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. Author manuscript; available in PMC: 2020 Sep 18.
Published in final edited form as: Sci Transl Med. 2019 Sep 18;11(510):eaax1880. doi: 10.1126/scitranslmed.aax1880

Immune correlates of the Thai RV144 HIV vaccine regimen in South Africa

Glenda E Gray 1,2,3,*, Ying Huang 3, Nicole Grunenberg 3, Fatima Laher 1, Surita Roux 4,†, Erica Andersen-Nissen 3,5, Stephen C De Rosa 3, Britta Flach 5, April K Randhawa 3, Ryan Jensen 3, Edith M Swann 6, Linda-Gail Bekker 4, Craig Innes 7, Erica Lazarus 1, Lynn Morris 8, Nonhlanhla N Mkhize 8, Guido Ferrari 9, David C Montefiori 9, Xiaoying Shen 9, Sheetal Sawant 9, Nicole Yates 9, John Hural 3, Abby Isaacs 3, Sanjay Phogat 10, Carlos A DiazGranados 10, Carter Lee 11, Faruk Sinangil 11, Nelson L Michael 12, Merlin L Robb 12, James G Kublin 3, Peter B Gilbert 3, M Juliana McElrath 3, Georgia D Tomaras 9, Lawrence Corey 3
PMCID: PMC7199879  NIHMSID: NIHMS1054745  PMID: 31534016

Abstract

One of the most successful HIV vaccines to date, the RV144 vaccine tested in Thailand demonstratedcorrelates of protection including cross-clade V1V2 IgG breadth, Env-specific CD4+ T cell polyfunctionality, and antibody-dependent cellular cytotoxicity (ADCC) in vaccinees with low IgA binding. The HIV Vaccine Trials Network (HVTN) 097 trial evaluated this vaccine regimen in South Africa, where clade C HIV-1 predominates. We compared cellular and humoral responses at peak and durability immunogenicity timepoints in HVTN 097 and RV144 vaccinee samples, and evaluated vaccine-matched and cross-clade immune responses. At peak immunogenicity, HVTN 097 vaccinees exhibited significantly higher cellular and humoral immune responses than RV144 vaccinees. CD4+ T cell responses were more frequent in HVTN 097 irrespective of age and sex, and CD4+ T cell Env-specific functionality scores were higher in HVTN 097. Env-specific CD40L+ CD4+ T cells were more common in HVTN 097, with individuals having this pattern of expression demonstrating higher median antibody responses to HIV-1 Env. IgG and IgG3 binding antibody rates and response magnitude to gp120 and V1V2 vaccine-matched antigens were higher or comparable in HVTN 097 than RV144 ADCC and ADCP functional antibody responses were elicited in HVTN 097. Env-specific IgG and CD4+ Env responses declined significantly over time in both trials. Overall, cross-clade immune responses associated with protection were better than expected in South Africa, suggesting wider applicability of this regimen.

One Sentence Summary

In South Africans, the RV144 HIV vaccine regimen elicited robust immune responses that have been associated with vaccine efficacy.

Introduction

The need for a vaccine to prevent HIV-1 acquisition remains evident, especially in the most burdened region of southern Africa, which is dominated by clade C infections. Even though the roll-out of treatment and prevention programs has contributed to efforts to stem the epidemic, in 2017 alone there were an estimated 800,000 new infections and 19.6 million people living with HIV in east and southern Africa (1). In the Republic of South Africa (RSA), the country with the largest HIV burden, the epidemic is generalized with heterosexual intercourse being the main mode of transmission.

RV144 was the first vaccine clinical trial to demonstrate any efficacy for preventing HIV-1 acquisition (2). Although estimated vaccine efficacy was as high as 60% at month 12, it waned thereafter to 31.2% by month 42 (3). Conducted in Thailand, with the clade B HIV-1 strain, CRF01_AE predominating, RV144 evaluated a heterologous prime-boost combination vaccination regimen. Four injections (months 0, 1, 3, 6) were given of ALVAC-HIV (vCP1521), a canarypox vector expressing envelope (Env) (clade E), group-specific antigen (Gag) (clade B), and protease (Pro) (clade B). Additionally, two booster injections (months 3, 6) were administered of alum-adjuvanted AIDSVAX B/E, a bivalent HIV glycoprotein 120 (gp120). The vaccine regimen induced HIV-specific humoral and cellular immune responses, some of which were found to be associated with reduced HIV infection risk, and included: the binding of plasma immunoglobulin G (IgG) antibodies to the variable 1 and 2 (V1V2) regions of gp120; the binding of IgA antibodies to Env; the avidity of IgG antibodies for Env in vaccinees with low IgA; antibody-dependent cellular cytotoxicity (ADCC) in vaccinees with low IgA; and the magnitude and polyfunctionality of Env-specific CD4+ T cells (4–8). Despite evidence of vaccine efficacy, neutralizing antibodies against circulating tier 2 HIV-1 strains from Thailand were undetectable in the RV144 trial, suggesting that the modest efficacy was largely attributed to non-neutralizing antibody effector functions (9–13). Additionally, virus sequence analyses and host genetic studies of RV144 revealed the interplay of vaccine-elicited responses, infecting viruses, and host factors. A genomic sieve analysis comparing breakthrough HIV-1 sequences between the infected vaccine and infected placebo groups, focusing on the V1V2 region of Env, identified two sites in the V2 loop associated with efficacy at amino acid positions 169 and 181. (14). A follow-up sieve analysis also identified potential immune pressure in the V3 loop of the HIV-1 envelope (15). Host genetic analyses identified associations of human leukocyte antigen (HLA) (16, 17) and FcγR polymorphisms (18) with immune response correlates of risk and/or vaccine efficacy, suggesting that host factors may influence vaccine immunogenicity and efficacy.

All of these studies investigating potential correlates of vaccine efficacy in RV144 involved a retrospective evaluation of HIV-infected and uninfected persons who received the vaccine (i.e., case-control studies) or genetic sieve analyses comparing breakthrough HIV infections between vaccine and placebo recipients. To evaluate prospectively whether these same immune response correlates of risk could be elicited in South Africans, we conducted a study immunizing with the RV144 regimen containing clade B and E inserts in RSA where clade C dominates. Particularly, we compared the magnitude and frequency of responses seen in South Africans to the Thai RV144 participants, as it pertains to the correlates of infection risk and potential cross-clade immune responses associated with these correlates. This study was a precursor to an adapted regimen, involving the subtype C ALVAC-HIV-1 and bivalent subtype C gp120/MF59 HIV-1 vaccine regimen HVTN 100 conducted in RSA to inform the advancement to efficacy testing (19). Our results provide critical insights about the potential extension of this vaccine approach to other regions of the world as well as the identification of non-neutralizing functional antibodies that are elicited by this vaccine regimen.

Results

Participant baseline characteristics

One hundred participants were enrolled into HVTN 097, a randomized controlled double-blind study, between 18 June 2013 and 12 December 2013 at three sites in RSA, with 91 participants receiving all four vaccinations (Fig. 1). Of the enrolled participants, 51% were male, 100% were Black African, and median age was 21.5 years with inter-quartile range (IQR) 20–25 years. Vaccination was discontinued in four participants, two of which were due to pregnancy. Table 1 shows the distributions of age and sex for HVTN 097 participants, similar to those in RV144 whose samples were selected for comparison. The vaccine was safe and well tolerated (fig. S1), with the side effect profile being equivalent to RV144 (2).

Fig. 1.

Fig. 1.

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CONSORT diagram.

Table 1.

Demographics of participants in HVTN 097 and RV144.

Modified intention to treat cohort Per-protocol cohort3
Measure HVTN 097 RV144 contemporaneous cohort1 (2015) RV144 case-control study2 (2010) HVTN 097 RV144 contemporaneous cohort1 (2015) RV144 case-control study2 (2010)
Treatment Placebo 20 24 20 18 24 20
Vaccine 80 212 205 73 201 195
Age 18–20 37 (37%) 66 (28%) 52 (23.1%) 34 (37.4%) 60 (26.7%) 47 (21.9%)
21–25 41 (41%) 112 (47.5%) 122 (54.2%) 37 (40.7%) 111 (49.3%) 118 (54.9%)
≥ 26 22 (22%) 58 (24.6%) 51 (22.7%) 20 (22%) 54 (24%) 50 (23.3%)
Sex Female 49 (49%) 98 (41.5%) 85 (37.8%) 42 (46.2%) 91 (40.4%) 75 (34.9%)
Male 51 (51%) 138 (58.5%) 140 (62.2%) 49 (53.8%) 134 (59.6%) 140 (65.1%)
BMI <25 66 (66%) NA NA 61 (67%) NA NA
25–30 19 (19%) NA NA 18 (19.8%) NA NA
≥31 15 (15%) NA NA 12 (13.2%) NA NA
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1

RV144- Thai subjects enrolled in RV144 selected in 2015 matched on sex and vaccine schedule to South Africans enrolled in HVTN 100. The 2015 cohort was used for the main analyses (binding antibody multiplex assay, intracellular cytokine staining).

2

RV144- Original case control cohort selected in 2010 used for the neutralizing antibody comparison.

3

Per-protocol for HVTN 097 is defined as receiving all four HIV vaccinations, regardless of tetanus and HBV vaccination status.

NA: Not applicable. BMI was not measured in RV144.

T cell responses

The vaccine regimen predominantly induced CD4+ T cells directed to HIV-1 Env at the peak immunogenicity timepoint (two weeks post second ALVAC/AIDSVAX vaccination), as measured by expression of IL-2 and/or IFN-γ (table S1). The Env-specific CD4+ T cell response rate was significantly higher in HVTN 097 than in RV144 vaccine recipients (RV144=36.4%; HVTN 097=51.9%, p=0.043), albeit overall magnitudes of the Env-specific CD4+ T cell frequencies among responders were similar in both trials, p=0.401 (Fig. 2A, table S1). Env-specific CD8+ T cell response rates though rarely detectable in both trials, when present, were significantly higher in HVTN 097 (p=0.031) (table S1). HIV-1 Gag-specific CD4+ and CD8+ T cell response rates were very low and not statistically different between HVTN 097 (3.8%) and RV144 (2.5%), as demonstrated from samples derived from a cohort of uninfected RV144 vaccinees (n=40) selected in 2010 (20). COMPASS analysis of the Env-specific CD4+ T cell single-cell functionality and polyfunctionality scores revealed that South African participants in HVTN 097 had a higher functionality score as compared to the Thai participants in RV144 (p=0.038) (Fig. 2B). The heatmaps in Fig. 2C illustrate the functional profiles induced in each study including the expression of CD40L (CD154), IFN-ℽ, TNF-α, IL-2, and IL-4. The HVTN 097 trial showed significantly higher response rates for CD40L (59.3% for HVTN 097 vs. 33.7% for RV144, p<0.001) and for IFN-ℽ (42.6% in HVTN 097 vs. 19.5% in RV144, p=0.001).

Fig. 2. T cell responses in vaccinees and placebo recipients in the HVTN 097 and RV144 per-protocol cohorts at the peak immunogenicity timepoint.

Fig. 2.

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T cell responses were measured by intracellular cytokine staining. (A) 92TH023-Env-specific IL-2 and/or IFN-γ CD4+ T cell responses among vaccinees in the HVTN 097 and RV144 per-protocol cohorts. Boxplots are based on positive responders only, with negative responders shown as gray triangles and response rates above the boxes. A response is considered positive if the p-value from a one sided Fisher’s exact test of whether the number of CD4+ T-cells positive for IL-2 and/or IFN-γ is higher in the peptide stimulated samples than in the negative control samples is > 0.00001. Fisher’s exact test p-values comparing response rates (p.rate) and Wilcoxon rank sum test p-values comparing magnitudes (p.mag) among positive responders between HVTN 097 and RV144 vaccine recipients are provided. (B) Functionality and polyfunctionality scores of 92TH023-Env specific CD4+ T cell subsets among vaccinees in the HVTN 097 and RV144 per-protocol cohorts. P-values compare functionality or polyfunctionality scores between HVTN 097 and RV144 vaccinees. (C) Heatmap of COMPASS posterior probabilities for 92TH023-Env specific CD4+ T cell responses among vaccine and placebo recipients in the HVTN 097 and RV144 per-protocol cohorts. White indicates the cytokine subset is not expressed, purple-shaded indicates it is expressed, ordered by degree of functionality. Rows correspond to participants, ordered by treatment group and by functionality score within each group. Each cell shows the probability [ranging from white (zero) to purple (one)] that the corresponding cell-subset (column) demonstrates an antigen-specific response in the corresponding participant (row).

Due to concerns from a previous vaccine trial conducted in RSA demonstrating that high body mass index (BMI) was associated with reduced vaccine-induced T cell responses (21), we stratified the groups by BMI. In this study, higher BMI was not associated with the CD4+ T cell response rate (p=0.153) or the magnitude of this response (p=0.712). Notably, CD4+ T cell responses were detected in 100% of vaccine recipients with BMI >30, compared to 53.3% and 45.7% with BMI 25–30 and <25, respectively (differences not statistically significant between all three groups) Fig. 3A). There was no difference in Env-specific CD4+ T cell responses between the trials when divided by sex (Fig. 3B). The higher response rate in HVTN 097 vs. RV144 effect was consistent across age groups; the 21–25-year-old age group showed the largest difference in response rate between South African volunteers (56%) compared to Thai volunteers (31.9%) (p=0.036; Fig. 3C).

Fig. 3. IL-2 and/or IFN-γ CD4+ T cell responses to Env among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 at the peak immunogenicity timepoint.

Fig. 3.

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(A) Stratified by sex. (B) Stratified by age. (C) Stratified by BMI.

By six months post second ALVAC/AIDSVAX vaccination (durability timepoint), the frequency of circulating 92TH023-Env specific CD4+ T cell responses among vaccine recipients in both studies had declined significantly when assessed by prevalence and magnitude; response rate dropped from 70.8% to 36.1% in HVTN 097 (p<0.001, Table 2) and from 36.1% to 27.5% in the n=40 uninfected vaccinees selected from RV144 samples in 2010 (p<0.001) (20).

Table 2.

Durability of immune responses among vaccine recipients in the per-protocol cohort of HVTN 097.

Measure1 Peak timepoint (V143) response rate Durability timepoint (V174) response rate McNemar’s test p-value V14 vs V17 (response rate) Peak timepoint (V14) median magnitude (among V14 responders) Durability timepoint (V17) median magnitude (among V14 responders) Durability timepoint median/peak timepoint median Wilcoxon signed-rank test p-value V14 vs V17 (magnitude)
CD4+ Env.92TH023.AE2 70.8% 36.1% <0.001 0.15% T cells expr. IL-2/IFN-γ 0.07% T cells expr. IL-2/IFN-γ 46.7% <0.001
BAMA IgG 92TH023_D11gp120.AE 100% 51.5% <0.001 17713.4 MFI-Blank 176.1 MFI-Blank 1.0% <0.001
BAMA IgG A244 D11gp120_avi.AE 100% 98.5% 1 20274.8 MFI-Blank 1098.8 MFI-Blank 5.4% <0.001
BAMA IgG Con 6 gp120/B 100% 62.7% <0.001 15022.3 MFI-Blank 372.4 MFI-Blank 2.5% <0.001
BAMA IgG MN gp120 gDneg/293F.B 100% 20.9% <0.001 9507.4 MFI-Blank 208.2 MFI-Blank 2.2% <0.001
BAMA IgG con_env03 140 CF.A1 100% 7.0% <0.001 1453 MFI-Blank 17.9 MFI-Blank 1.2% <0.001
BAMA IgG 01_con_env03 gp140CF_avi.AE 100% 67.2% <0.001 14395.1 MFI-Blank 251.5 MFI-Blank 1.8% <0.001
BAMA IgG Con S gp140 CFI 100% 33.3% <0.001 17069.3 MFI-Blank 449 MFI-Blank 2.6% <0.001
BAMA IgG A244 V1V2 Tags/293F.AE 100% 62.7% <0.001 25793.7 MFI-Blank 680.5 MFI-Blank 2.6% <0.001
BAMA IgG CaseA2_gp70_V1V2.B 96.8% 19.1% <0.001 4145.6 MFI-Blank 23.5 MFI-Blank 0.6% <0.001
BAMA IgG3 92TH023 gp120.AE 66.7% 1.4% <0.001 475.8 MFI-Blank 1.8 MFI-Blank 0.4% <0.001
BAMA IgG3 A244 D11gp120_avi.AE 72.2% 2.8% <0.001 412 MFI-Blank 5.1 MFI-Blank 1.2% <0.001
BAMA IgG3 Con 6 gp120/B 62.5% 0% <0.001 246.9 MFI-Blank 4.1 MFI-Blank 1.7% <0.001
BAMA IgG3 MN gp120 gDneg/293F.B 70.8% 1.4% <0.001 741.4 MFI-Blank 2.8 MFI-Blank 0.4% <0.001
BAMA IgG3 con_env03 140 CF.A1 9.7% 0% 0.016 172 MFI-Blank 3.8 MFI-Blank 2.2% 0.016
BAMA IgG3 01_con_env03 gp140CF_avi.AE 45.8% 1.4% <0.001 295 MFI-Blank 3.6 MFI-Blank 1.2% <0.001
BAMA IgG3 Con S gp140 CFI 75.0% 0% <0.001 348.7 MFI-Blank 2.1 MFI-Blank 0.6% <0.001
BAMA IgG3 A244 V1V2 Tags/293F.AE 88.9% 4.2% <0.001 1464.8 MFI-Blank 6.5 MFI-Blank 0.4% <0.001
BAMA IgG3 1086 V2 tags/293F.C 29.2% 1.4% <0.001 241.1 MFI-Blank 9.2 MFI-Blank 3.8% <0.001
BAMA IgG3 1086_V1V2_Tags.C 72.2% 1.4% <0.001 727 MFI-Blank 6.6 MFI-Blank 0.9% <0.001
BAMA IgG3 CaseA2_gp70_V1V2.B 28.6% 0% <0.001 400.9 MFI-Blank 2.1 MFI-Blank 0.5% <0.001
BAMA IgG3 MN V3 gp70.B 74.3% 0% <0.001 491.8 MFI-Blank 1 MFI-Blank 0.2% <0.001
BAMA IgG3 gp41 5.6% 5.6% 1 286.6 MFI-Blank 62 MFI-Blank 21.6% 0.125
BAMA IgG3 p24 70.8% 33.8% <0.001 3013.3 MFI-Blank 177 MFI-Blank 5.9% <0.001
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1

The clade of the antigen is indicated by the last letter(s) of the name (e.g., 92TH023_D11gp120.AE is clade AE).

2

This analysis uses 12-color flow cytometric panel originally used for the protocol, whereas the comparisons with RV144 elsewhere in this paper used an updated 16-color panel to match contemporaneous RV144 sample studies.

3

Visit 14 (V14) corresponds to the peak immunogenicity timepoint (two weeks post second ALVAC/AIDSVAX vaccination).

4

Visit 17 (V17) corresponds to the durability immunogenicity timepoint (six months post second ALVAC/AIDSVAX vaccination).

MFI-Blank = background-subtracted mean fluorescent intensity (MFI), where background refers to a plate level control (i.e., a blank well run on each plate).

Humoral responses

The anti-gp120 IgG binding antibody response rates in vaccinees were close to 100% for all participants in both RV144 and HVTN 097 but were of higher magnitude in HVTN 097 than RV144 (Fig. 4). Both South African and Thai participants generated cross-clade (clades AE, B, and C) antibody responses as measured by the binding antibody multiplex assay (BAMA). Interestingly, South Africans exhibited a higher prevalence and magnitude of antibody responses to a panel of clade C antigens to both gp120 and gp140 (Fig. 4C). IgG antibodies recognizing the V1V2 region, the primary correlate of decreased HIV-1 risk in RV144, were also significantly higher in HVTN 097 (p-value=0.004 comparing magnitude among positive responders to any V1V2) as can be seen in Fig. 5A, fig. S2 and table S3. Significantly higher titers of V1V2 antibodies to a wide variety of clade B and clade C V1V2 antigens were seen in HVTN 097 versus RV144 participants. This included titers to the CaseA2_V1V2.B antigen, the primary correlate of reduced risk in RV144 (Fig. 5A and Fig. 4C). Overall, the magnitude-breadth curves to a diverse panel of clade C isolates were higher in HVTN 097 vs. RV144 participants (Fig. 5B). Linear epitope analyses of the C1 to V2 region of Env revealed a very high prevalence of epitope-specific responses to the TH023.AE and A244.AE vaccine strains in HVTN 097 recipients (Fig. 5C). In HVTN 097, we observed a 100% response rate to the A244 linear V2 epitope that correlated with lower risk in RV144 (22). This increased response to both vaccine immunogens and cross-clade responses was also seen in the IgG3 responses. IgG3 binding antibody responses to both gp120 and V1V2 antigens were also higher or comparable in prevalence and magnitude among HVTN 097 participants (Fig. 5D, table S3 and fig. S3). This pattern of greater or comparable IgG or IgG3 antibody responses in HVTN 097 vs. RV144 was observed consistently within sex and age subgroups and across panels of gp120 and V1V2 antigens evaluated (table S4). We looked at the relationship between BMI and immune responses in HVTN 097 vaccine recipient positive responders and observed larger magnitudes of gp120 and V1V2 responses associated with higher BMI: p-values comparing different BMI subgroups were <0.001, 0.015, and 0.0063 across the panels of clade C V1V2, multi-clade V1V2, and gp120 antigens, respectively, presented in table S4. In contrast to males, female vaccine recipients in HVTN 097 had borderline significantly higher response magnitudes to clade C V1V2, multi-clade V1V2, and gp120 panels: p-values were 0.046, 0.10, and 0.048, respectively. The median magnitude of the readout (net mean fluorescent intensity) to the clade C V1V2 panel was 3969 (95% CI: 1911; 5557) in females and 1938 (95% CI: 1122; 2900) in males, p=0.046. No significant difference was seen in the magnitude of responses based on age.

Fig. 4. IgG binding antibody responses in vaccine recipients to Env gp120 vaccine-insert antigens in the per-protocol cohorts of HVTN 097 and RV144 at the peak immunogenicity timepoint.

Fig. 4.

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IgG binding antibody multiplex assay (BAMA) responses to Env gp120 vaccine-insert antigens (92TH023.AE and A244.AE) at the 1:50 dilution among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 two weeks after the second ALVAC/AIDSVAX vaccination are shown by boxplot (A) and histogram (B). The net response magnitude (MFI-blank) is background-subtracted mean fluorescent intensity (MFI), where background refers to a plate level control (i.e., a blank well run on each plate). A post-enrollment sample is considered to be positive if the net magnitude is ≥ an antigen-specific cutoff, the net magnitude is > 3 times the baseline net magnitude, and the MFI is > 3 times the baseline MFI. Fisher’s exact test p-values comparing response rates (p.rate) and Wilcoxon rank sum test p-values comparing magnitudes (p.mag) among positive responders between HVTN 097 and RV144 vaccine recipients are provided in (A). Geometric mean IgG binding antibody responses among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 at the peak immunogenicity timepoint (C). Fisher’s exact test p-values comparing response rates (p.rate) and Wilcoxon rank sum test p-values comparing magnitudes (p.mag) among positive responders between HVTN 097 and RV144 vaccine recipients are provided. Boxplots are based on positive responders only represented by the green and blue circles for HVTN 097 and RV144, respectively; negative responders are shown as gray triangles.

Fig. 5. IgG and IgG3 binding antibody responses to V1V2 antigens and magnitude-breadth plot of IgG binding antibody responses to clade C V1V2 antigens among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 at the peak immunogenicity timepoint.

Fig. 5.

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(A) IgG binding antibody responses to V1V2 antigens among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 two weeks after the second ALVAC/AIDSVAX vaccination. (B) Magnitude-breadth plot of IgG binding antibody responses to clade C V1V2 antigens among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 two weeks after the second ALVAC/AIDSVAX vaccination. Solid curves are average breadth across individuals for HVTN 097 and RV144 vaccine recipients with breadth defined by the proportion of antigens in the panel with log10 (MFI – blank) greater than the threshold on the x-axis. Clade C V1V2 antigens = gp70–001428_2_42 V1V2.C, gp70–7060101641 V1V2.C, gp70–96ZM651_02 V1v2.C, gp70-BF1266_431a_V1V2.C, gp70-CAP210_2_00_E8 V1V2.C, gp70-TV1_21 V1V2.C. (C) Binding to peptides in V1V2 region of 3 vaccine strains. Magnitude of binding to overlapping peptides in V1V2 region of vaccine strains by serum IgG at two weeks after the second ALVAC/AIDSVAX vaccination. Thin lines represent individual participants. Thick lines represent weighted means. (D) IgG3 binding antibody responses to V1V2 antigens among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 two weeks after the second ALVAC/AIDSVAX vaccination. Boxplots are based on positive responders only with negative responders shown as gray triangles with positive response rates above the boxes. Fisher’s exact test p-values comparing response rates (p.rate) and Wilcoxon rank sum test p-values comparing magnitudes (p.mag) among positive responders between HVTN 097 and RV144 vaccine recipients are provided.

Similar to the cellular responses, the response rate and magnitude of IgG or IgG3 responses to gp120 and V1V2 declined significantly over time (p-value<0.001 for TH023.AE, A244.AE and CaseA2_V1V2.B) (Table 2); the median magnitude responses of IgG or IgG3 gp120, gp140, V1V2, or V3 antibodies at the durability timepoint (six months post second ALVAC/AIDSVAX vaccination) varied from 0.20% to 5.4% of those at the peak timepoint (two weeks post second ALVAC/AIDSVAX vaccination) across various antigens among participants who were positive responders at peak (Table 2). The considerable decline in IgG response over time was also observed in RV144 (20, 23, 24). Interestingly, a significant difference in decline was seen between the two studies with regard to IgG gp70 scaffolded clade B CaseA2_V1V2. The decrease in response rate from peak to the durability timepoint was greater in RV144 (from 97.3% to 7.9%) compared to HVTN 097 (from 96.8% to 19.1%) (p=0.043) and the fold decline in response magnitude was also greater in RV144 among positive responders at the peak timepoint (p=0.0011).

Differences in sample type, serum (used in HVTN097) vs. plasma (used in RV144), for the binding antibody assays were previously assessed (19). Although serum antibody responses were of slightly higher magnitude (at most, 0.10 log10 net MFI) compared to plasma antibody responses to certain gp120 antigens, no significant differences were detected for antibodies recognizing V1V2 antigens between sample types. A sensitivity analysis with a mean location-shift of −0.10log10 applied to the HVTN 097 responses led to almost identical results as what observed in the main analyses regarding the comparison with RV144; the conclusion regarding the comparison between the two studies remains the same (19).

Antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and neutralization

One of the aspects of our study was the examination of both ADCC and ADCP immune responses post vaccination. Vaccine-mediated protection may be achieved through the elicitation of polyfunctional immune responses, including ADCC and ADCP, aimed at the lysis or phagocytosis of target cells, respectively.The partial efficacy of RV144 has been correlated with ADCC mediated via the induction of antibodies that targeted the V1V2 region of the HIV-1 envelope and has led to speculation that ADCC and other antibody-dependent cellular effector functions, such as ADCP might provide a role in preventing mucosal acquisition of HIV-1, with ADCP acting through engagement on monocytes, macrophages and dendritic cells, leading to phagocytosis of the opsonized virus and infected cells (25). In addition, both of these functional antibody responses have been reported to be associated with protection in nonhuman primate (NHP) studies (26, 27). The response rate for ADCC antibodies as measured by the granzyme B assay utilizing recombinant gp120-coated target cellswas higher in HVTN 097 than RV144: 72.6% (53/73) vs 58.5% (114/195) (p= 0.04) (Fig. 6A). This assay was chosen above the IFN-ℽ assay, which does not assess cell-mediated toxicity directly. Granzyme B is a key mediator of target cell death, therefore release of Granzyme B may be a more specific indicator of ADCC activity. We also evaluated the magnitude of the ADCC responses as area under the curve (AUC) and we observed similar ranges (7.17–62.46 for HVTN 097 and 4.24–62.76 for RV144) among the positive responders (p=0.45) (Fig. 6A). Vaccine-elicited antibodies were also functional for ADCP activity (Fig. 6B). At the peak timepoint (two weeks post second ALVAC/AIDSVAX vaccination), 100% of vaccine recipients had a positive ADCP response to HIV-1 envelope (1086_gp140) compared to 0% of placebo recipients (p<0.001). Among the vaccinees with positive responses, the range of mean phagocytosis score was between 7.0 and 26.9 for 1086_gp140 and were also significantly different between vaccine and placebo recipients (p<0.001). The prevalence of ADCP antibodies to clade C gp140 immunogens was 100% and 77% to clade C V1V2 in HVTN 097.

Fig. 6. Antibody responses in vaccine recipients in HVTN 097 and RV144.

Fig. 6.

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(A) ADCC responses were evaluated against subtype AE 92TH023_gD-neg and A244_gD-neg recombinant gp120-coated CEM.NKRCCR5 target cells for the HVTN 097 (n=73) and RV144 (n=195) samples collected at the peak immunogenicity timepoint (two weeks after the second ALVAC/AIDSVAX vaccination), as indicated on the x-axis. PBMC from one normal healthy HIV seronegative donor were used as source of effector cells. The y-axis represents the values of the area under the curve (AUC) to represent the magnitude of responses. Boxplots are based on positive responders only represented by the green and blue circles for HVTN 097 and RV144, respectively; negative responders are shown as gray triangles. Response rates and number of responders over total are reported above each group boxplot. P-value comparing response rates among positive responders between HVTN 097 and RV144 vaccine recipients is provided. (B) IgG-mediated ADCP was tested at study baseline and two weeks after the second ALVAC/AIDSVAX vaccination for a subset of per-protocol participants, with 63 vaccine recipients (46 in Group T1 and 17 in Group T2) and 5 placebo recipients. ADCP score using the human THP-1 cell line is shown. (C) Neutralizing antibody responses (magnitude-breadth curves) to tier 1 viruses among vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 two weeks after the second ALVAC/AIDSVAX vaccination. Solid curves are average breadth across individuals for HVTN 097 and RV144 vaccine recipients with breadth defined by the proportion of antigens in the panel with log10 ID50 titer greater than the threshold on the x-axis.

Neutralizing antibody activity at the peak timepoint (two weeks post second ALVAC/AIDSVAX vaccination) was seen only to viruses with a highly sensitive tier 1A neutralization phenotype (MN.3.B, TH023.6.AE, MW965.26.C, and SF162.LS.B) (fig. S4). Similar to the other cellular and humoral responses described above, the magnitude and breadth of neutralization of tier 1A viruses was higher in HVTN 097 vaccine recipients (mean AUC=1.74) than in RV144 (mean AUC=1.45) (p<0.001; Fig. 6C). In particular, among positive responders the magnitude was significantly higher for each of MN.3.B (p<0.001), TH023.6.AE (p<0.001), MW965.26.C (p<0.001), and SF162.LS.B (p=0.022) (table S3 and Fig. S8). No neutralizing activity was detected against a global panel of 5 heterologous tier 2 CRF01_AE circulating strains (fig. S4B), suggesting that any protection this vaccine affords is not due to broadly reactive tier 2 HIV-1 neutralization.

Interaction between CD4+ T cells expressing CD40L and antibody responses

Because of the major role of CD40L in providing T cell help to B cells, including B cell proliferation, isotype switching and memory B cell function (28), we separately analyzed the association between percent of T cells expressing CD40L and binding antibody responses in RV144 and HVTN 097. As seen in Fig. 7, there was a weak association between CD4+ envelope-specific T cells to 92TH023-Env and a variety of binding antibodies. This included IgG and IgG3 to gp120 antigens (Fig. 7A, B) and IgG and IgG3 AUC to V1V2 antigens from a variety of HIV-1 strains (Fig. 7C, D).

Fig. 7. Association between percent of CD4+ T cells expressing CD40L reactive to HIV-1 envelope vaccine strain 92TH023 and binding antibodies at the peak immunogenicity timepoint.

Fig. 7.

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(A) IgG responses to A244.AE gp120. (B) IgG3 response to A244.AE gp120. (C) IgG response (AUC) to clade C V1V2 panel. (D) IgG3 response (AUC) to clade C V1V2 panel. X-axis is percent of CD4+ T cells expressing CD40L to 92TH023-ENV, and a histogram of that distribution is shown in green. Along the y-axis a histogram of the corresponding y-variable is shown in red.

Primary component analyses

Primary component analyses (PCA) revealed that HVTN 097 and RV144 vaccine recipients displayed overall similar immunological profiles as seen by the mixture of individual responses by trial in Fig. 8A, with the variability in immune responses explained primarily by IgG binding antibodies and Env-specific CD4+ T cells (both highly correlated with the first principal component that explains 50.2% variability of the immunological profile) (Fig. 8A, B). Although CD40L responses to Env were related to binding antibody, Fig. 8B illustrates that T cell polyfunctionality per se was not well correlated with the antibody immune responses, indicating the importance of evaluating the specific cytokine expression patterns of the T cells to each vaccine regimen.

Fig. 8. Multi-assay principal components analysis (PCA).

Fig. 8.

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(A) Biplot and (B) Spearman correlation heatmap for vaccine recipients in the per-protocol cohorts of HVTN 097 and RV144 at the peak immunogenicity timepoint (two weeks post second ALVAC/AIDSVAX vaccination). In Panel A, the x-axis is the value from the first principal component and the y-axis is the second principal component, where each axis label includes the percentage of variation in the total set of readouts captured by the principal component. Points on the plot represent the values of the principal components of each observation. Points that are close together correspond to observations that have similar values on the components displayed in the plot. The top axis is the first principal component loadings and the right axis is the second principal component loadings, where loadings are the weights by which each original immunogenicity endpoint score should be multiplied to get the value of the first or second principal component. An arrow (vector) is drawn for each immunogenicity endpoint from the origin to the point defined by its first two principal component loadings. Vectors that point in the same direction correspond to endpoints that have similar response profiles on the basis of the first two PCs. The observations whose points project furthest in the direction in which the vector points are the observations that have the most weight of whatever the endpoint measures. Those points t hat project at the other end have the least weight of whatever the endpoint measures. The angle between two arrows conveys information about the correlation of the two endpoint scores, with a zero degree angle denoting perfect correlation and a 90 degree angle denoting no correlation. In the legend of panel A: gp120_IgG3 stands for 92TH023_gp120_IgG3 and A244_gp120_IgG3; gp120_IgG stands for 92TH023_gp120_IgG and A244_gp120_IgG; PFS(FS) stands for Polyfunctionality score (Functionality score).

Discussion

Findings in this study demonstrate that the immune responses associated with reduced risk of HIV-1 infection in the RV144 trial can be elicited, and often at greater frequency and magnitude, among HIV-1 seronegative South Africans. This was seen, irrespective of sex, age, and locale, in both Env-specific antibody and CD4+ T cell responses. In addition, the RV144 vaccine regimen although designed for the Southeast Asian clade A/E epidemic, elicited substantial cross-clade immune responses to antibodies and T cell antigens derived from the predominantly clade C epidemic in sub-Saharan Africa, indicative of this regimen’s potential for global coverage. Importantly, we demonstrate several interesting functional antibody responses associated with protection of infection in both NHP and human vaccine trials, including high frequencies of ADCP, ADCC, and CD40L+CD4+ T cell responses to HIV-1 Env.

Our data are encouraging because several other HIV vaccine studies have demonstrated a differential effect of sex and BMI on vaccine-induced immune responses. In South Africa, the HVTN 503/Phambili study demonstrated an inverse relationship of MRK Ad5 HIV-1 gag/pol/nef vaccine-induced CD4+ T cell immune response with BMI: overweight and obese participants had more muted responses compared to participants with low/normal BMI (21). In contrast, there were no negative effects of BMI on vaccine-induced CD4+ T cell immune responses or on IgG binding antibody responses in HVTN 097. As BMI data were not collected in RV144, a limitation of our study was our inability to compare the impact of BMI on immune responses across the two studies. Of the 100 participants enrolled in HVTN 097, only 15% of the cohort had a BMI > 31, reducing the generalizability of our finding. Given the regional differences, BMI assessments in RV144 would have provided valuable insights in the role that body mass plays on vaccine-induced immune responses.

Notably, HVTN 097 showed cellular responses to vaccination to be similar in both sexes. There have been inconsistent results for CD4+ T cell responses by sex in studies of a recombinant pox vector, NYVAC-C, where some studies have demonstrated no differences by sex, whilst in another study, females were more likely to be responders, as compared to males (21).

The observation that South Africans had stronger immune responses than Thais may be due to an interplay between race, ethnicity, genetic factors, pathogen exposure, the microbiome or factors such as smoking or alcohol use that have impacted immune responses to other vaccines (29, 30). A further limitation of our study was the limited baseline demographic information we had for both populations that may have assisted us further in understanding the differences we saw in vaccine-induced immune responses. The AIDSVAX B/E protein boost immunogen was identical between the two studies (same lot). Although the manufacturing lot of the ALVAC prime was different, the release assays were similar, making manufacturing in our opinion an unlikely explanation for the differences observed here. Although binding antibody responses measured in serum are slightly higher than those measured in plasma (31), our detailed statistical analyses indicate this did not account for the differences observed in this study.

Different genetic background may explain the differences in immune response seen between these two populations: race

Supplementary Material

Supplemental Materials

Acknowledgments

The authors wish to thank the trial participants and staff, the study teams, community members, the HVTN Core staff, the Statistical Center for HIV/AIDS Research and Prevention (SCHARP), the HVTN laboratories, the product developers, and the NIH product development team. We thank Vicki Ashley, DeAnna Tenney, Derrick Goodman, R. Glenn Overman, Judith Lucas, Allan DeCamp, Alexander Chao, One B. Dintwe, Yong Lin, and Tandile Hermanus for technical and analytical expertise; Drs. Bette Korber, Abe Pinter, and Bart Haynes for envelope sequences and proteins; and Dr. Marcella Sarzotti-Kelsoe for QAU oversight. We thank Ashley Clayton for assistance with manuscript preparation. We thank Erika Rudnicki for assistance with the preparation of manuscript figures and tables and Drs. Lindsay Carpp and Mindy Miner for technical editing of the manuscript and figures.

Funding: The HVTN 097 clinical trial was supported by the National Institute of Allergy and Infectious Diseases (NIAID) U.S. Public Health Service Grants UM1 AI068614 [LOC: HIV Vaccine Trials Network], UM1 AI068618 [LC: HIV Vaccine Trials Network], UM1 AI068635 [SDMC: HIV Vaccine Trials Network], UM1 AI069453 [Soweto-Bara Clinical Research Site], UM1 AI069519 [Cape Town–Emavundleni Clinical Research Site], and UM1 AI069469 [Klerksdorp Clinical Research Site]. Within the terms of the Grant Award of the Cooperative Agreement with the HVTN, NIAID, as protocol sponsor, contributed to, reviewed and approved the HVTN 097 study design, NIAID contributed to review and analysis of data, and preparation of the manuscript, and concurred with the decision to submit for publication, but was not involved in the data collection, and did not perform statistical analyses. NIAID provided support in the form of salary for author Edith Swann. No pharmaceutical company or other agency paid for the writing of this article. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. We thank the James B. Pendleton Charitable Trust for their generous equipment donation. Additional funding for laboratory assays was provided by the South African Medical Research Council (SAMRC) and we thank the Bill & Melinda Gates Foundation for their generous contribution to the Cape Town HVTN Immunology Laboratory facility. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID or National Institutes of Health (NIH). The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense.

Competing interests: GEG: reports grants from NIH/NIAID during the conduct of the study. YH: reports grants from NIH/NIAID during the conduct of the study. NG: reports grants from NIH/NIAID during the conduct of the study. FL: reports grants from NIH/NIAID during the conduct of the study. SR: reports grants from NIH/NIAID during the conduct of the study. XS: reports funding from Fred Hutchinson Cancer Research Center via a sub-award agreement, during the conduct of the study. EAN: reports grants from NIH/NIAID, SAMRC and The Bill & Melinda Gates Foundation during the conduct of the study. SCD: reports grants from NIH/NIAID during the conduct of the study. BF: reports grants from NIH/NIAID, SAMRC and The Bill & Melinda Gates Foundation during the conduct of the study. AKR: reports grants from NIH/NIAID during the conduct of the study. RJ: reports grants from NIH/NIAID during the conduct of the study. EMS: is employed by the NIAID, the study sponsor. GEG, YH, NG, FL, SR, XS, EAN, SCD, BF, AKR, RJ, LGB, CI, EL, LM, NNM, GF, DCM, SS, NY, JH, AI, JGK, PBG, MJM, GDT and LC are recipients of NIAID funding, and this publication is a result of activities funded by NIAID. EMS was not involved with the process of funding these awards, nor in their administration or scientific aspects, and, in accordance with NIAID policies, is deferred from decisions regarding funding of coauthors for a requisite period. LGB: reports grants from NIH/NIAID during the conduct of the study. CI: reports grants from NIH/NIAID during the conduct of the study. EL: reports grants from NIH/NIAID during the conduct of the study. LM: reports funding from Fred Hutchinson Cancer Research Center via a sub-award agreement, during the conduct of the study. NNM: reports funding from Fred Hutchinson Cancer Research Center via a sub-award agreement, during the conduct of the study. GF: reports funding from Fred Hutchinson Cancer Research Center via a sub-award agreement, during the conduct of the study. DCM: reports grants from NIH/NIAID during the conduct of the study, as well as funding from Fred Hutchinson Cancer Research Center via a sub-award agreement. SS: reports funding from Fred Hutchinson Cancer Research Center via a sub-award agreement, during the conduct of the study. NY: reports funding from Fred Hutchinson Cancer Research Center via a sub-award agreement, during the conduct of the study. JH: reports grants from NIH/NIAID during the conduct of the study. AI: reports grants from NIH/NIAID during the conduct of the study. SP: is a full-time employee of Sanofi Pasteur and holds stocks/shares with Sanofi Pasteur. CAD: is a full-time employee of Sanofi Pasteur and holds stocks/shares with Sanofi Pasteur. CL: is a full-time employee of GSID. FS: is a full-time employee of GSID. NLM: reports grants from the U.S. Army (Intramural Army funds) during the conduct of the study. MLR: reports grants from the U.S. Army (Intramural Army funds) during the conduct of the study. JGK: reports grants from NIH/NIAID during the conduct of the study. PBG: reports grants from NIH/NIAID during the conduct of the study. MJM: reports grants from NIH/NIAID during the conduct of the study. GDT: reports funding from Fred Hutchinson Cancer Research Center via a sub-award agreement, during the conduct of the study. LC: reports grants from NIH/NIAID during the conduct of the study.

Footnotes

Data and materials availability: All data related to this study are present in the paper or Supplementary Materials. Requests for specimens from the HVTN 097 study should be submitted to vtn.research@hvtn.org and will be made available under a material transfer agreement with the University of Cape Town.

Publisher's Disclaimer: This manuscript has been accepted for publication in Science Translational Medicine. This version has not undergone final editing. Please refer to the complete version of record at www.sciencetranslationalmedicine.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior written permission of AAAS.

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