The scientific consensus is that safe and highly effective vaccines are required to end the ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A new study, released as a preprint on the bioRxiv* server, describes a new vaccine that presents two viral antigens to the human immune system, in order to elicit a more efficacious response.

Optimized viral antigens and vector platform

The current vaccine is developed on an adenovirus vector platform, and expresses the viral spike (S) and nucleocapsid (N) antigens, both modified to enhance immune responses.

The spike (S) protein was optimized for increased expression on the cell surface, to increase antibody production. The N protein was modified with an Enhanced T-cell Stimulation Domain (N-ETSD) so that it would migrate to the endosomal/lysosomal compartment within the cell, thus promoting increased antigen presentation via the major histocompatibility complex (MHC) I and II molecules.

The human adenovirus serotype 5 (hAd5) vector was modified by E1, E2b and E3 gene region deletions. These suppressed adenovirus-specific immune responses, so that the vaccine would be immunogenic even in individuals with pre-existing adenovirus immunity. This would allow durable antigen expression without the danger of inducing immunity to the vector itself.

This novel viral vector has the added advantage of safety, which was confirmed in a set of over 125 cancer patients in phase I and II studies. The findings of these studies showed that pre-existing immunity to the adenovirus did not prevent the induction of robust adaptive cellular immunity, in the form of specific CD4+ and CD8+ T cells.

Advantages of dual-antigen framework

The vaccine uses a dual antigen framework to minimize the risk of vaccine resistance, especially from the novel variants of concern (VOCs) that are rapidly emerging. These include the triple mutation-bearing South African variant (E484K, K417N, and N501Y); the UK variant (N501Y); and the Cal.20.C L452R variant.

The mutations in all three cases affect the receptor-binding domain (RBD) and may thus cause the virus to be resistant to the vaccines based on earlier strains that were in circulation during the first phase of the pandemic.

The N antigen has several advantages. Firstly, it is the most abundantly expressed viral antigen. Secondly, it is highly conserved among coronaviruses. Thirdly, almost all SARS-CoV-2 infections cause anti-N antibody production. Finally, the great majority of COVID-19 survivors had N-directed CD4+ T cell responses.

Importance of T cell responses

Since the adaptive cellular response to the virus appears to be at least as important as the humoral response in preventing severe COVID-19, these features of the N antigen assume great importance in vaccine design.

Antibody titers are closely associated with T cell responses, as are neutralizing antibody titers. This indicates that the T cell response is an essential prerequisite for a successful humoral response. Indeed, CD4+ T cell activation leads to the subsequent activation of antibody-producing specific B cells.

Both CD4+ and CD8+ T cells are commonly found to be elicited against the virus in COVID-19 patients, and may persist for up to 17 years, indicating their fundamental importance to long-term protection. Such T cell responses primarily involve the N antigen.

Earlier studies have shown that not only do memory T cells from prior SARS-CoV infection 17 years before the current pandemic recognize the viral proteins, but that some patients recovered from COVID-19 without detectable antibody responses, but with T cell responses.

Some studies seem to imply a rapid waning of neutralizing antibodies at about three months post-COVID-19, further buttressing the need to elicit T cell responses, and to include both S and N antigens in a COVID-19 vaccine.

Easier vaccine storage

The current study also focused on producing injectable and intranasal vaccines capable of being stored at -20^oC (up to one year) or 2-8^oC for up to one month, unlike the ultra-cold temperatures required to store the currently available mRNA-based vaccines. A formulation intended for oral use was also produced, that can be stored at room temperature.

Mucosal, humoral and T cell immunity

Besides storage ease, the intranasal and oral vaccines allow for the induction of mucosal IgA-mediated immunity, which indicates that they can reduce transmission more effectively than systemically administered vaccines.

Whether used intranasally, injected subcutaneously, or used by both routes together, the vaccine-induced a Th1-dominant neutralizing antibody response targeting the S and N antigen. In addition, both N- and S-directed CD4+ and CD8+ T cells were elicited, though cytokine levels were greater following stimulation by N proteins in all cases.

CD4+ and CD8+ T cell responses were stronger, respectively, following stimulation by N and S peptides, respectively, following subcutaneous injection. However, the N protein elicited a greater T cell response in both categories relative to the spike.

The T cell responses also displayed a Th1 bias, and their multifunctional nature indicated a higher degree of protection.

Viral neutralization in mice was confirmed to occur in all assays using serum from vaccinated animals.

What are the implications?

Perhaps the most significant and compelling finding was that a single prime administration by combined SC and IN dosing generated immune responses that were at least as great as dosing regimens that included a boost.”

The researchers have an interesting postulate: the higher CD4+ T cell responses and the Th1-skewed immune response were due to, firstly, the higher spike expression on the cell surface and secondly, the N optimization for localization to the endosomal/lysosomal compartment, which predisposes to MHC I and II presentation.

The latter resulted in a greater dendritic cell presentation, cross-presentation, B cell activation and therefore a massive neutralizing response, even at high dilution. It also leads to the simultaneous activation of CD4+ and CD8+ T cells, favoring the production of memory T and B cells.

CD+ T cells are induced by the subcellular localization of the N-ETSD construct. These cells, in turn, induce memory T cells and helper cells, to activate B cells for antibody generation. This agrees with earlier studies showing the lysosomal localization of the N protein is essential for the highest T cell interferon (IFN)-γ and cytotoxic T cell responses to occur.

The polyfunctional T cell responses to the N antigen include the induction of IFN-γ, TNF-a, and IL-2, which are features of immunity to bacterial and microbial infection. These are also characteristic of recovered COVID-19 patients, indicating the greater protection afforded by this vaccine candidate.

The hAd5 S-Fusion + N-ETSD vaccine has completed phase I trials in subcutaneous prime-boost regimens.

To our knowledge, our vaccine is currently the only one available in SC, thermally-stable oral,51 and IN formulations that offer the expanded possibilities for efficient, feasible delivery across the globe, particularly in developing nations.”

Combined subcutaneous and intranasal priming doses seem to be capable of providing broad-based immunity, including IgA-mediated mucosal immunity, against the virus. These findings provide the basis for further animal and clinical testing of this vaccine platform.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.