Effects of gp120 Inner Domain (ID2) Immunogen Doses on Elicitation of Anti-HIV-1 Functional Fc-Effector Response to C1/C2 (Cluster A) Epitopes in Mice

Abstract

Fc-mediated effector functions of antibodies, including antibody-dependent cytotoxicity (ADCC), have been shown to contribute to vaccine-induced protection from HIV-1 infection, especially those directed against non-neutralizing, CD4 inducible (CD4i) epitopes within the gp120 constant 1 and 2 regions (C1/C2 or Cluster A epitopes). However, recent passive immunization studies have not been able to definitively confirm roles for these antibodies in HIV-1 prevention mostly due to the complications of cross-species Fc-FcR interactions and suboptimal dosing strategies. Here, we use our stabilized gp120 Inner domain (ID2) immunogen that displays the Cluster A epitopes within a minimal structural unit of HIV-1 Env to investigate an immunization protocol that induces a fine-tuned antibody repertoire capable of an effective Fc-effector response. This includes the generation of isotypes and the enhanced antibody specificity known to be vital for maximal Fc-effector activities, while minimizing the induction of isotypes know to be detrimental for these functions. Although our studies were done in in BALB/c mice we conclude that when optimally titrated for the species of interest, ID2 with GLA-SE adjuvant will elicit high titers of antibodies targeting the Cluster A region with potent Fc-mediated effector functions, making it a valuable immunogen candidate for testing an exclusive role of non-neutralizing antibody response in HIV-1 protection in vaccine settings.

Keywords: ADCC; HIV-1; dosing; fc-mediated effector functions; inner domain (ID2) immunogen; isotype; non-neutralizing antibody response.

Figure 1

Kinetics and titer of ID2-specific IgG. Kinetics and titers of ID2-specific IgG induction over the course of ID2 + GLA-SE immunization using the vaccination scheme shown in (A). Throughout the immunization protocol, sera were collected two weeks post vaccination for each time point and analyzed for the induction of IgG against ID2 in an ELISA format. Sera were analyzed 2 weeks following (B) one immunization, (C) two immunizations, (D) three immunizations, and (E) the fourth and final immunization. n = 6 mice for each group, sera were evaluated for each mouse in triplicate and displayed as mean ± SEM.

Figure 2

RFADCC capabilities of sera from ID2 immunized mice. The RFADCC potential of pooled sera from ID2 immunized mice was assayed against a number of targets. (A) RFADCC against gp120_93TH057 core coated (B) full-length gp120_93TH057 and (C) gp120_BaL coated EGFP-CEN-NKR-CCR5SNAP target cells. The left panels show the % RFADCC over a number of sera of A32 positive control antibody concentrations over a 4-fold dilution. The middle panels display the maximum % lysis against each target and the right panels display the overall area under curve (AUC) for each immunization group. Sera were pooled for each dosing group and analyzed 2 weeks following the final immunization. n = 6 mice for each group, pooled sera were evaluated in triplicate and displayed as mean ± SEM. Max % lysis and AUC were analyzed for statistical significance using a one-way ANOVA with Tukey’s test for multiple comparisons. Significance values are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

Ability of ID-2 immunized sera to compete with A32 and N5-i5 for ID2 binding. Competition ELISA to determine the level of competition of immunized sera with (A) A32 and (B) N5-i5. Sera were mixed 1:1 with a biotinylated antibody at a concentration equivalent to the half-max binding against ID2 and analyzed for % competition. Sera from each mouse were analyzed in triplicate over a 10-fold dilution beginning at 1:100 (Left panels) with % competition at 1:100 dilution displayed in right panels. n = 6 mice for each group, sera were evaluated for each mouse in triplicate and displayed as mean ± SEM. Statistical significance was determined using a one-way ANOVA with Tukey’s test for multiple comparisons. Significance values are indicated as * p < 0.05, ** p < 0.01.

Figure 4

Titer of ID2-specific isotypes induced by varying doses of ID2. Titers of ID2-specific isotypes at the termination of ID2 + GLA-SE immunization. Terminal sera were collected two weeks post final vaccination and analyzed for (A) IgG1, (B) IgG2a, (C) IgG2b, (D) IgG2c, (E) IgA and (F) IgG3. n = 6 mice for each group, sera were evaluated for each mouse in triplicate and displayed as mean ± SEM for area under the curve (AUC) over the dilution range displayed in Figure 1. Statistical significance was determined using a one-way ANOVA with Tukey’s test for multiple comparisons. Significance values are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

RFADCC capabilities of individual sera from 2.2 µg ID2 immunized mice. The RFADCC potential of individual sera from ID2 immunized mice was assayed against gp120_93TH057 core coated EGFP-CEN-NKR-CCR5SNAP target cells. (A) RFADCC of 2.2 µg ID2 immunized mice over a broad concentration range of sera dilutions for each individual mouse. (B) A32 and (C) N5-i5 competition displayed segregated by potency of RFADCC response. (D) IgG2a AUC value and (E) ratio of IgG1:IgG2a + IgG2b as segregated by potency of RFADCC response. (F) Correlation of IgA to RFADCC showing R squared and p calculated using Pearson’s two-tailed correlation. n = 6 mice, individual sera were evaluated in triplicate and displayed as mean ± SEM. Statistical significance was analyzed using an unpaired T-test.