Development of a vaccine for HIV is necessary as 38 million people are now infected worldwide, including 1.1 million in the US. The annual estimated HIV incidence is 1.5 million global infections per year, with 38,000 in the US. Moreover, adolescent girls and women are heavily impacted by the virus as they are both biologically more susceptible to HIV-1 infection than men, and often socially and culturally more vulnerable as well. Approximately 5,000 women aged 15-24 are infected with HIV every week, and in sub-Saharan Africa most new HIV infections in adolescents occur in. While advances in treatment options have helped to reduce the impact of HIV where available, these resources have limited reach, and the development of an effective HIV vaccine is urgently needed to fully curb the epidemic. Of the ten clinical HIV vaccine trials carried out to date, only the Canarypox based (ALVAC) vaccine combined with a bivalent gp120 protein boost in Alum adjuvant in the RV144 phase III clinical trial has demonstrated a modest degree of efficacy, reducing virus acquisition by 31.2% in both men and women. Most of the preclinical work that preceded RV144 was performed by my group in the macaque model of SIV infection. Following the conclusion of the trial in 2009, we redesigned our ALVAC-SIV/gp120 vaccine modalities to better mirror RV144 immunogens (ALVAC-Gag-pro/gp120-TM and two gp120 proteins) based on SIV founder variants and evaluated vaccine efficacy by exposing vaccinated macaques to low repeated doses of the most pathogenic and genetically complex SIV variant propagated in primary macaque cells, SIVmac251. In this model, the read out of vaccine efficacy is the reduction of average/exposure risk of mucosal SIVmac251 acquisition in vaccinated animals versus naïve controls. The refreshed animal model successfully recapitulated the efficacy of the RV144 vaccine modality in independent studies and predicted the futility of the ALVAC-HIV vaccine tested with the MF59 adjuvant (rather than alum) in the South African HVTN-702 trial validating its utility as an accurate parallel of HIV infection and serving as the basis for our ongoing work. This rigorous model permitted us to down-select two promising immunization approaches through the methodical comparison of envelope immunogens (gp120 wild type, gp120/CD4 fused protein and V1-deleted [DV1] gp120); priming strategies (ALVAC, Ad26 and DNA), and adjuvants (Alum, MF59, IGF-1 and ALFQA). Both selected approaches are constituted by co-administration of DNA plasmids producing Virus Like Particles (VLPs) in the priming, followed by 2 immunizations with VLPs produced by recombinant ALVAC-SIV, one alone and the other in combination (in contralateral limb) with V1-deleted gp120 protein (DV1gp120) formulated either in Alum or in ALFQA. Together, these vaccine regimens decreased the risk of mucosal SIVmac251 acquisition by up to 79% and protected 60% of macaques from infection. Occult SIVmac251 infection in vaccinated macaques was ruled out by simultaneous depletion of CD8+, NK cells, and monocytes. Of note, these vaccine regimens induce negligible CD8+ T-cell responses and no antibodies able to neutralize this SIVmac251 challenge stock, suggesting that non-canonical immunity may mediate protection. By combining functional analyses on freshly produced mucosal samples from large cohorts of vaccinated animals and banked, cryopreserved PBMCs and plasma, we were able to investigate correlates of risk of virus acquisition across vaccinated groups and build a plausible model for the ability of these regimens to inhibit virus seeding and consequent persistent infection. VLPs containing V1-deleted gp120 (delivered by DNA priming) favor induction of antibodies recognizing the native helical conformations of V2 that bind and kill infected cells by ADCC. V2-specific ADCC is a highly reproducible correlate of decreased risk of infection in all our studies. However, we demonstrated that ADCC alone is not sufficient to protect from SIV infection by passively administering NCI-05 monoclonal antibodies that recognize the coil-helical conformation of V2 and kill infected cells . Importantly, we found that DNA priming increases CCL2 and IL-18, respectively engaging monocytes and mucosal NKp44+ IL17+. In addition, recombinant ALVAC immunization synergizes with DNA by further harnessing monocytes and induces inflammasome activation. Importantly, ALVAC is distinct from other poxvirus vectors for its ability to induce IL-10. Our working hypothesis is that the combination of ALVAC with gp120 and alum adjuvant, both of which are also able to induce IL-10, results in engagement of the CCR2/CCL2 monocyte anti-inflammatory axis, activation of the AMP/CREB1 pathway, M2-like monocyte polarization, and efferocytosis, a pro-resolution monocyte function essential for the clearance of apoptotic cells. This hypothesis is further supported by the finding that, following immunization, the frequency of both mucosa macrophages and dendritic cells expressing CD73, an ecto-5'nucleotidase enzyme that cleaves AMP to generate the highly immune suppressing adenosine, correlated with decreased risk of virus acquisition. Thus, it could be envisioned that virus entry at the mucosal site of immunized animals results in apoptosis of infected cells (by ADCC), prompt clearance of apoptotic cells by macrophages (efferocytosis), reduced CD4+ cells activation and low or no expression of CCR5 (by CD73 expression), and reduced target cells for the virus, enabling eradication of infected cells. Indeed, CD4+ mucosal cells low or negative for CCR5 expression are associated with a decreased risk of virus acquisition. To translate this vaccine approach to humans, we have obtained support from the NIH Office of AIDS Research (OAR) to produce in GMP conditions HIV clade B p55gag DNA, HIV clade A/E A244DV1gp160 DNA, and clade A/E DV1gp120 protein, which will be tested in the First in Human CLEAR trial in 2025 at NCI and Walter Reed Army Institute of Research. This phase I trial will test the safety of all vaccine components, compare the immunogenicity of the V1-deleted immunogens with Alum or ALFQA, and that of V1-deleted immunogens to V1-replete immunogens with Alum. RNAseq studies on whole blood from immunized macaques revealed that the transcript level of the zinc finger protein ZC3H7A5 correlated strongly with vaccine futility (after accounting for False Discovery Rate). This finding prompted us to test whether vaccination could synergize with the zinc protein inhibitor SAMT-247, a virucidal compound that targets the HIV/SIV nucleocapsid protein. We found that topical administration of gel or vaginal rings (unpublished) releasing SAMT-247 synergizes with vaccination decreased risk of infection up to 92% with up to 80% of animals remaining uninfected. Here, we uncovered that SAMT-247 modulates NK, myeloid, and CD4+ cell immunity.