Adaptive Immune Response
Adaptive immune response is the specific response that the immune system exerts against pathogens. This mechanism is particularly active toward viral infections where the immune system produces specialized lymphocytes (to fight the virus), called memory B cells (to be effective in case of new infections) and antibodies (corresponding to the humoral response).135 The stimulation of adaptive responses in case of specific infections can be induced artificially through the introduction of attenuated pathogens, stimulating the production of specific antibodies. This is the principle of vaccination, which is the most common procedure for immunization of large areas of population against many kinds of lethal viruses.135 Besides, the use of viral proteins as antigens in the vaccine formulation leads to neutralizing antibodies, but, due to the low immunogenicity of isolated proteins, does not always stimulate sufficiently the immune system to reach total protection. More recently, nanotechnology has been applied to develop more efficient vaccines (e.g., nanovaccines). The use of nanostructures with a size similar to virus (virus-like nanoparticles) sensibly enhances the response helping to reach immunity.135 HNMs can adsorb viral particles and present them to the immune system.136,137 This method of vaccination has been successfully applied in vivo for the challenge of herpes simplex 2 virus. HSV-2 starts its spreading in vaginal tissues and then diffuses to the neurons causing death in mice. ZnO NPs (teardrop morphology), after vaginal inoculation with HSV-2, are able not only to prevent viral cell adhesion but also to expose viral antigens to T cells and DCs, leading to immunization. The preclinical trials against HSV-2 showed a survival to infection higher than 90%. This approach highlights the possibility to couple cell mimicking NMs to other co-adjuvants for the formulation of large spectrum nanovaccines (Figure 16).136,137
Figure 16  (A) Scanning electron microscopy images of ZnO tetrapod nanoparticles (ZOTEN) synthesized by flame transport synthesis. (B) Mice were challenged intravaginally with HSV-2 333 with or without ZOTEN. (C) To monitor progression of infection, mice were observed daily for the development of lesions around the vaginal opening, and base of the tail. Representative images from three independent experiments are shown. Reproduced with permission from ref (137). Copyright 2016 the American Association of Immunologists, Inc. HNMs have been also applied to the delivery of antigens, exposing them to the immune system. Fullerenes were found as suitable carriers for the delivery of drugs or nucleic acids.138 Functionalized fullerene can also self-assemble into virus-sized NPs.139 Investigated as vaccines in cancer immune therapy,140 polyhydroxy fullerenes (called fullerenols) display interesting properties for antiviral therapy, based on their capacity to self-assemble into virus-like particles (VLPs) and so to enhance the immunogenicity of the antigens.141 The great advantage of this strategy relies on the easy encapsulation process during the self-assembly making fullerenols versatile for the formulation of different kinds of vaccines. These VLPs were investigated against HIV-1141 and hepatitis C viruses.142 Compared to conventional protein- or peptide-based vaccines intended to induce antigen-specific adaptive immune responses, DNA vaccines are more stable, cost-effective, easy to manufacture, and safe in handling.143 However, DNA vaccines have the disadvantage of being poorly immunogenic.144 Fullerenol VLPs allow to avoid the use of other adjuvants. In the case of a vaccine against HIV-1, fullerenol VLPs penetrated easily into the cells resulting in an enhancement of DNA transfection. This was proved in a study using fullerenol encapsulating DNA encoding the HIV-1 envelope protein gp145 (Figure 17).141In vitro assays were performed in human embryonic kidney cells line (HEK293) showing good transfection ability. Following various immunization routes (e.g., activation of Toll-like receptor signaling or effector memory T cell immune response), fullerenol VLPs can induce an innate and a cellular immunity. A similar study was performed for hepatitis C using the HCV recombinant protein as antigen,142 confirming the potential efficacy of using fullerenols as antiviral vaccines. Nevertheless, these results require further mechanistic investigations. Indeed, in vitro studies also evidenced a suppressive effect of acquired immune response of C60 pyrrolidine tris-acid and fullerenol C60(OH)36.145 The fullerenol had a dose-dependent effect on T cell receptor-mediated activation and antibody production by B cells under anti-CD40/IL-4 stimulation. However, the molecular mechanism is still unknown.
Figure 17  Use of fullerenol as co-adjuvant for vaccination. (a) The structure of fullerenol, red balls represent O and white for H on the fullerene surface, and green balls represent C atoms. (b) The schematic diagram of HIV Env plasmid DNA encapsulated during the self-assembly of fullerenol. (c) TEM image of Env entrapped by fullerenol. (d) Compared to naked Env immunization group, IFN-γ production (immunospot) was significantly enhanced when mice were immunized with the formulation via various immunization routes, including intradermal (i.d.), intramuscular (i.m.), subcutaneous (s.c.), and intranasal (i.n.) injections. Fullerenol could decrease the antigen dosage (e) and immunization times (f). Reproduced with permission from ref (141). Copyright 2013 John Wiley & Sons, Inc. Other HNMs including AuNPs (two subcutaneous injections in guinea pigs) and nanodiamonds (three subcutaneous injections in Balb/C mice) were explored as carriers of viral proteins for immunization of swine transmissible gastroenteritis virus and H7N9 influenza, respectively, with good preliminary results.146,147 In these cases, the vaccine formulation relies on the adsorption of the viral antigen onto the surface of the nanoparticles. However, an effective vaccination depends on several factors. First of all, the size of the NPs plays a key role on the immune system. For instance, size-dependent vaccination efficacy has been reported in mice immunization against the foot-and-mouth disease virus (intraperitoneal and subcutaneous injection, every 7 days for 7 weeks) using AuNPs as antigen carriers. In this study, the most effective activity to stimulate the immune system was exerted by particles with a diameter in the range of 8 nm.148 Both smaller or bigger particles evidenced a drop-off of the immunization effect. This aspect cannot be ascribed to the antigen concentration, but must be associated only to the nanoparticle size; however, the mechanism of interaction remains unknown. The selected antigen plays also a crucial role in the preparation of wide spectrum vaccines. For instance, in the case of influenza, two major membrane glycoproteins, hemagglutinin and neuraminidase, are generally used as antigens. However, the antibodies produced by this vaccination strategy are selective to the dominant epitope which has a low effectiveness or is totally ineffective against other epitopes or other kinds of influenza viruses. M2 (a viral protein responsible for the budding and scission of the influenza virus) is commonly expressed in different types of influenza viruses with a high rate of conservation but with low antigenicity. It has been shown that AuNPs functionalized with M2e protein have a high immunization capacity in comparison to the antigen alone. Mice immunized with AuNPs (two intranasal injections), and then challenged, showed a survival rate higher than 90% to California-H1N1pdm, Victoria-H3N2, and Vietnam-H5N1 infections.149 This strategy shows that HNMs can be used to boost the immune response of low immunogenic molecules, providing a wide spectrum vaccination potential. Unfortunately, it has not been determined if the budding process in SARS-Cov-2 is mediated by viral proteins or via the host cell’s endosomal sorting complex, thus more research on the SARS-Cov-2 viral machinery is highly desirable.
All these approaches are based on the capacity of the nanoparticles to adsorb the antigens and expose them to the immune system in the appropriate conformation to produce the neutralizing and protective antibodies. However, the adsorption is not an easy process to control. For instance, AuNPs have been ineffective in the immunization against SARS-Cov, when S viral proteins were used as antigens.150 In particular, the immunization with the protein alone was more efficient than when it was adsorbed onto the AuNP surface. This failure was associated with a conformational change or denaturation of the antigen, which did not lead to the production of the specific antibody.150
Covalent chemistry strategies offer a higher control on the antigen quantification with higher reproducibility and possibility to bind different groups onto the surface of HNMs. For example, calcium phosphate nanoparticles (CaPNPs) were successfully covalently functionalized with Hen Egg lysozyme as model antigen showing an immunization 100 times higher in vivo compared to antigens alone using (one subdermal injection in mice).151 A similar approach was used with iron oxide NPs using mannose (to target DCs) and hepatitis B antigen showing good immunological activity in vitro (two subdermal injections in mice at 14 days distance).152 More recently, other types of vaccination strategies have been applied using HNMs. A smart example has been reported using multifunctional CaPNPs on herpes virus. In this study, CaPNPs have been covalently functionalized with alum/MPL as the adjuvant and two peptides as antigens selected via reverse vaccination. The NM stimulated the immune system generating highly efficient antibodies able to block cell-to-cell infection of herpes virus in vivo (three intramuscular injections in mice every 14 days), increasing the survival rate of immunized mice to 100% against the controls (20% survival, Figure 18).153
Figure 18  Scheme of CaPNPs used for vaccine. Left: CaPNPs functionalized with CpGm adjuvant and two different peptides as antigens. Top right: CaPNPs were able to reduce cell-to-cell virus spread in vitro. Bottom right: CaP nanovaccines were able to immunize mice against HSV-1. Reproduced with permission from ref (153). Copyright 2019 Elsevier B.V.