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To prevent SARS-CoV-2 infections and generate long-lasting immunity, vaccines need to generate both, a strong humoral immune response as well as viral-specific CD4 and CD8 T cells. Previous results from our lab have shown that immunization in the presence of an agonist antibody targeting OX40 led to higher antibody titers and increased numbers of antigen-specific CD4 and CD8 T cells. Using the same strategy, we explored the effect of OX40 co-stimulation in the prime and boost together with the SARS-CoV-2 spike protein + adjuvant in C57Bl/6 mice. Our results show that OX40 engagement led to a significant increase in long-lived antibody responses when compared to mice that did not receive additional co-stimulation. In addition, spike protein and peptide-specific proliferation were greatly increased for both CD4 and CD8 T cells, with augmented secretion of IFN-γ, TNF-α and IL-2. Booster immunizations (7 months post prime) did not lead to anergy, but instead further increased the antibody and T cell responses. In initial experiments, the self-amplifying RNA (saRNA) vaccination generated lower antibody titers, independent of OX40 co-stimulation. However, the saRNA vaccine did induce a robust expansion of antigen-specific CD8 T cells, that expressed high levels of GrzmB, which was further increased by OX40 administration. The strong immune responses and the differential effects of the protein and saRNA vaccines suggest that heterologous prime-boost approaches, as currently approved in adults, might be beneficial in boosting different arms of the anti-viral immune response against SARS-CoV2, with OX40 agonists enhancing both approaches. Further studies in animals will aid defining the effects and benefits of this approach.

Background hetIL-15 is evaluated in on-going immunotherapy clinical trials. We studied the mechanism of hetIL-15 antitumor function in several mouse models (MC38, TC-1, 4T1 and EO771). Methods We evaluated tumor immune infiltrate by flow cytometry, multi-color immunohistochemistry (IHC), transcriptomics and proteomics. Results hetIL-15 treatment delayed primary tumor growth, induced tumor regression and reduced metastases. Flow cytometry and IHC showed increased intratumoral NK and CD8+ T cell infiltration. Tumor-resident NK and CD8+ T cells were activated with enhanced IFN-γ production, increased proliferation (Ki67+), expression of survival factors (Bcl-2) and cytotoxic potential (Granzyme B+). hetIL-15 therapy resulted in increased IFN-γ and XCL1 levels and enhanced tumor accumulation of conventional type 1 dendritic cells (cDC1) expressing XCR1, IRF-8 and CD103. Production of CXCL9 and CXCL10 chemokines by tumor infiltrating cDC1 was boosted in an IFN-γ-dependent manner. Increased frequency of circulating NK and CD8+ T cells expressing CXCR3 was found, suggesting their migration toward tumors following the CXCL9/10 chemokine gradient. Conclusions hetIL-15 inhibits tumor growth by triggering lymphocyte-myeloid cell interactions modifying the tumor chemokine and cytokine milieu. hetIL-15 causes induction of XCL1 by lymphocytes, inducing DC intratumoral number, subsequent IFN-γ dependent CXCL9/10 production by DC and increased attraction of effector lymphocytes.

Previously, we reported that while a single vaccination effectively generated therapeutic tumor-specific T cells for adoptive immunotherapy (AIT), repeated vaccination with the poorly immunogenic B16BL6 melanoma, modified to secrete GM-CSF, induced high levels of CD4+FoxP3+ T regulatory (Treg) cells and AIT with spleen cells was non-therapeutic. The effect could be overcome by transient depletion of CD4+ T cells, confirming the effect of Treg cells. Here experiments were performed to test whether this suppressive effect was observed in active-specific protection in 2 sarcoma models, MCA-304 and MCA-310. Mice were vaccinated once or thrice with either irradiated parental sarcoma or that sarcoma expressing GM-CSF. We did not observe significant changes in percentage or total count of splenic Treg cells under any conditions, however, tumor-specific IFN-γ release decreased in mice thrice vaccinated with GM-CSF-secreting tumors compared to parental (p < 0.001). Importantly, multiple vaccination offered tumor protection that was as good or better than single vaccination. In the most strongly immunogenic tumor, MCA-304, both single and triple vaccination with parental or GM-CSF-secreting tumors provided 100% protection. However, in the weakly immunogenic, MCA-310 sarcoma, multiple vaccinations improved effectiveness with the greatest effect seen with the GM-CSF-secreting MCA-310. Our results provide a rationale for the continued use of GM-CSF as a cancer vaccine adjuvant.

TCR-gene-modified CD8 T cells are used for cancer treatment. We examined if tumor-specific CD4 T cells would enhance persistence and function of adoptively transferred CD8 T cells. RAG1-/- mice bearing 3-day B16-D5 melanoma pulmonary metastases received both naïve tumor-specific Trp-1 TCR CD4 T cells and effector gp100-specific TCR CD8 transgenic T cells or CD8 T cells alone. Significantly fewer mice had pulmonary metastases when treated with both CD4 and CD8 T cells compared to CD8 T cells alone at day 10 (3/20 vs. 14/14 p

Vaccination with MCA sarcoma cells protects from a subsequent challenge with the same, but not other MCA sarcomas. This observation of unique tumor-specific protection is a well-established paradigm (Prehn and Main 1957). We postulated that all MCA sarcomas overexpress common mutated gene products with a short half-life (SLiPs), but only the unique tumor-rejection antigens are stable enough to be cross-presented and induce anti-tumor immunity. We recently reported that with proteosomal blockade, SLiPs could be isolated in autophagic-containing (LC-3) vesicles (termed DRibble) that were efficient for cross-presentation of antigens (Li, Y. et al). Here we examined if vaccination with DRibbles from one MCA sarcoma would cross-protect from a challenge with an antigenically distinct MCA sarcoma. While whole tumor cell vaccines provide protection to unrelated tumor in 0 of 9 comparisons, DRibble vaccines provided significant (p