Background: The majority of antigen-based SARS-CoV-2 (SCV2) vaccines utilized in the clinic have had the Spike protein or domains thereof as the immunogen. While the Spike protein is highly immunogenic, it is also subject to genetic drift over time that has led to a series of variants of concern that continue to evolve, requiring yearly updates to the vaccine formulations. In this study, we investigate the potential of the N-terminal ectodomain of the ORF3a protein encoded by the orf3a gene of SCV2 to be an evolution-resistant vaccine antigen. This domain is highly conserved over time, and, unlike many other SCV2 conserved proteins, it is present on the exterior of the virion, making it accessible to antibodies. ORF3a is also important for eliciting robust anti-SARS-CoV-2 T-cell responses. Methods: We designed a DNA vaccine fusing the N-terminal ectodomain of orf3a to macrophage-inflammatory protein 3α (MIP3α), which is a chemokine utilized in our laboratory that enhances vaccine immunogenicity by targeting antigen to its receptor CCR6 present on im-mature dendritic cells. The DNA vaccine was tested in mouse immunogenicity studies, vaccinating by intramuscular (IM) electroporation and by intranasal (IN) with CpG adjuvant administrations. We also tested a peptide vaccine fusing amino acids 15-28 of the ectodomain to immunogenic carrier protein KLH, adjuvanted with Addavax. Results: The DNA IM route was able to induce 3a-specific splenic T-cell responses, showing proof of principle that the region can be immunogenic. The DNA IN route further showed that we could induce ORF3a-specific T-cell responses in the lung, critical for potential disease mitigation. The peptide vaccine elicited a robust anti-ORF3a antibody response systemically as well as in the mucosa of the lungs and sinus cavity. Conclusions: These studies collectively show that this evolutionarily stable region can be targeted by vaccination strategies, and future work will test if these vaccines alone or in combination can result in reduced disease burden in animal challenge models.

Background: The majority of antigen-based SARS-CoV-2 (SCV2) vaccines utilized in the clinic have had the Spike protein or domains thereof as the immunogen. While the Spike protein is highly immunogenic, it is also subject to genetic drift over time, which has led to a series of variants of concern that continue to evolve, requiring yearly updates to the vaccine formulations. In this study, we investigate the potential of the N-terminal ectodomain of the ORF3a protein encoded by the orf3a gene of SCV2 to be an evolution-resistant vaccine antigen. This domain is highly conserved over time, and, unlike many other SCV2 conserved proteins, it is present on the exterior of the virion, making it accessible to antibodies. ORF3a is also important for eliciting robust anti-SARS-CoV-2 T-cell responses. Methods: We designed a DNA vaccine by fusing the N-terminal ectodomain of orf3a to macrophage-inflammatory protein 3α (MIP3α), which is a chemokine utilized in our laboratory that enhances vaccine immunogenicity by targeting an antigen to its receptor CCR6 present on immature dendritic cells. The DNA vaccine was tested in mouse immunogenicity studies, vaccinating by intramuscular (IM) electroporation and by intranasal (IN) with CpG adjuvant administrations. We also tested a peptide vaccine fusing amino acids 15–28 of the ectodomain to immunogenic carrier protein KLH, adjuvanted with Addavax. Results: The DNA IM route was able to induce 3a-specific splenic T-cell responses, showing proof of principle that the region can be immunogenic. The DNA IN route further showed that we could induce ORF3a-specific T-cell responses in the lung, which are critical for potential disease mitigation. The peptide vaccine elicited a robust anti-ORF3a antibody response systemically, as well as in the mucosa of the lungs and sinus cavity. Conclusions: These studies collectively show that this evolutionarily stable region can be targeted by vaccination strategies, and future work will test if these vaccines, alone or in combination, can result in reduced disease burden in animal challenge models.

Mycobacterium tuberculosis remains a global health crisis, ranking among the deadliest infectious diseases worldwide. In response to the WHO’s call for therapeutic vaccines to complement antibiotic regimens and reduce tuberculosis (TB) treatment duration, we developed an intranasal DNA vaccine fusing the M. tuberculosis stringent response gene relMtb with the gene encoding the DC-targeting chemokine Mip3a (also known as CCL20 ). Administered alongside the first-line regimen, this vaccine accelerated a stable cure in immunocompetent murine TB models, reducing lung inflammation and eliciting robust and sustained RelMtb-stimulated T cell responses systemically and locally. The Mip3a/relMtb vaccine enhanced DC recruitment, activation, and spatial coordination with T cells, suggesting improved innate-adaptive immune synergy. Notably, it augmented the efficacy of a novel drug-resistant TB regimen as well. Critically, the vaccine induced analogous antigen-stimulated T cell immunity in nonhuman primates, the gold standard for preclinical TB vaccine evaluation, with responses detected in blood and bronchoalveolar lavage mirroring those observed in the murine models. These findings underscore the potential of this strategy to advance therapeutic TB vaccine development targeting M. tuberculosis persisters while providing a framework to define correlates of vaccine-mediated protection.