Th1 Dominant Nucleocapsid and Spike Antigen-Specific CD4+ and CD8+ Memory T Cell Recall Induced by hAd5 S-Fusion + N-ETSD Infection of Autologous Dendritic Cells from Patients Previously Infected with SARS-CoV-2

Abstract

To address the need for a safe, efficacious vaccine against SARS-CoV-2 infection with the critical properties of enabling both blocking viral entry into cells and clearing virus from cells already infected, we have developed a bivalent, human adenovirus serotype 5 (hAd5) SARS-CoV-2 S-Fusion + N-ETSD vaccine that is currently in clinical testing. This vaccine uses the next-generation hAd5 [E1-, E2b-, E3-] platform previously used successfully in cancer patients with pre-existing adenovirus immunity, engineered to express both SARS-CoV-2 spike (S) protein modified to improve the generation of neutralizing antibodies to block entry of the virus, and nucleocapsid (N) protein with an Enhanced T cell Stimulation Domain (ETSD) to activate CD4+ and CD8+ T cells to clear the virus and block replication by killing infected cells. The targeting of N to endosomes and lysosomes to enhance CD4+ and CD8+ T-cell responses distinguishes our vaccine. In our previously reported pre-clinical studies we showed that in mice, the hAd5 S-Fusion + N-ETSD vaccine elicits both humoral and T-cell responses that are robust and T helper cell 1 (Th1) dominant. Here we report that the hAd5 S-Fusion + N-ETSD vaccine is recognized by anti-sera and T cells from previously SARS-CoV-2 infected patients, and that the presence of N is vital for T-cell recall. The findings presented herein: i. demonstrate specific recognition of hAd5 S-Fusion + N-ETSD infected cells by plasma antibodies from previously SARS-CoV-2 infected patients, but not antibodies from virus-naive subjects; ii. show enhanced binding of plasma SARS-CoV-2 antibodies from previously infected patients to monocyte-derived dendritic cells (MoDCs) expressing the hAd5 S-Fusion + N-ETSD vaccine as compared to hAd5 S-Fusion alone; iii. reveal N-ETSD localizes to vesicles associated with MHC class II antigen presentation, including endosomes, lysosomes, and autophagosomes in MoDCs; iv. demonstrate endosome/lysosome-targeted N-ETSD elicits higher interferon-gamma T-cell responses than cytoplasm-localized N; and v. N-ETSD alone or in the hAd5 S-Fusion + N-ETSD construct induces both CD4+ and CD8+ T cell memory recall. This recognition of hAd5 S-Fusion + N-ETSD vaccine antigens by T cells from previously SARS-CoV-2 infected patients, together with the ability of this vaccine candidate to elicit de novo immune responses in naive mice suggests that it re-capitulates the natural immune response to SARS-CoV-2 to activate both B and T cells towards viral neutralization and recognition of infected cells, critical for prevention of COVID-19 disease. Intriguingly, our hAd5 S-Fusion + N-ETSD T-cell biased vaccine has the potential to not only provide protection for uninfected individuals, but also to be utilized as a therapeutic for already infected patients to induce rapid clearance of the virus by activating T cells to kill the virus-infected cells, thereby reducing viral replication and lateral transmission.

Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, potentially resulting in an escape mutant

“…Although not the subject of the investigation described here, we are developing a dual-antigen human adenovirus serotype 5 (Ad5) platform-based vaccine that delivers both a spike protein with a linker to increase cell surface expression and humoral responses (S-Fusion) and the highly antigenic and conserved nucleocapsid (N) protein with a signal sequence (an Enhanced T-cell Stimulation Domain, ETSD) to direct it to subcellular compartments that enhance MHC I and II responses 18 . It is our belief that the vaccine, hAd5 S-Fusion + N-ETSD, due to its ability to elicit cell-mediated in addition to humoral immune responses, as shown in both a rodent model 19 and non-human primates 20 , offers hope to those regions such as South Africa wherein dangerous variants of SARS-CoV-2 have swept the country.…”

Complete Protection of Nasal and Lung Airways Against SARS-CoV-2 Challenge by Antibody Plus Th1 Dominant N- and S-Specific T-Cell Responses to Subcutaneous Prime and Thermally-Stable Oral Boost Bivalent hAd5 Vaccination in an NHP Study

“…Importantly, we have previously shown that the hAd5 S-Fusion + N-ETSD vaccine elicits T helper cell 1 (Th1) dominant antibody responses to both S and N as well as T-cell activation after vaccination of a murine (CD1) pre-clinical animal model (5). We have also shown that the SARS-CoV-2 antigens expressed by the hAd5 S-Fusion + N-ETSD construct are recognized by T cells from previously SARS-CoV-2 infected individuals when expressed by autologous monocytederived dendritic cells (6). These studies provide evidence that vaccination with the hAd5 S-Fusion + N-ETSD vaccine (Fig.…”

The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice

“…The hAd5 S-Fusion + N-ETSD vaccine we utilized the hAd5 [E1-, E2b-, E3-] comprises a wildtype spike (S) sequence [accession number YP009724390] modified with a proprietary linker peptide sequence as well as a wildtype nucleocapsid (N) sequence [accession number YP009724397] with a an Enhanced T-cell Stimulation Domain (ETSD) signal sequence to direct translated N to the endosomal/lysosomal pathway. 2 The SARS-CoV-2 S protein is found on the viral surface 11 and the N protein is found in the interior of the virus 23,56 (Fig. S1B).…”

“…The vaccine comprises the SARS-CoV-2 spike (S) protein optimized for cell surface expression (S-Fusion) 1 to increase humoral responses and the nucleocapsid protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to target N to the endosomal/lysosomal cellular compartment to enhance MHC I and II presentation. 2…”

“…It was our goal in the studies presented herein to confirm enhanced cell surface expression of S-Fusion as compared to S-WT (the localization of N-ETSD to endo/lysosomes is demonstrated in Sieling et al . 2020 2 ) in in vitro studies, then assess humoral and T cell responses in vivo studies in CD-1 mice. In mice, first the immune responses to SC prime and SC boost vaccination were determined, then SC and IN prime delivery were compared.…”