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Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8 + T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8 + T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8 + T cells were not recognized by VACV-reactive CD8 + T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R 70-78 variant of the dominant VACV B8R 70-78 epitope. C4R 70-78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels of Nr4a1 -encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77 tg ) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearranged Vα14-Jα18 TCR-α chain gene into the Nur77 tg (Nur77 tg ;Vα14 tg ) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4-producing NKT2 cell subset but not IFN-γ-producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions., Competing Interests: The authors declare no competing interest.

Tissue-resident memory T cells (T RM ) patrol nonlymphoid organs and provide superior protection against pathogens that commonly infect mucosal and barrier tissues, such as the lungs, intestine, liver, and skin. Thus, there is a need for vaccine technologies that can induce a robust, protective T RM response in these tissues. Nanoparticle (NP) vaccines offer important advantages over conventional vaccines; however, there has been minimal investigation into the design of NP-based vaccines for eliciting T RM responses. Here, we describe a pH-responsive polymeric nanoparticle vaccine for generating antigen-specific CD8 + T RM cells in the lungs. With a single intranasal dose, the NP vaccine elicited airway- and lung-resident CD8 + T RM cells and protected against respiratory virus challenge in both sublethal (vaccinia) and lethal (influenza) infection models for up to 9 weeks after immunization. In elucidating the contribution of material properties to the resulting T RM response, we found that the pH-responsive activity of the carrier was important, as a structurally analogous non-pH-responsive control carrier elicited significantly fewer lung-resident CD8 + T cells. We also demonstrated that dual-delivery of protein antigen and nucleic acid adjuvant on the same NP substantially enhanced the magnitude, functionality, and longevity of the antigen-specific CD8 + T RM response in the lungs. Compared to administration of soluble antigen and adjuvant, the NP also mediated retention of vaccine cargo in pulmonary antigen-presenting cells (APCs), enhanced APC activation, and increased production of T RM -related cytokines. Overall, these data suggest a promising vaccine platform technology for rapid generation of protective CD8 + T RM cells in the lungs.