The strategies above involve several steps that together comprise a push-pull approach, to optimize antigen structure, improve quantity and quality of the response, and remove regulatory barriers. Blocking negative regulation in HIV and vaccine strategies: We carried out epitope enhancement (sequence modification to improve MHC binding) to increase MHC binding of epitopes from several viral antigens including HIV, and showed these improved vaccine efficacy. We have pioneered the use of anti-TGFb as a novel checkpoint inhibitor, given that TGFb is one of the most immunosuppressive cytokines known. We even showed preliminary evidence of efficacy and safety in a clinical trial in melanoma patients. We have also compared combinations of checkpoint inhibitors and anti-TGF-b in enhancing vaccine efficacy in mice, and found preliminary data for synergy among anti-TGFb, anti-LAG3, anti-TIGIT and anti-PDL1. These strategies are applicable to both HIV and cancer. Cytokines as vaccine adjuvants for HIV and induction of high avidity T cells: Our earlier work first showed that high avidity T cells were more effective at clearing viral infections. We found ways to induce them with cytokines and TLR ligands, including expressing IL-15 in the vaccine, and inclusion of costimulatory molecules, in collaborations with Tom Waldmann and with Jeff Schlom, respectively. The quality of response proved more important than the quantity. We recently found, using a novel adjuvant, CAF09, that we could lower the vaccine dose sufficiently to induce higher avidity CD4 T cells to better clear HIV-gp160-expressing virus infection in mice. We also found that IL-1b induces Th17 helper cells that do not help Tc1 CD8 T cells that protect against vaccinia virus expressing HIV gp160. Rather, they skew the CD8 response to Tc17 cells that make IL-17 and do not protect. TGF-b blockade can prevent this problem, as TGF-b is involved in Th17 induction. We are examining the epigenetic and transcriptional mechanisms behind high avidity T cells by carrying out a single-cell RNAseq study of high vs low avidity T cell lines generated against the same antigen. T cells from OT-I TCR-transgenic mice were grown with high or low concentrations of antigen to produce low or high avidity lines, respectively, with the identical TCR, to exclude effects of intrinsic TCR affinity. These were stimulated with the same SIINFEKL peptide, and subjected to RNAseq. Also, T cells were generated against the same SIINFEKL peptide and subjected to concurrent TCR and RNAseq analysis, and high and low avidity lines with the same clonotype were compared. Promising genes are being tested in knock-down studies. We also found that IL-21 synergizes with IFN-g to induce IFN-stimulated genes and clear Citrobacter colitis through an effect on STAT1. We also examined combinations of cytokines and TLR ligands as vaccine adjuvants and found greatest efficacy of IL-15 + TLR3 and TLR9 agonists, but also some efficacy of IL-12 + GM-CSF. We are comparing these for qualitative differences in the immune response as well as quantity. Mucosal immunity, microbiome and HIV/SIV vaccines: About 85% of HIV transmission is mucosal. We found that a mucosal T cell vaccine can impact the initial mucosal nidus of infection. We are studying induction and trafficking of T cells, DCs, and MDSCs among mucosal compartments to optimize mucosal vaccine efficacy. In mice, we found that T cells could be directly primed in the vaginal mucosa, despite lack of organized lymphoid structures, contrary to textbook dogma. We also discovered that colonic DCs can imprint CD8 T cells to home back to the colon preferentially, based on differential retinoic acid expression vs. small intestine DCs. We are currently examining the mechanism of this effect in terms of chemokine receptor expression and homing activity, and by using an RNAseq comparison of clonal OT-1 T cells stimulated by DCs from the small vs large intestines, as well as single-cell RNAseq of DCs from the colon vs small intestine. We discovered that altering a cathepsin S cleavage site could protect an immunodominant epitope of HIV gp120 from degradation in endosomes during cross-presentation, providing proof of concept for a novel mechanism of virus escape for HIV that infects mostly non-APCs. Using NHP models, we found that activated mucosal T cells determine susceptibility to SIV/HIV infection (transmission), eclipse time prior to systemic viral detection, and acute viral load. We found that even in naive animals, gut microbiota can strongly affect susceptibility to transmission, by immune activation, and also affect vaccine efficacy. We are exploring the mechanisms by testing the effect of antibiotics on multiple cell subsets in the large compared to the small intestine. Further, we found that vaccines can induce MDSCs that counteract vaccine protection, and also infection can affect trafficking of MDSCs. We also demonstrated for the first time that MDSCs could be infected by SHIV in vivo. As most HIV transmission is through mucosal surfaces and HIV homes to the gut mucosa, we have designed and invented a nanoparticle vaccine delivered orally in mice but coated to pass through the stomach intact and to be released selectively in the large intestine where it induces CTL responses in the large intestine and vaginal mucosa and protects against rectal or vaginal challenge with a virus. We translated our oral nanoparticle (NP) approach to macaque SIV vaccines, finding reduced risk against SHIV rectal acquisition in 2 studies. Surprisingly, we discovered that protection against SIV acquisition in 3 studies can occur without anti-envelope antibodies. While T cell immunity was induced, it did not correlate with protection, and protection was not abrogated by CD8 T cell depletion. Rather, protection correlated with trained innate immunity, involving monocyte "memory" for SIV in induction of cytokines maintained by epigenetic changes. We have carried out RNAseq to determine what changes occur in monocytes after immunization, leading to new mechanistic hypotheses including an unexpected role for platelets and their production of platelet factor 4 (PF4), also known as CXCL4. This is manifested by platelet-cell aggregates in which platelets actually coat cells. Also, we are combining an SIV vaccine and mucosal NP boost to increase mucosal immunity with a microbicide to reduce the viral inoculum in an OAR-funded study. Our hypothesis is that priming and boosting with mucosally delivered nanoparticles containing V2 loop antigens will both increase mucosal immunity to protect against intrarectal SIV challenge, and selectively expand immune responses against the V2 loop that have been shown to correlate with protection in the RV144 phase III human trial. Initial results show that priming and boosting orally with NPs containing a V2-loop pentamer can increase protection compared to the base vaccine alone consisting of SIV gag and env DNA, ALVAC expressing gp120, and deltaV1 gp120 protein in alum. Immune correlates of improved protection, include mucosal IgA, stronger ADCC especially targeting the V2 loop, mucosal gp120-specific plasma cells and plasmablasts, and T cell responses. However, some aspect of empty control NPs counteracted some of the protective benefits, in part by activating gut mucosal CD4 T cells that can be targets for SIV infection. Thus, the NP strategy needs to be adjusted to avoid this unwanted counterproductive effect.