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

Abstract

In response to the need for an efficacious, thermally-stable COVID-19 vaccine that can elicit both humoral and cell-mediated T-cell responses, we have developed a dual-antigen human adenovirus serotype 5 (hAd5) COVID-19 vaccine in formulations suitable for subcutaneous (SC), intranasal (IN), or oral delivery. The vaccine expresses both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins using an hAd5 platform with E1, E2b, and E3 sequences deleted; hAd5(E1-, E2b-, E3-); that is effective even in the presence of hAd5 immunity. In the vaccine, S is modified (S-Fusion) for enhanced cell surface display to elicit humoral responses and N is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal pathway to increase MHC I and II presentation. Initial studies using subcutaneous (SC) prime and SC boost vaccination of CD-1 mice demonstrated that the hAd5 S-Fusion + N-ETSD vaccine elicits T-helper cell 1 (Th1) dominant T-cell and humoral responses to both S and N. We then compared SC to IN prime vaccination with either an SC or IN boost post-SC prime and an IN boost after IN prime. These studies reveal that IN prime/IN boost is as effective at generating Th1 dominant humoral responses to both S and N as the other combinations, but that the SC prime with either an IN or SC boost elicits greater T cell responses. In a third study to assess the power of the two routes of delivery when used together, we used a combined SC plus IN prime with or without a boost and found the combined prime alone to be as effective as the combined prime with either an SC or IN boost in generating both humoral and T-cell responses. The findings here in CD-1 mice demonstrate that combined SC and IN prime-only delivery has the potential to provide broad immunity, including mucosal immunity, against SARS-CoV-2 and supports further testing of this delivery approach in additional animal models and clinical trials.

Prime hAd5 Spike + Nucleocapsid Vaccination Induces Ten-Fold Increases in Mean T-Cell Responses in Phase 1 Subjects that are Sustained Against Spike Variants

“…Here we show that our strategy of including N-ETSD, confirmed to be directed to the endo-/lysosomal compartment with S optimized for cell-surface display 1,47 results in recognition of the N-ETSD and S-Fusion + N-ETSD antigen(s) by previously SARS-CoV-2 patient T cells and in generation of S- and N-reactive T cells in participants receiving the hAd5 S-Fusion + N-ETSD vaccine. Not only does the enhancement of T-cell responses by the addition of N in this vaccine confer a greatly likelihood of sustained protection against variants as our T-cell epitope HLA binding prediction analysis would suggest, it presents the possibility that this vaccine can be effective as a ‘universal’ booster.…”

“…Here we show that our strategy of including N-ETSD, confirmed to be directed to the endo-/lysosomal compartment with S optimized for cell-surface display 1,47 results in recognition of the…”

Mucosal and transdermal vaccine delivery strategies against COVID-19

“…This vaccine candidate has entered in phase 1 trial [ClinicalTrials.gov Identifier: NCT04732468; ClinicalTrials.gov Identifier: NCT04591717]. The company recently reported a pre-clinical study in mice where they demonstrated significant immunogenicity when the vaccine candidate was used intranasally [23].…”

Dual-Antigen COVID-19 Vaccine Subcutaneous Prime Delivery With Oral Boosts Protects NHP Against SARS-CoV-2 Challenge

“…To address the ongoing COVID-19 pandemic ( 1 ), particularly in the face of viral evolution and evidence of viral variant resistance to antibodies and convalescent plasma ( 2 – 5 ), we have developed a vaccine anticipated to protect individuals from SARS-CoV-2 that has the potential to not only elicit robust humoral responses but also activate T cells. The dual-antigen vaccine ( Figure 1A ) comprises the SARS-CoV-2 spike protein fused to a signal sequence (S-Fusion) that, as predicted based on reports for similar sequences ( 6 , 7 ), in our previous in vitro studies enhances cell-surface expression of the spike receptor binding domain (S RBD) as compared to S wildtype ( 8 , 9 ). The vaccine also delivers the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) that directs N to the endo/lysosomal subcellular compartment as confirmed by immunohistochemistry ( 10 ).…”

“…In our preliminary studies of the hAd5 S-Fusion + N-ETSD vaccine in a murine model, we demonstrated that the vaccine not only elicits T helper cell 1 (Th1)-biased antibody responses to both S and N, it activates T cells ( 8 , 9 ). We have also confirmed that the vaccine antigens re-capitulate T-cell activation prompted by natural SARS-CoV-2 infection by demonstrating activation of COVID-19 convalescent patient CD4+ and CD8+ T cells upon exposure to homologous monocyte-derived dendritic cells (MoDCs) that had been transduced with the vaccine ( 10 ).…”