The SARS-CoV-2 virus has caused the most severe pandemic in the world. The development of a safe and effective vaccine is essential to manage the disease. We optimized an inactivated virus vaccine using the gamma irradiation method as an alternative to classical chemical inactivation methods. Previous studies have shown that gamma irradiation can induce immunogenicity more effectively than conventional inactivation methods [6, 27]. The use of gamma radiation process (due to a cobalt-60 source) has been developed for the production of effective vaccines [28, 29]. Gamma irradiation preserves antigen assembly, can reduce free radical damage due to water radiolysis, and can be used in a frozen state [20, 30]. Gamma irradiation is a cost-effective and faster method of viral inactivation used to produce many inactivated viral vaccines, such as human and avian influenza vaccines. The current study reports on the efficacy of a gamma-irradiated virus vaccine that rather than formalin inactivated virus.
The spike protein consists of two subunits, S1 and S2, which are cleaved by host cell furin-like proteases such as cathepsins during assembly and release of virus particles from infected cells. The S1 subunit has a variable sequence across coronavirus genera and species. The sequence of the spike protein in the genera of coronaviruses alpha and delta is very similar. The S1 subunit contains the receptor-binding domain (RBD) and plays an important role in binding to host receptors. Therefore, considering the important antigenic property of the spike protein in inducing immune responses in the host, the anti-S1 spike protein monoclonal antibody was used by ELISA method to compare the antigenic properties of the irradiated and un-irradiated virus samples. The spike (S) protein of the virus, which contains the major neutralizing epitopes in the receptor-binding domain (RBD) and the N-terminal domain (NTD), has been shown to be the most promising immunogen. Thus, most recently approved vaccines use full-length S (with or without modification) or whole virus (inactivated) as the target antigen [31]. Also in this study, evaluation of the antigenic properties of the spike protein using an ELISA kit showed no significant differences between irradiated and un-irradiated SARS-CoV-2 virus samples with and without trehalose (P > 0.05). However, the optical density of the irradiated and un-irradiated virus samples plus 20% trehalose is higher than that of the samples without trehalose. It may depend on the disaccharide playing an important role in maintaining the properties of proteins, which of course should be further investigated when evaluating the inactivated virus antigen in animal models.
Neutralizing Antibody
Offersgaard reported that increasing the dose of inactivated SARS-CoV-2 improved the induction of neutralizing antibodies when tested in mice. Although a second immunization in both mice and hamsters resulted in a large increase in nAbs titers compared with a single immunization, a third immunization in mice had little effect on nAb titers and S-specific antibody endpoint titers compared with two immunizations [32]. At times when the Delta or Omicron variant is prevalent, a third dose of vaccination has been shown to be highly effective in protecting individuals from severe COVID-19-related sequelae and preventing COVID-19-associated hospitalizations [33, 34]. Furthermore, higher levels of neutralizing antibodies are associated with lower risk of symptomatic infection, and immune protection depends on levels of neutralizing antibodies [35, 36]. Therefore, the vaccine-mediated antibody response against SARS-CoV-2 is required for higher efficacy of the SARS-CoV-2 vaccine [41].
Inactivated SARS-CoV − 2 vaccines based on a traditional platform show high safety and efficacy and prevent COVID-19 serum antibody response to currently commercially available inactivated vaccines has been studied in detail [38, 39, 40, 41]. However, the profile of serum or plasma antibody response elicited by inactivated vaccines against all circulating variants of concern (VOCs) (alpha, beta, gamma, and delta) and circulating variants of interest (VOIs) (lambda, mu, kappa, eta, iota v1, iota v2, epsilon, and zeta) is less well defined. Also, the efficacy of vaccine-induced neutralizing antibodies is rarely reported. Therefore, a comprehensive analysis of the characteristics of antibody responses to inactivated vaccines and characterization of potent and broadly neutralizing antibodies is informative for optimizing and updating vaccine design and immunization strategies and therapeutics [37]. Passive antibodies administered are one of the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. To date, the most potent monoclonal antibodies (mAbs) isolated from infected and vaccinated individuals were often dominant by those targeting RBD while many isolated NTD mAbs failed to reach 100% potency in neutralizing activity [39]. In this study, a gamma-irradiated vaccine against SARS-CoV-2 was used as an inactivated vaccine, and the neutralising antibody response showed a significant increase in four vaccinated hamster groups as early as three weeks after the first vaccination. However, it reached the peak antibody titer three weeks after the third vaccination. Therefore, we can suggest that the gamma-irradiated inactivated vaccine SARS-COV-2 can be vaccinated in three doses, one primer and two booster doses three weeks apart.
Mucosal antibodies
IgA can be enriched up to threefold in upper respiratory tract secretions compared to IgG, while IgG is the most common isotype in the blood and lower respiratory tract. The higher concentration of sIgA compared to IgG has been shown to result in greater avidity and higher neutralising capacity [25, 42, 43, and 44]. Mucosal IgG is usually derived from plasma by transduction but can also be produced locally by mucosal B cells in the lamina propria. In addition to directly neutralising viruses, non-neutralising antibodies can also mediate the clearance of viruses and virus-infected cells via interactions of the Fc domain of the antibody with complement [45], thus enabling antibody-dependent cellular cytotoxicity (ADCC) [46]. Recently, survival after moderate-severe SARS-CoV-2 infection was associated with antibody responses with robust Fc effector activity, suggesting that such immunity may contribute to protection against respiratory disease. IgA can be expressed at mucosal surfaces in both monomeric and dimeric forms as secretory IgA (sIgA) and occurs in humans in two isotypes, with IgA1 present in both systemic and mucosal secretions and IgA2 predominantly in mucosa [25]. Although sIgA has been reported to be elicited at oral and nasal mucosal surfaces after intramuscular vaccination in both clinical and animal studies for influenza and SARS-CoV-2, titers are generally modest and variable. In contrast, mucosal immunisation readily produces robust sIgA responses in upper and lower respiratory tract mucosa [25]. One of the most informative methods for evaluating effective immune responses against SARS-CoV-2, whether triggered by natural infection or intranasal immunization, is the determination of mucosal sIgA in nasal secretions or saliva synthesized by IgA-secreting plasma cells. In this study, the concentration of sIgA in nasal washings and NALT was evaluated and it increased significantly when the irradiated vaccine plus trehalose was administered via the intranasal route three weeks after the second and third vaccinations (P < 0.05).
Spleen Lymphocyte proliferation as the T cell response (ref5)
Previous experience with SARS-CoV-1 and MERS suggests that T cells may be the most important immune response for disease control [48]. However, T-cell immunity against coronaviruses is an important aspect of a successful vaccine, and it is a long-lived vaccine. In contrast to antibodies, cytotoxic T cells against coronaviruses persist and can be detected several months after an infection. Therefore, an ideal SARS-CoV-2 vaccine should stimulate both B and T cell immunity to provide optimal protection against COVID-19 [27].
Gamma radiation, as a superior inactivation method, can preserve T cell immunogenicity compared with other inactivation methods. Gamma rays can strongly penetrate the virus, resulting in direct damage to the genetic material without altering the structural proteins [49]. Mullbacher has previously shown that alphaviruses [50] and bunyaviruses [51] can be rendered noninfectious by gamma irradiation and yet have the ability to elicit cytotoxic T-cell responses. Gamma-irradiation can be used to produce an experimental influenza vaccine and reported that gamma-irradiated Influenza virus preparations promoted T-cell immunity [52, 53, 54, 55, and 56]. Viral replication can be eliminated during gamma irradiation, while immunogenicity and viral protein structure are preserved, so that viral proteins are naturally presented to the immune system, facilitating the induction of both T cells and humoral immunity. In the current study, splenic lymphocyte proliferation was increased in all vaccinated hamsters and was higher in the irradiated vaccine groups. In addition, we may introduce an irradiated inactivated vaccine SARS-CoV-2 plus disaccharide trehalose via the intranasal route of administration and another irradiated inactivated vaccine SARS-CoV-2 plus alum via the subcutaneous route of administration as safe and efficient vaccines against COVID-19. Furthermore, cytokine assay will be done in the future study to evaluate exact immune cells.