Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

Women, especially adolescent girls in sub-Saharan Africa, remain disproportionately affected by HIV, underscoring the urgent need for effective prevention, including vaccines. Prior human HIV vaccine efficacy trials have largely failed except for the ALVAC/gp120/alum (RV144) regimen, which showed modest efficacy (~31%). The macaque model has reproduced RV144 correlates and performance, and improvements (DNA prime, V1 deletion to expose V2) increased protective immune responses, particularly V2-specific ADCC, NK cell and monocyte functions, and reduced CCR5 expression on CD4 T cells. However, overall efficacy of the AVIDNA/ALVAC/AV1gp120/alum regimen remains suboptimal (average ~70% reduction per-exposure risk, protecting ~50% of animals). SAMT-247, a zinc-finger inhibitor targeting the HIV/SIV nucleocapsid protein with virucidal activity and intracellular recycling, has shown prevention potential in vitro, ex vivo, and animal models. The research question was whether adding topical SAMT-247 to an engineered V1-deleted envelope vaccine could synergistically enhance protection against vaginal SIVmac251 acquisition and elucidate underlying immune mechanisms, particularly the role of zinc-dependent pathways.

• Human trials: Of nine efficacy trials, only RV144 (ALVAC + gp120/alum) showed modest efficacy (31.2%). HVTN-702 using MF59 failed, consistent with macaque predictions.
• Macaque studies: ALVAC/SIV + gp120 reproduced RV144, with improvements via DNA prime, simplified regimens, and V1 deletion to better expose V2 epitopes, enhancing protective correlates (V2-specific ADCC; NKp44+ IL-17+ mucosal cells; CCR2/CCL2 axis; CREB1 activation; monocyte efferocytosis; decreased CCR5 on Th1/Th2).
• Microbicides: SAMT compounds target mutationally intolerant NC zinc fingers causing zinc ejection and virucidal inactivation, with intracellular recycling. Prior studies show inhibition in cell-based and explant models, protection in mice and macaques, but variable efficacy.
• Rationale: Combining a clinically translatable RV144-like vaccine (with V1 deletion) and SAMT-247 could yield additive/synergistic protection, especially at mucosa, where innate effectors and reduced T-cell activation are key.

Study design: Female Indian rhesus macaques (n=50, age 2–3 years) were randomized into four groups: vaccine-only (n=18), vaccine + SAMT-247 (n=20), SAMT-247-only (n=6), concurrent gel controls (n=6). An additional 31 historical naïve controls challenged identically with the same SIVmac251 stock were included for statistical power. MHC typing and randomization balanced age, weight, haplotypes.

Vaccine regimen: Weeks 0 and 4: DNA prime with SIVgp160ΔV1 (2 mg/dose) and SIV239gag (1 mg/dose), intramuscularly in both thighs. Week 8: ALVAC-SIV encoding gag/pro/env (10^6 pfu). Week 12: ALVAC-SIV boost plus AV1 gp120 protein (400 µg) formulated in alum (Alhydrogel). Week 17 onward: weekly low-dose intravaginal SIVmac251 challenges (1 ml, 4,000 TCID50/ml) up to 14 exposures until confirmed infection (droplet digital PCR for viral DNA/RNA, plasma RNA persistence).

Microbicide dosing: 0.8% SAMT-247 in hydroxyethyl cellulose (HEC) gel (2 ml) applied intravaginally 4 h before each challenge to the vaccine+SAMT-247 group and the SAMT-247-only group; corresponding groups received HEC gel alone.

Primary endpoint and statistics: Vaccine efficacy measured as per-exposure risk reduction using log-rank (Mantel-Cox) tests within a discrete-time proportional hazards model. Average per-challenge risk estimated as total infections divided by administered challenges. Non-parametric tests used for continuous variables; Spearman correlations for immune correlates. Exploratory study; nominal P values without multiple-comparisons adjustment.

Immunological assays:

• Humoral responses: Plasma IgG to AV1 gp120 by ELISA; linear epitope mapping (Pepscan). ADCC assessed with EGFP-CEM-NKr-CCR5-SNAP target cells coated with AV1 gp120 and human PBMC effectors; endpoint titers and V2-specific ADCC determined via competitive inhibition with NC105/NC109 F(ab')2.
• NK cell function: Intracellular granzyme B, perforin, IFN-γ, TNF-α in blood and rectal mucosa NKG2A+ cells after stimulation (PMA/ionomycin or gp120 peptides ± SAMT-247). Mucosal NKp44+ IL-17+ cells quantified.
• Monocyte efferocytosis: CD14+ monocytes isolated pre-vaccination and at week 14; efferocytosis of apoptotic neutrophils quantified (percentage and MFI). Parallel in vitro SAMT-247 addition (100 µM) assessed effect on efferocytosis.
• T-cell phenotyping: CD4+ Th1 (CXCR3+CCR6−) and Th2 (CXCR3−CCR6+) memory subsets; expression of CCR5 and α4β7; activation/proliferation/exhaustion markers (OX40, CD40L, CD69, Ki67, CTLA-4, PD-1, PD-L1, LAG-3) using AIM assays upon gp120 ± SAMT-247 stimulation. Cytokines (IFN-γ, TNF-α, IL-10) assessed in blood and rectal mucosa.
• Zinc dependency: Effects of zinc chelation with TPEN (5 µM) on NK, monocyte, and T-cell functions after gp120 ± SAMT-247. Confocal microscopy measured intracellular zinc intensity in human NKG2A+ NK cells after PMA ± SAMT-247, with/without chelator.

Ethics: All animal and human procedures approved by appropriate ACUC/IRB committees; animals housed in AAALAC-accredited facilities; healthy donor PBMCs under NIH protocol (NCT00001846).

• Efficacy against SIV acquisition:

  • Vaccine-only: 65% reduction in per-exposure risk vs all controls (P=0.0074); trend vs concurrent controls; significant vs historical controls (P=0.0061). 40% (7/18) remained uninfected.
  • Vaccine + SAMT-247: 92.7% reduction in per-exposure risk vs all controls (P<0.0001); significant vs concurrent (P=0.0002) and vs historical (P<0.0001). Protected 16/20 (80%) animals. Significantly better than vaccine-only (P=0.006).
  • SAMT-247-only: No significant protection vs controls (P=0.27; also NS vs concurrent or historical separately).
  • Among vaccinated animals that became infected, early plasma viral RNA (2 weeks post-infection) was lower regardless of SAMT-247, but not sustained.

• Humoral immunity and ADCC:

  • Binding IgG to AV1 gp120 and V2 peptides, and overall ADCC titers did not differ between vaccine-only and vaccine+SAMT-247 groups at week 17.
  • In vaccine-only animals, ADCC activity and titers correlated with decreased risk (R=0.67, P=0.002; R=0.60, P=0.009). V2-specific ADCC (NC105 and NC109 competition) also correlated (R=0.75, P=0.0003; R=0.77, P=0.0002).
  • In vitro, treating effector PBMCs with SAMT-247 significantly increased ADCC mediated by vaccinated plasma (P<0.0001); SAMT-247–induced increment correlated with delayed infection in vivo in the vaccine+SAMT-247 group (R=0.50, P=0.024).

• NK cell modulation by SAMT-247:

  • In macaque mucosal NKG2A+ cells, SAMT-247 increased granzyme B and perforin (P=0.02 for both) and decreased IFN-γ (P=0.02) after PMA stimulation.
  • Frequencies of mucosal Env-specific NKp44+ IL-17+ cells pre-challenge (week 13) did not differ between groups; however, their baseline frequency correlated with decreased risk in vaccine-only animals (R=0.77, P=0.0002). In vitro SAMT-247 increased PMA-induced NKp44+ IL-17+ cells in rectal mucosa (P=0.04).

• Monocyte efferocytosis:

  • Vaccine-induced CD14+ efferocytosis (week 14) correlated with reduced risk in vaccine-only animals (R=0.62, P=0.01).
  • In vitro SAMT-247 increased both the percentage of efferocytosing CD14+ cells and efferocytosis MFI at pre- and post-immunization timepoints (P<0.0001).
  • In vaccine+SAMT-247 animals, the SAMT-247-induced increase in efferocytosis trended with protection (R=0.42, P=0.065).

• T-cell activation and cytokines:

  • At week 17, gp120 + SAMT-247 decreased vaccine-induced (Ki67+) αβ CCR5+ Th1 and Th2 frequencies (P=0.04; P=0.03) and increased total αβ CCR5+ Th1/Th2 frequencies (P=0.008; P=0.004). The percentages of αβ CCR5+ Th1 and Th2 after gp120 + SAMT-247 stimulation correlated with delayed acquisition (Th1: R=0.82, P=0.012; Th2: R=0.76 (reported 0.78 in figure text), P=0.020).
  • SAMT-247 did not enhance expression of activation/proliferation/exhaustion markers (OX40, CD40L, CD69, Ki67, CTLA-4, PD-1, PD-L1, LAG-3) following gp120 stimulation.
  • In mucosal Th cells, SAMT-247 with PMA reduced TNF-α (Th2 P=0.004; Th1 P=0.04) and increased IL-10 in Th1 (P=0.04).

• Zinc dependency:

  • Confocal microscopy showed higher intracellular zinc staining in PMA + SAMT-247–treated human NK cells vs unstimulated (P=0.04).
  • Zinc chelation with TPEN decreased IFN-γ and TNF-α production in NK cells after gp120 or gp120+SAMT-247; granzyme and perforin expression were variably affected depending on condition.
  • TPEN reduced frequency and IL-10 production of CD14+ monocytes and broadly compromised Th1/Th2 cytokine production (IFN-γ, TNF-α, IL-10) across CCR5/α4β7 subsets, indicating zinc dependence of these functions.

Overall, SAMT-247 synergized with vaccination by enhancing NK cytotoxicity and V2-specific ADCC, boosting monocyte efferocytosis and NKp44+ IL-17+ mucosal responses, and promoting anti-inflammatory cytokine profiles, likely via modulation of zinc availability.

The addition of topical SAMT-247 to a V1-deleted, RV144-like DNA/ALVAC/AV1 gp120/alum vaccine markedly enhanced protection against vaginal SIVmac251 acquisition, reducing per-exposure risk by ~93% and protecting 80% of animals. SAMT-247 alone was insufficient under the dosing window used, indicating synergy rather than simple additivity. Immune correlate analyses suggest SAMT-247 augments key mucosal and systemic innate effector functions—enhancing NK cell cytotoxic machinery (granzyme/perforin) and ADCC, increasing mucosal NKp44+ IL-17+ cells that support epithelial integrity, and boosting monocyte efferocytosis—while simultaneously tempering pro-inflammatory T-cell cytokines (lower TNF-α, higher IL-10). These changes likely improve mucosal homeostasis and reduce availability of activated CCR5+ CD4+ targets at exposure, aligning with established correlates of decreased HIV/SIV acquisition risk. Zinc chelation experiments implicate zinc mobilization as a mechanistic axis through which SAMT-247 influences immune function: multiple NK, monocyte, and T-cell activities were zinc-dependent, and SAMT-247 increased intracellular zinc signal under stimulation. Collectively, the findings support that effective prevention may hinge on potentiating innate effector mechanisms and maintaining low T-cell activation in concert with vaccine-elicited humoral responses. The results delineate a plausible translational path to enhance vaccine efficacy in women using a topical microbicide adjunct.

This study demonstrates that combining a V1-deleted envelope DNA/ALVAC/AV1 gp120/alum vaccine with the topical zinc-finger inhibitor microbicide SAMT-247 confers strong protection against vaginal SIV acquisition in macaques, outperforming vaccination alone. Mechanistically, SAMT-247 appears to enhance vaccine-induced protection by modulating zinc-dependent pathways that increase NK cell cytotoxicity and ADCC, augment monocyte efferocytosis, and promote anti-inflammatory T-cell cytokine profiles while reducing T-cell activation. These data highlight underappreciated roles of monocytes and NK cells as effectors of vaccine-mediated protection and underscore the importance of pro-resolution responses at mucosal sites. Future work should evaluate safety, pharmacodynamics, dosing schedules, and efficacy of the vaccine–microbicide combination in human trials, further dissect zinc-mediated mechanisms, and explore optimization of mucosal delivery and broader applicability across diverse viral strains and populations.

• Use of historical controls, although no significant difference was observed compared with concurrent controls, introduces potential biases.
• Lack of mucosal samples from vaccinated animals at time of exposure limited direct in vivo assessment of mucosal ADCC/NK functions; some conclusions relied on in vitro stimulation or surrogate tissues.
• Exploratory nature with multiple immune comparisons; P values were nominal without correction for multiple testing.
• SAMT-247 was evaluated with a specific timing (4 h pre-challenge) designed to reduce standalone efficacy; generalizability to other timing/dosing regimens requires study.
• Nonhuman primate SIV model and female-only cohorts may limit direct extrapolation to humans and to males.
• Some mechanistic assays had small sample sizes in subgroups (for example, n=2–4 in certain zinc chelation experiments).