Vaccine induction of antibodies and tissue-resident CD8+ T cells enhances protection against mucosal SHIV-infection in young macaques

Abstract

Antibodies and cytotoxic T cells represent 2 arms of host defense against pathogens. We hypothesized that vaccines that induce both high-magnitude CD8+ T cell responses and antibody responses might confer enhanced protection against HIV. To test this hypothesis, we immunized 3 groups of nonhuman primates: (a) Group 1, which includes sequential immunization regimen involving heterologous viral vectors (HVVs) comprising vesicular stomatitis virus, vaccinia virus, and adenovirus serotype 5-expressing SIVmac239 Gag; (b) Group 2, which includes immunization with a clade C HIV-1 envelope (Env) gp140 protein adjuvanted with nanoparticles containing a TLR7/8 agonist (3M-052); and (c) Group 3, which includes a combination of both regimens. Immunization with HVVs induced very high-magnitude Gag-specific CD8+ T cell responses in blood and tissue-resident CD8+ memory T cells in vaginal mucosa. Immunization with 3M-052 adjuvanted Env protein induced robust and persistent antibody responses and long-lasting innate responses. Despite similar antibody titers in Groups 2 and 3, there was enhanced protection in the younger animals in Group 3, against intravaginal infection with a heterologous SHIV strain. This protection correlated with the magnitude of the serum and vaginal Env-specific antibody titers on the day of challenge. Thus, vaccination strategies that induce both CD8+ T cell and antibody responses can confer enhanced protection against infection.

Keywords: AIDS vaccine; AIDS/HIV; Adaptive immunity; Vaccines.

Figure 1. Vaccination that induces both antibody and tissue-resident CD8⁺ T cell responses confer enhanced protection against mucosal SHIV infection in young macaques.

(A) Vaccination groups and immunogens: 65 female RMs of ages 5–15 years were divided into 3 experimental groups. Animals in Group 1 were sequentially immunized with replication competent recombinant heterologous viral vectors (HVV) VSV, VV, and Ad5 each encoding SIVmac239 Gag protein. Animals in Group 2 were immunized with recombinant gp140 C.1086 K160N trimeric Env protein adjuvanted with the TLR7/8 agonist, 3M-052, encapsulated in PLGA nanoparticles (NP). Group 3 animals received immunizations with both HVV and adjuvanted Env protein, according to schedule indicated. (B) Study overview. Animals were bled 4 weeks before primary immunization for baseline analysis. Immunization was performed with each viral vector or adjuvanted Env protein on the weeks indicated by arrows. At week 54, 4 animals in each group were sacrificed to evaluate prechallenge immune responses. Starting at week 58, the remaining animals were challenged weekly by the intravaginal route a total of 10 times or until infected with SHIV-1157ipd3N4, which expresses a heterologous Clade C Env. (C) Rate of infection acquisition in all vaccinated animals in comparison with the 15 unvaccinated controls. The gray section highlights SHIV acquisition up to 5 challenges. (D) Acquisition of infection in animals <8 years old (dotted line) and animals >8 years old (solid line). When compared with young unvaccinated controls, younger animals (<8 years) given the HVV, Env + NP vaccine regimen were found to be significantly protected using the Mantel-Cox Log-rank test or Gehan-Breslow Wilcoxon test for early time points.

Figure 2. High magnitude and persistent SIV Gag–specific CD4⁺ and CD8⁺ T cell responses induced by immunization with heterologous viral vectors.

(A) Flow cytometry dot plots illustrating expansion of CM9 tetramer⁺CD8⁺ T cells after VSV-, VV-, and Ad5-SIV Gag immunizations. (B) Kinetics of CM9 tetramer⁺CD8⁺ T cell responses in Mamu-A*01⁺ animals immunized with HVV alone (red) or with HVV, Env + NP (blue). (C and D) Kinetics of SIV Gag–specific IFN-γ–producing CD4⁺ and CD8⁺ T cells measured by ICS after stimulation of PBMC with SIVmac239 Gag peptides. (E and F) Kinetics of HIV Env–specific CD4⁺ and CD8⁺ T cells producing IFN-γ measured by ICS after stimulation of PBMC with an Env peptide pool. Gray lines represent the threshold cut-off value (0.01) after background subtraction. In all graphs, geometric mean values and ± SD are presented. Asterisks denote significant differences between groups. ****P ≤ 0.0001 by 2-tailed Mann-Whitney rank sum test.

Figure 3. High magnitude and persistent antibody responses induced by immunization with Env protein and TLR7/8 ligand containing PLGA nanoparticles.

(A) Concentrations of gp140 C.1086 K160N–specific IgG binding antibodies were measured by ELISA in serum 2 weeks after the first, second, third, and fourth Env protein immunization in animals vaccinated with Env + NP alone (green) or HVV, Env + NP (blue). Geometric mean values and ± SD are shown. (B) Levels of C. 1086 K160N gp140–specific vaginal IgG antibodies in 1 month before challenge (week 54). Bars represent medians. (C) Avidity indices of serum IgG antibodies specific for the gp140 C.1086 K160N immunogen or gp120 1157ipd3N4 protein measured at week 58 (day of challenge). Bars represent medians. (D) Titers of neutralizing antibodies measured against MW965.26 Tier 1A virus (solid line), 6644.v2.c33 Tier 1B virus (dotted line), and the SHIV-1157ipd3N4 Tier 2 challenge virus (thin dotted line). (E) ADCVI activity measured in serum on the day of challenge. Bars represent medians. Significance differences between groups were determined using the 2-tailed Mann-Whitney test. Open triangles and diamonds represent animals younger than 8 years old; closed triangles and diamonds represent animals > 8 years.

Figure 4. Induction of Env-specific plasmablasts in blood and long-lived plasma cells in BM by adjuvanted Env protein immunizations.

(A and B) C. 1086 K160N gp140–specific IgG and IgA plasmablasts measured by ELISPOT in blood collected on day 4 and day 7 after the 2nd (week 21), 3rd (week 29), and 4th (week 39) Env protein immunization in animals vaccinated with Env + NP (green) or HVV, Env + NP (blue). Bars represent medians. (C and D) Gp140 C.1086 K160N–specific IgG and IgA plasma cells measured 5 weeks (week 44) and 8 weeks (week 49) after the last Env protein immunization. Bars represent medians. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by 2-tailed Mann-Whitney.

Figure 5. Sustained activation of monocyte subsets following Env + NP immunization.

(A) Study overview. Activated (CD86⁺) CD14⁺CD16^–, CD14⁺CD16⁺, and CD14^-CD16⁺ monocytes in blood after vaccination with viral vectors or Env protein. (B) Appearance of blood monocytes expressing the CCR7 chemokine receptor for homing to lymphoid organs after vaccination. Data represents the mean and ± SEM of the geometric mean difference (Δ) between isotype control and antibody staining values. Within each group, significant increases in cell levels relative to baseline were found using the Wilcoxon matched-pairs signed rank test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Statistical differences between vaccine groups were analyzed using the Mann-Whitney test and are indicated by a square bracket.

Figure 6. Associations between prechallenge antibody responses and rate of infection acquisition in HVV, Env + NP immunized animals.

(A–G) Using the 2-tailed Spearman rank test, the rate of infection acquisition in HVV, Env + NP–vaccinated animals was found to be significantly correlated with concentrations of serum IgG antibodies against the gp140 immunogen, gp41 protein, and C1 peptide (A–C), levels of C.1086 gp140–specific vaginal IgG antibodies (D), numbers of anti–gp140 IgG– secreting plasma cells in BM (E), ADCVI activity against SHIV-1157ipd3N4 in serum (F), and titers of serum-neutralizing antibodies to Tier 1 Clade C viruses MW965.26 and SHIV-1157ipEL-p on the weeks indicated (G). In all graphs, open and closed triangles denote animals <8 and >8 years old, respectively. Correlation tests were performed using results for all animals in the HVV, Env + NP vaccination group.

Figure 7. Distribution of SIV-specific CD8⁺ T cells in blood, iliac lymph node, vagina, and cervix.

Representative dot plots illustrating frequencies of CM9 tetramer⁺CD8⁺ T cells in blood, iliac lymph node, vagina, and cervix of an HVV-vaccinated animal. (B) Numbers of CM9 tetramer⁺CD8⁺ T cells in iliac lymph node, vagina, and cervix of 2 HVV- and 2 HVV, Env + NP–vaccinated animals at necropsy (week 54). Results represent the mean ± SD determined after imaging multiple regions within sectioned tissue. (C) Confocal microscopy of vaginal tissue at week 54 (top) and zoomed-in area (bottom). Blue, CD8; red, tetramer; green, CD4. (D) Representative dot plots showing frequencies of memory CD8⁺ T cells expressing the CD107a/b degranulation marker and IFN-γ after CM9 peptide stimulation of lymphocytes isolated from blood, iliac lymph node, vagina, and cervix.