Abstract

INTRODUCTION

The Thai phase 3 (RV144) vaccine regimen, ALVAC prime/bivalent clade B/E recombinant gp120 boost, provided an estimated 31.2% efficacy against the acquisition of HIV-1 infection at 42 months after vaccination (1). V1–V2 immunoglobulin G (IgG) antibody responses correlated with decreased risk of HIV-1 infection (2, 3), and a virus sieve analysis demonstrated immune pressure at position 169 in the V2 loop of gp120 (169K) (4). Notably, there was 60.5% estimated efficacy at 6 months after vaccination (5), suggestive of an early vaccine effect that wanes over time. This highlights the need for a better understanding of the quality of the antibody responses that are induced but not durable, so that further vaccine studies can be designed to specifically boost particular immune responses. The VAX003 clinical trial, in a high-risk injection drug use cohort, containing the same bivalent clade B/E gp120 protein immunogen as RV144, without the ALVAC prime did not show protection (6) despite higher vaccine-elicited neutralizing antibodies (nAbs) compared to RV144 (7). Identifying potential markers of infection risk, as well as determining the differences in the quality of the antibody responses among different vaccine regimens, is critical for designing further immunogens to test specific hypotheses in future efficacy studies.

Antibody subclasses (IgG1 to IgG4) have distinct affinities for Fc receptors (8). Thus, antibodies with the same epitope specificity, but of different subclasses, can have different functional attributes. In particular, IgG3 antibodies can fix complement, have high affinity for FcγRI, FcγRII, FcγRIIa, and FcγRIII, and also have the longest and most flexible hinge region of the IgG subclasses. There is precedence for the role of IgG3 antibodies in immune-mediated pathogen control. Antigen-specific IgG3 antibodies were associated with long-term control of malaria caused by the Plasmodium falciparum parasite (9), as well as clearance and long-term clinical protection from Chikungunya virus (CHIKV) (10). IgG3 antibodies were responsible for monocyte-mediated cellular inhibition of P. falciparum (11) and were associated with CHIKV neutralization (10). Little is known about the potentially protective role of vaccine-elicited IgG3 antibodies for HIV-1, other than that some HIV-1 broadly nAbs [for example, 2F5 and 4E10 monoclonal antibodies (mAbs)] are of IgG3 origin (12) and are associated with different effector functions [for example, antibody-dependent cellular cytotoxicity (ADCC), neutralization (13), and complement fixation (14)].

Here, we demonstrate that Env IgG3 responses mark a qualitative difference in immune response between two HIV-1 vaccine regimens with divergent efficacy outcomes in human volunteers. We show that Env IgG3 responses correlate with decreased infection risk in a correlates analysis and are part of an immediate vaccine-induced humoral response that quickly wanes.

RESULTS

IgG subclass profiles between RV144 and VAX003 vaccine regimens are distinct

It was previously reported that nAbs (7) were higher in a protein boost vaccine strategy (VAX003) compared to vector prime/protein boost (RV144). Thus, we examined whether there was a form of antibody response that might be higher in RV144 that was not apparent when measuring total IgG. We examined each of the IgG subclass responses (IgG1 to IgG4) to HIV-1 envelope proteins (vaccine strain and consensus envelope proteins) for both RV144 [ALVAC prime and two protein boosts, visit 8 (V8)] and VAX003 [four protein boosts, visit 9 (V9)] and also after two protein boosts for VAX003 [visit 5 (V5)]. In addition to examining vaccine strain responses, we also included group M and clade AE consensus envelopes. ConS is a consensus of the consensus sequences of each subtype, central to all circulating clades, and reacts well with sera from all subtypes including AE (15–17). Moreover, the group M consensus envelope was similar to autologous envelopes in detection of the initial antibody response in acute infections and is sensitive for determining vaccine immunogenicity in vaccine trials (2, 18). Env IgG1 response rates were generally higher in VAX003 (V9) compared to RV144 (V8) (Fig. 1). We examined vaccine strain antigens (A244 gp120, MN gp120, and 92TH023 gp120), a clade B envelope (GNE8 gp120), and consensus envelope antigens [AE Con gp140 and ConS gp140 (group M)] to determine the quality of the vaccine-induced antibody response. At V9, the VAX003 vaccine induced significantly higher IgG2 and IgG4 response rates (Fig. 1). In contrast, the frequency of some envelope IgG3 responses was higher in RV144 compared to VAX003, most predominantly when compared at V9. Although Env IgG3 responses were induced by VAX003 and the frequency of responders was elevated at V5, the Env IgG3 responses declined by V9 and were neither maintained nor elevated, after two additional protein boosts. This decline in the IgG3 response rate was in stark contrast to those for IgG1, IgG2, and IgG4 that were generally boosted to higher response rates and magnitudes by V9 in VAX003.

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Fig. 1

Higher HIV-1 Env IgG3 but lower HIV-1 Env IgG1, IgG2, and IgG4 antibody response rates in RV144 compared to VAX003 vaccinees

(A to D) HIV-1 Envelope antibody response rates for proteins from the vaccine strain immunogens in the prime (92Th023) and boost (A244, MN) as well as against group M and clade AE consensus proteins (ConS gp140 and AE Con gp140, respectively) (percent responders are shown for RV144 V8 and VAX003 V5 and V9) for IgG1 (A), IgG2 (B), IgG3 (C), and IgG4 (D). ***P < 0.0005; **P < 0.005; *P < 0.05. P values were calculated with the two-tailed Fisher’s exact test and adjusted with the Benjamini-Hochberg correction.

Concentration ranges for HIV-1 antigen–specific IgG subclasses were 0.1 to 55.3 μg/ml (IgG1), 0.6 to 10.5 μg/ml (IgG2), 6.0 × 10⁻⁴ to 0.15 μg/ml (IgG3), and 3.0 × 10⁻³ to 6.5 μg/ml (IgG4) for RV144. In both studies, the concentrations of IgG1 were the highest among the IgG subclasses for both RV144 and VAX003, consistent with relative total concentrations in the blood and HIV-1–specific concentrations found in infected individuals (14, 19) (Fig. 2A). The concentrations of IgG1 specific for the vaccine strain immunogens and to HIV-1 AE and group M consensus antigens were significantly lower in RV144 compared to VAX003 (Fig. 2A). Among the Env-specific IgG subclass responses in RV144, IgG2 and IgG4 were less frequently elicited, with IgG2 being the least frequently detected subclass. The concentrations of Env IgG2 were, overall, lower in RV144 than in VAX003 (Fig. 2B). Similarly, Env IgG4 response rates and concentrations were profoundly lower in RV144 than in VAX003 (Figs. 1D and ​and2D2D).

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Fig. 2

HIV-1 Env IgG subclass response magnitude: RV144 versus VAX003

(A) HIV-1 Env IgG1 antibody concentrations were lower in RV144 compared to VAX003. Antibody response magnitude in mean fluorescence intensity (MFI) is shown for positive responders only. (B) HIV-1 Env IgG2 antibody responses were higher in VAX003 than in RV144. (C) HIV-1 Env IgG3 antibody responses were elevated after two protein boosts (V5) in VAX003 and then declined after four protein boosts (V9). (D) HIV-1 Env IgG4 responses were lower in RV144 compared to VAX003. (E) IgG avidity to the vaccine strain antigen, A244, and also to the group M consensus gp140 was measured by surface plasmon resonance (SPR). ***P < 0.0005; **P < 0.005; *P < 0.05. P values for (A) to (D) were calculated with two-tailed t tests. P values for (E) and (F) were calculated with the Wilcoxon rank-based test.

IgG3 response rates were significantly higher (P = 0.0002 to 0.0084, two-tailed Fisher’s exact test) in RV144 compared to VAX003 (for ConS gp140, GNE8 gp120, AE Con gp140, and 92TH023 gp120) at the peak immunogenicity time point based on maximum overall humoral responses (V8 for RV144 and V9 for VAX003) for each trial (Fig. 1C and Table 1A). Among the vaccinees with HIV-1 Env IgG3 responses, the concentrations of Env-specific IgG3 were similar at the peak immunogenicity time point for RV144 and VAX003 (Fig. 2C). Response rates to A244 gp120 and MN gp120, the two proteins used in the RV144 and VAX003 boost, were more similar between the two regimens. We next examined the IgG subclass distribution after two protein boosts to compare to RV144 (ALVAC prime + two protein boosts). The magnitude of the Env IgG3 response was generally elevated after two protein boosts in VAX003 and then declined after four protein boosts (Fig. 2C). This suggests that continued protein/alum boosting may help drive the subclass response toward lower IgG3 and higher IgG4. The IgG antibody avidity score to the vaccine strain immunogens was overall lower in RV144 compared to VAX003 (Fig. 2E). Although each time point was after four immunizations for both VAX003 and RV144, the difference in avidity is likely due to four doses of protein in VAX003 compared to two doses in RV144. Both the resonance units and dissociation rates (K_d) are lower in RV144 compared to VAX003. Therefore, the difference in avidity is not solely due to higher binding responses. Because these measurements were performed on purified polyclonal samples, certain specificities of higher avidity may predominate.

Table 1

Rates of IgG3 antibodies are higher in RV144 compared to the VAX003 vaccine regimens

IgG3 HIV-1 Env binding antibody differences between RV144 and VAX003. P values <0.05 are in bold.

Type of Env protein	HIV-1 Env protein	RV144-VAX003 V5		RV144-VAX003 V9
		P*	% Difference†	P*	% Difference†
Group M consensus envelope protein	ConS gp140	0.687	3.4	0.0084	20.6
Clade B‡	GNE8 gp120	0.235	9.1	0.0002	29.8
Circulating strain in Thailand (AE) consensus	AE Con gp140	0.112	13.8	0.0019	25.3
RV144 vaccine prime	92TH023 gp120	0.087	12.3	0.0002	27.2
RV144 and VAX003 vaccine boost	A244 gp120	1.000	0.1	0.075	13.7
RV144 and VAX003 vaccine boost	MN gp120	0.845	1.3	0.394	5.6

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^*For IgG subclasses, response rates for each antigen were compared with the two-tailed Fisher’s exact test. To correct for family-wise α inflation, P values were adjusted with the Benjamini-Hochberg correction.

^†Difference in percent response rates.

^‡IgG3 binding antibody profiles for 92TH023 and GNE8 gp120 responses were highly correlated (0.9), Spearman rank-based correlation coefficient (P = 3.26 × 10⁻⁹⁷).

V1–V2 IgG3 responses are higher in vector prime/protein boost compared to protein-only immunization

Given that V1–V2 IgG antibodies correlated with decreased risk of infection in the RV144 correlates analysis (2), we next tested whether V1–V2 IgG3 binding antibody responses were greater in RV144 vaccine recipients compared to VAX003 vaccinees. RV144 elicited significantly higher V1–V2 IgG3 response rates than VAX003 at both time points [P = 0.039 (V5) and P < 0.0001(V9), two-tailed Fisher’s exact test] (Fig. 3A). However, among those that were positive, the magnitude of the response was higher in VAX003 at V5 (Fig. 3, B and C) (P = 0.0116 to <0.0001, two-tailed t test).

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Fig. 3

V1–V2 IgG3 antibody responses differ between RV144 and VAX003 and correlate with a decreased HIV-1 infection risk in a RV144 case-control study

(A) Responders to V1–V2 antigens in RV144 and VAX003. (B and C) Magnitude of V1–V2 responses to RV144 at V8 and VAX003 at V5 and V9. (D and E) Magnitude of V1–V2 IgG3 responses in the RV144 case-control study. (F and G) Cumulative incidence plots for V1–V2 IgG3 responses. ***P < 0.0005; **P < 0.005; *P < 0.05. P values in (A) were calculated with the two-tailed Fisher’s exact test. P values in (B) and (C) were calculated with two-tailed t tests.

Overall, vaccine-induced IgG3 and IgG responses for either the HIV-1 envelope or V1–V2 were not strongly predictive of one another (figs. S2 and S3), indicating that the measurement of the IgG3 response is a unique measurement that cannot be inferred from measuring total IgG. Thus, we determined whether V1–V2–specific IgG3 antibodies correlated with decreased risk of HIV-1 transmission in a blinded follow-up RV144 case-control study. Rolland et al. (4) reported that position 169 in the V2 sequence was a site of immune pressure, and vaccine-elicited V2-directed antibodies were sensitive and/or dependent on this residue (21, 22). We tested whether IgG3 antibodies specific to the V1–V2 sequence that matched the major circulating strains in Thailand, at a known site of immune pressure (gp70 B.CaseA2 V1–V2 169K), would correlate with decreased risk of infection. We modified the V1–V2 fusion protein used in the primary RV144 correlates analysis (2), with a mutation at site 169 (V169K). In a blinded case-control study, we tested plasma drawn 2 weeks after the last vaccination for 205 vaccinees, 41 cases (infected vaccinees), and 40 HIV-1 uninfected participants enrolled in the placebo arm of the trial by a validated binding multiplex assay (2, 18) for binding to different V1–V2 antigens (Fig. 3, D to G, and fig. S4). Both the response rate (Table 3A) and magnitude (Table 3B) to the indicated V1–V2 antigens resulted in a lowered odds ratio (OR), trending toward decreased risk of infection (table S1). The analysis was performed with both univariate models (analysis of one immune variable at a time) and IgA-adjusted models, which include the IgA score variable (an important independent predictor of infection risk), in addition to the immune variable of interest. In the IgA-adjusted models, the magnitude of the IgG3 response to three different V1–V2 antigens significantly correlated with decreased risk of infection (P < 0.05, logistic regression), suggesting that some of the response could target multiple V1–V2 sequences. Remarkably, IgG3 antibodies to gp70 B.CaseA2 V1–V2 169K significantly correlated with decreased risk of infection in both the univariate and IgA-adjusted models (P = 0.006 and 0.003, respectively, logistic regression) (Table 3A and Fig. 3, D to G).

Table 3

V1–V2 Env IgG3 response rates (A) and magnitude (B) are associated with lower ORs and correlate with decreased risk of infection

Plasma HIV-1–specific IgG3 responses from 205 vaccinees, 41 infected vaccinees (cases), and 40 placebos at 2 weeks after the last vaccination were analyzed with clades A, B, and C V1–V2 antigens for risk of infection. (A) Case-control results from the categorical analysis (responder/nonresponder) for V1–V2 antigens. (B) Case-control results from the quantitative analysis of the magnitude of the response for V1–V2 antigens. P values <0.05 are in bold.

A. Correlation of IgG3 response rate with risk of infection in the RV144 case-control study
Sequence description	HIV-1 Env scaffold	Univariate		IgA-adjusted
		OR	P	OR	P
V1–V2 from RV144 vaccine boost (A244 gp120)	AE.A244 V1–V2 tags	0.494	0.162	0.391	0.075
V1–V2 from clade C	C.1086 V1–V2 tags	0.564	0.142	0.511	0.093
V1–V2 from Haynes et al. (2)	gp70 B.CaseA2 V1–V2	0.522	0.131	0.419	0.054
V1–V2 with mutation (169K)	gp70 B.CaseA2 V1–V2 169K	0.354	0.006	0.317	0.003

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B. Correlation of IgG3 magnitude with risk of infection in the RV144 case-control study
Type of Env protein/sequence information	HIV-1 Env scaffold	Univariate		IgA-adjusted
		OR	P	OR	P
V1–V2 from RV144 vaccine boost (A244 gp120)	AE.A244 V1–V2 tags	0.791	0.185	0.682	0.045
V1–V2 from Haynes et al. (2)	gp70 V1–V2 CaseA2	0.749	0.088	0.689	0.034
V1–V2 from clade C gp120	gp70.C. V1–V2	0.753	0.104	0.703	0.049

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We measured the durability of Env and V1–V2 IgG and IgG3 antibody responses in a longitudinal follow-up study for RV144 from week 26 to week 182 after vaccination (Table 4). A previous report showed that V2 peptide-specific antibodies declined after RV144 vaccination (23). We found that the IgG response rate to the vaccine strain A244 gp120 monomer declined from 100 to 29% between weeks 26 and 182, whereas IgG responses to the V1–V2 antigens declined from 97 to 0 to 11% (depending on the antigen) (Table 4A). There was a trend toward a greater decline in total IgG antibody responses to the V1–V2 antigens compared to the envelope protein gp120. This may reflect the higher overall responses to the gp120 protein compared to the V1–V2 region, allowing these responses to remain above the limit of detection longer than the V1–V2 responses. The IgG3 envelope response rate declined from 79 to 0% (weeks 26 to 182) (Table 4B). Notably, IgG3 responses showed a larger decline than the total IgG responses to the vaccine strain gp120 (45% IgG versus 0% IgG3 gp120 at week 52), which is also consistent with the shorter apparent half-life of each IgG3 response. The RV144-induced Env V1–V2 IgG3 apparent response half-life is 1.6- to 3.7-fold shorter than IgG (Table 4C). Moreover, envelope IgG4 responses either remained the same or were elevated at the later visits (table S2), in contrast to the decline in the IgG3 responses. IgG3 responses to V1–V2 antigens also declined from 50 to 84% to 3% between weeks 26 and 52. This is in contrast to IgG responses to V1–V2 that declined from 97 to 11% to 50% between weeks 26 and 52. Thus, in both vaccine studies, we identified differential kinetics in the IgG subclass responses to vaccination over time, with a vaccine-induced IgG3 response that waned early after vaccination.

Table 4

RV144-induced Env V1–V2 IgG3 apparent half-life is 1.6- to 3.7-fold shorter than IgG.

Longitudinal HIV-1 Env and V1–V2 IgG (A) and IgG3 (B) binding antibody response were determined longitudinally from the peak antibody response at weeks 26 to 182 (seven time points). The percent responders for each antigen at each time point after vaccination are shown. (C) Apparent half-life was calculated with an exponential decay model from the binding (MFI) of IgG and IgG3 to specific antigens at a series of sequential time points after vaccination.

A
IgG response rates		Weeks after vaccination
Type of Env protein	HIV-1 Env protein	26	52	78	104	130	156	182
RV144 Env boost (monomeric form)	A244 gp120	100	79	45	34	34	34	29
V1–V2 from clade C	C.1086 V1–V2 tags	97	50	24	21	13	8	11
V1–V2 from Haynes et al. (2)	gp70 B.CaseA2 V1–V2	97	11	3	3	3	0	0
V1–V2 with mutation (169K)	gp70 B.CaseA2 V1–V2 169K	97	13	3	3	3	3	3

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B
IgG3 response rates		Weeks after vaccination
Type of Env protein	HIV-1 Env protein	26	52	78	104	130	156	182
RV144 Env boost (monomeric form)	A244 gp120	79	0	0	0	0	0	0
V1–V2 from clade C	C.1086 V1–V2 tags	84	3	5	3	3	3	3
V1–V2 from Haynes et al. (2)	gp70 B.CaseA2 V1–V2	37	3	3	3	3	3	3
V1–V2 with mutation (169K)	gp70 B.CaseA2 V1–V2 169K	50	3	3	3	3	3	3

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C. Apparent half-life of Env-specific IgG and IgG3 in RV144
Type of Env protein/sequence information	HIV-1 Env protein	Apparent half-life (weeks)		Fold difference
		IgG	IgG3
RV144 vaccine boost (monomer)	A244 gp120	18.0	8.4	2.1
V1–V2 from clade C	C.1086 V1–V2 tags	23.7	10.2	2.3
V1–V2 from Haynes et al. (2)	gp70 B.CaseA2 V1–V2	11.7	7.5	1.6
V1–V2 with mutation (169K)	gp70 B.CaseA2 V1–V2 169K	17.6	4.7	3.7

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DISCUSSION

Multiple HIV-1 vaccine efficacy studies have been completed, yet only one trial in Thailand resulted in partial efficacy (RV144) (1). Further understanding of the differences in the quality of the immune response induced by different vaccine regimens with divergent outcomes will allow for specific hypotheses to be tested in further vaccine trials. Another trial in Thailand (VAX003) that used a protein component of the RV144 vaccine showed no efficacy (6). We identified that the Env IgG subclass profile was markedly different between the two Thailand efficacy studies, and we further identified that V1–V2 IgG3 correlated with decreased infection risk. Moreover, this IgG3 response waned quickly, corresponding with the observed waning vaccine efficacy in RV144. Thus, these data provide a rationale for further testing in HIV-1 vaccine trials for induction and maintenance of specific Env IgG3 responses, as well as testing in other studies whether the IgG3 response is a correlate of infection risk. Here, we found higher IgG3 Env antibody response rates to the gp70 V1–V2 clade B–CaseA2 Env fusion protein (gp70 B.CaseA2 V1–V2), as well as to ALVAC 92TH023 Env protein, and consensus AE and group M (ConS gp140) Env proteins in RV144 compared to VAX003 vaccinees. These IgG3 response rates at peak immunogenicity were the only humoral responses that were more frequently detected in RV144 than in VAX003; all other antibody responses tested, as well as the previously reported nAb responses, were lower in RV144 as compared to VAX003 (7). In our analyses of antibody binding, we used the envelope proteins that matched the RV144 vaccine immunogen. We previously reported that these antigens contain an N-terminal Env sequence deletion that altered the antigenicity of these proteins compared to other proteins (24). Likely, the differences in the MN gp120 results obtained in the Chung et al. (20) study and our study are due to differences in epitope exposure on the different types of MN proteins (with and without the N-terminal deletion sequence).

In addition to the lower number of protein boosts, the ALVAC prime component of the RV144 vaccine regimen may have stimulated different antibody subclasses and specificities compared to those stimulated in the protein-only vaccine (VAX003), presumably through differential antigen presentation and B cell maturation pathways. The hypothesis that the ALVAC prime drove the preferential induction of particular IgG3 specificities is consistent with the finding that the most significant difference in the IgG3 responses between the two vaccines was identified with 92TH023 envelope (sequence present in the ALVAC prime) but not with MN and A244 envelopes (present in the protein boost of both vaccine regimens). Alternatively, differences in immune status, including activation state, of the two populations participating in the clinical trials (injection drug users for VAX003 versus community-based for RV144) could have also contributed to the observed differences in vaccine-elicited IgG subclass responses. The finding of higher Env IgG4 responses in the protein-only vaccine regimen compared to the vector prime/protein boost regimen is consistent with previous reports that found that MN gp160/gp120 prime boost protein vaccination elicited high levels of Env IgG4 (25), whereas the ALVAC (vcp152) prime with an MN gp120 boost elicited lower Env IgG4 relative to IgG1 and IgG3 responses (26). Repeated protein boosting may induce higher levels of nAbs; however, our study also indicates that the IgG subclass distribution can be skewed away from IgG3 with additional protein boosts. Moreover, there may be a delayed kinetics of induction of IgG4 after vaccination such that the time after initial vaccination may also contribute to the differences in antibody subclasses.

We previously tested IgG3 antibody responses to different HIV-1 envelopes as part of the secondary immune-correlates analyses in RV144 and found that only one IgG3 Env response rate, but not magnitude, to A244 gp120 weakly correlated with decreased risk of infection (P = 0.03) (2). Additional envelope antigens tested here (table S1) also showed lower OR but did not reach statistical significance. Here, we tested the IgG3 response to the V1–V2 sequence, matching the major circulating strains in Thailand and a site of immune pressure, and report that this IgG3 response significantly correlated with a decreased risk of infection. We also show that RV144 induced a breadth of IgG3 V1–V2 responses such that clades AE and C V1–V2 (represented by Envs C.1086 and C.97ZA012 in the analysis) also correlated with reduced transmission risk. Thus, our study also enables a direct comparison of vaccine-induced antibody responses in efficacy trials including HVTN 505 (27) and planned South African trials. The clade C.1086 V1–V2 tags (Env subunit protein) (21) contain the sequences from the subtype C.1086 envelope that has been chosen as the envelope immunogen for planned HIV-1 efficacy studies in South Africa on the basis of the antigenicity of the envelope protein as well as originating from a clade C transmitted founder virus (28). Thus, responses to these specific V1–V2 antigens in this study may be used as a minimum benchmark for antibody response rates and magnitudes in further HIV-1 efficacy trials. The blinded follow-up case-control study, together with the RV144 and VAX003 comparison study, demonstrates that the Env IgG3 responses may distinguish the quality of vaccine-induced humoral responses.

Together, these findings suggest that the recombinant canary pox–based priming immunogen ALVAC-HIV (vCP1521) may have affected the adaptive immune response elicited by AIDSVAX B/E rgp120 subunit formulation through innate signaling or differential stimulation of the CD4⁺ T cell response, and thereby generated a qualitatively different Env-specific IgG subclass profile. The prime-boost RV144 vaccine efficacy trial regimen could have elicited a biased class switch recombination to IgG3 by affecting T cell cytokine profiles or activated a different population of responder B cells (and marginal zone B cells). Further studies will examine whether CD27⁻IgG⁺ B cells that show a preference for IgG3 expression (and that have low somatic mutation) (29) were preferentially elicited in the RV144 prime/boost regimen compared to protein-only regimens. These B cell populations could represent a first wave of memory B cells in response to vaccination that need to be stimulated or redirected to a long-lived memory B cell population (30). The role of effector cells (with different FcR expression) in mediating antiviral activity together with vaccine-elicited HIV-1 Env IgG3 responses is an important area to explore in future HIV-1 vaccine trials. Peripheral blood mononuclear cells (PBMCs) were not stored for VAX003; however, a follow-up study with the VAX003 regimen (RV328) will collect PBMCs, bone marrow aspirates, and mucosal biopsies. Similarly, follow-up studies to RV144 (that is, RV305 and RV306) will collect similar samples, allowing further comparisons between RV144 and VAX003 regimens to understand the induction and ontogeny of HIV-1 envelope–specific IgG3 responses. Vaccine-induced IgG3 responses correlated with ADCC, indicating that this antibody subclass may have effector function. Notably, the association of Env IgG3 with medium/high ADCC was significant in RV144, but not in VAX003, suggesting that the IgG3 responses are, overall, of a different quality in RV144. ADCC-mediating antibodies in RV144 targeted multiple epitopes (V2, V3, conformation C1) that could all contribute to the ADCC responses measured in RV144 plasma (31). It will be important to investigate the relative contribution of different epitope-specific ADCC responses induced by further protective versus nonprotective vaccine regimens (like VAX003).

A limitation of our study on RV144 correlates of risk is that we did not define mechanistic correlates of protection (32). Thus, additional studies are needed to determine whether the antibody responses identified here are a marker of HIV-1 risk or whether they play a mechanistic role in decreased HIV-1 risk. Hence, the role of IgG3 compared to other IgG subclasses in other functional assays such as assays that measure antiviral functions at mucosal sites or virion capture (21, 33) will be important to investigate. Another limitation of our study is that our analyses did not include interrogating the different antibody binding epitopes comprised within the V1–V2 antigens or protein envelopes for the IgG subclass response. Differences in epitope recognition on HIV-1 virions or infected cells may also play a role in the different quality of the IgG3 response between RV144 and VAX003, as well as affecting the functional profile of the antibodies. Finally, whether there is a relationship between the early vaccine effect in RV144 (60% early at 12 months waning to 31.2% later at 42 months) and the initial peak and decline in the vaccine-induced envelope IgG3 response is unknown. The rapid decline in the vaccine-induced IgG3 response in both the RV144 and VAX003 regimens may indicate that this response will be difficult to maintain. Thus, it will be important to test different adjuvants and immunogens to determine whether vaccine-induced Env IgG3 durability can be improved. Together, these data provide a rationale to investigate IgG3 responses as correlates of transmission risk in future HIV-1 vaccine trials and to explore whether this response could be tested as a correlate of protection (32).

MATERIALS AND METHODS

Supplementary Material

Acknowledgments

Footnotes

References