T499

82191-82199

Sentence

denotes

Funding.

T500

82200-82431

Sentence

denotes

The authors have received funding from the Canadian Institutes of Health Research, the Cancer Research Society, and the Ontario Student Assistance Program through a Queen Elizabeth II Graduate Scholarship in Science and Technology.

T501

82432-82517

Sentence

denotes

The Ottawa Hospital Academic Medical Organization and The Ottawa Hospital Foundation.

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