In this study, we combined two vaccine technologies for intranasal antigen delivery using nanoparticle delivery system and CpG adjuvant (TLR-9 agonist) to design a novel needle-free SARS-CoV-2 vaccine platform. Currently, only eight of the 170 vaccines that are under clinical studies are administered intranasally and in that only two of them are subunit vaccines (World Health Organization, 2022a). The remaining intranasal vaccines are vector-based for which there is a risk associated with pre-existing immunity against the vector that impair post-vaccination immunity. A nanoparticle-based vaccine formulation from mannose-conjugated chitosan polymer was prepared at the Ohio State University (OSU, USA) using an optimized strategy followed for swine influenza virus (Dhakal et al., 2018; Renu et al., 2021) and avian salmonellosis (Han et al., 2020) NP vaccines. Our candidate NP vaccine called NARUVAX-C19/Nano is listed by the WHO as a Covid-19 vaccine under preclinical studies (World Health Organization, 2022a). Based on previous data (Borges et al., 2008; Zuo et al., 2021; Malik et al., 2018) on the effectiveness of CpG oligodeoxynucleotide adjuvant in enhancing the immunogenicity of several intranasal vaccines, a NP-vaccine formulation containing the adjuvant CpG55.2 (Vaxine Pty Ltd, Adelaide, Australia) was tested in our NP formulation. This adjuvant has its own unique sequence and was used in intramuscular subunit SARS-CoV-2 vaccine (Li et al., 2021; Li et al., 2022; Tabarsi et al., 2022) called Covax-19/SpikoGen®, which has passed Phase III clinical trials and received approval for emergency use in Iran.
In this study, intranasal administered NP-vaccine formulations elicited a different immunogenicity profile from that of intramuscular alum-adjuvanted RBD vaccine. Our both the NP-vaccine formulations in rodent models did not induce measurable serum neutralizing antibodies, while induced mainly sIgA and cell mediated immune responses. This feature of our NP vaccine is a cardinal difference from other intranasal subunit RBD- or Spike-based vaccines conjugated with Diphtheria toxoid (EcoCRM®) (Wong et al., 2022) or outer membrane vesicles (OMVs) from Neisseria meningitidis (van der Ley et al., 2021), respectively, which were shown to induce both systemic neutralizing antibodies and local IgA responses. However, despite the absence of serum neutralizing antibodies, a crucial component of anti-SARS-CoV-2 infection immunity (Buchholz et al., 2004; Jiang et al., 2020), our NP-vaccine formulations in vaccinated hamsters provided significant protection against SARS-CoV-2 infection. The NP-vaccine significantly reduced the challenge virus load in both the upper and lower respiratory tract, and, most importantly, reduced the degree of lung damage in hamsters after challenge to a greater extent than the intramuscular vaccine group. It appears likely that protection with NP-vaccine formulations was due to both anti-RBD sIgA and Th1 cellular immune responses, consistent with findings of others (Weiskopf et al., 2020; Moss, 2022; Aleebrahim-Dehkordi et al., 2022). Although the addition of CpG to the NP-vaccine formulation induced production of IL-17, a Th17 cytokine involved in protective immunity against many pathogens (Mills, 2022), this did not translate to increased protective efficacy of the intranasal NP vaccine.
The intramuscular administered alum-adjuvanted Spike-RBD vaccine, despite inducing a systemic humoral and Th2 cellular immune responses, it failed to provide protection against SARS-CoV-2 infection. We attribute this to the use of monomeric RBD protein, which when compared to the full-length Spike trimer induces significantly lower titers of neutralizing antibodies according to our earlier studies (Tabynov et al., 2022c). In general, the use of Spike-RBD protein in any COVID-19 vaccine without the NP platform requires large doses of antigen and multiple immunizations to achieve the protective efficacy (Yang et al., 2021). Alum is a highly Th2-biased adjuvant, and in our earlier study (Tabynov et al., 2022b) with a veterinary COVID-19 vaccine called NARUVAX-C19 (pets) studied in juvenile cats, the alum adjuvant did not enhance neutralizing antibody titers compared to antigen alone.
Another important part of the present research was evaluation of vaccines in protecting against virus transmission from vaccinated challenged animals to naïve sentinels. Despite significantly lower titers of SARS-CoV-2 than control group in NP-vaccinates in nasal turbinates and oropharyngeal swabs of hamsters, that was not seen with other intranasal vaccine candidates (Wong et al., 2022; van der Ley et al., 2021), the infected NP-vaccinates continued to transmit virus to sentinel animals. This finding is consistent with others showing hamsters or humanized mice immunized with viral vector (Bricker et al., 2021; Tostanoski et al., 2020) or genetic (Kalnin et al., 2021) vaccines, after challenge still transmit infectious virus. Previously, our intramuscular subunit spike-trimer based squalene emulsion-adjuvanted NARUVAX-C19 vaccine protected against SARS-CoV-2 virus transmission in a hamster model, showing that blocking of transmission is achievable with some vaccines (Tabynov et al., 2022c). Many RBD-based vaccines require at least 3 doses to achieve maximum efficacy, so a limitation of our study is that we only tested a 2-dose vaccine regimen. Another limitation is that we only tested challenge with the 100% homologous virus strain.
Future studies will explore how the NP-vaccine can be modified to increase its systemic immunogenicity and protective efficacy, like reported in pigs vaccinated with chitosan-NP swine influenza virus vaccines (Dhakal et al., 2018; Renu et al., 2021). This could include testing a combination vaccine scheme where our other subunit spike-based oil-adjuvanted NARUVAX-C19 vaccine is administered intramuscularly followed by intranasal NP-vaccine as a mucosal booster. It would also be interesting to test whether the efficacy could be improved by loading the NP with full spike trimer rather than RBD protein alone.