Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

Abstract

A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses.IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

Keywords: HIV-1; antibody function; gp140; human immunodeficiency virus; long-term; multivalent; neutralizing antibodies; vaccine; vaccines.

FIG 1

Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and >50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.

FIG 2

Mapping of polyclonal binding antibody responses by competition ELISA. Competition ELISAs showing the ability of polyclonal guinea pig sera to outcompete binding of 3BNC117 IgG (A), PG9 IgG (B), PGT121 IgG (C), and 447-52D IgG (D) human monoclonal antibodies to HIV-1 Env gp140 coating antigens. Guinea pig vaccination regimes, as shown in Fig. 1A, are indicated at the top. Data for different vaccination time points are shown as indicated in the key.

FIG 3

Binding antibodies to linear peptides elicited in longitudinally vaccinated guinea pigs. (A and B) Magnitudes of antibody responses in sera from vaccinated guinea pigs to linear peptides in variable loops 1, 2, and 3. (A) Magnitudes of antibody binding to peptide microarray as defined by mean fluorescence intensities (MFI) of signals. Envelope regions targeted are shown at the top. (B) Binding antibody titers to variable loop 1 and 2 scaffolds as determined by endpoint ELISAs. Dotted line indicates background signal, and error bars indicate standard deviations. Pre, naive sera. Asterisks indicate results for ABCM- or 4C-vaccinated animals that were statistically significantly different from the results for C97-vaccinated animals at the matched time points (P < 0.05, Mann-Whitney U test). (C) Percentages of positive peptides as measured by peptide microarray. Percentage of positive peptides is defined as follows: [(positive peptides within a region/total number of peptides within a region) × 100]. Black bars indicate results for ABCM- or 4C-vaccinated guinea pigs that were statistically significantly different from the results for C97-vaccinated animals at the matched time points (P < 0.05, Mann-Whitney U test). (D) Binding distribution of binding antibodies across V2 peptides as determined by peptide microarray. (E) Binding distribution of binding antibodies across select clades and circulating recombinant forms (CRF) of HIV-1 Env V2 peptides as determined by microarray. Error bars show standard deviations. Asterisks indicate results for ABCM- or 4C-vaccinated guinea pigs that were statistically significantly different from the results for C97-vaccinated animals at a matched time point and clade/CRF (P < 0.05, Mann-Whitney U test).

FIG 4

Magnitudes of functional neutralizing and functional non-neutralizing antibody responses. (A) Magnitudes of heterologous tier 1 NAb titers elicited in vaccinated guinea pigs. Guinea pig sera obtained prevaccination (week 0) and at weeks 16, 62, 78, 104, and 120 were tested against a multi-clade panel of tier 1 isolates in the TZM.bl neutralization assay. Groups were vaccinated with the ABCM (n = 4), 4C (n = 5), or C97 (n = 5) regimen. The test pseudovirus, its tier, and clade are shown above each panel. The MuLV background signal for each animal-matched MuLV control was subtracted from neutralization data for all data points. Error bars show the standard deviation for each group at each time point. The limit of detection for the assay is a 50% infective dose (ID₅₀) titer of 20. Asterisks denote statistically significant differences from the results for C97 vaccination alone (P < 0.05, Mann-Whitney U test). (B) Magnitudes of antibody-dependent neutrophil phagocytosis (ADNP) for guinea pig samples. Guinea pig sera obtained prevaccination (naive) and at week 120 were tested in an ADNP assay as described in Materials and Methods. Groups were vaccinated with the ABCM (n = 4), 4C (n = 5), or C97 (n = 5) regimen. Each dot represents a sample tested in triplicate, and error bars show standard deviations for each vaccination group. Controls included human HIV-positive serum, human HIV-negative serum, and no-antibody samples. Phagocytic score was determined using the following formula: (% bead-positive SSC^high, CD11R1⁺, and CD4⁻ cells × MFI of bead-positive cells)/10,000.

FIG 5

Magnitudes of heterologous tier 2 NAb activities in purified IgGs elicited in vaccinated guinea pigs. Purified polyclonal IgGs from vaccinated guinea pigs were evaluated against select tier 2 pseudoviruses and MuLV (negative control) at week 120 against the full tier 2 global panel (A) and at weeks 16, 78, 138, and 200 against select tier 2 viruses in the global panel (B). Horizontal black lines indicate mean titers, and gray dots indicate responses below the limit of detection of the assay. Red, blue, and black dots represent responses that were both detectable and greater than the value for the MuLV control. Each dot represents the result for a single guinea pig, colored according to vaccination regimen. In the C97 vaccination group, one guinea pig died at week 134 and one at week 177 due to age. Horizontal brackets denote statistically significantly different results (P < 0.05, Student's t test).

FIG 6

Mapping of tier 2 neutralizing antibody responses. (A) Purified polyclonal IgGs from vaccinated guinea pigs were evaluated against the tier 2 pseudovirus JRCSF. Horizontal black lines indicate mean titers, gray dots indicate responses below the limit of detection of the assay, and blue and black dots represent responses that were both detectable and greater than the limit of detection. Each dot represents the result for a single guinea pig. (B to D) Mapping was conducted with CD4 binding site competition by RSC3 (B), on a V3 glycan knockout virus (S334A) (C), and with V3 peptide competition (D). Tables show neutralization potencies against wild-type virus compared to mutant virus or against a virus with and without competition. +, increased neutralization potency; −, decreased neutralization potency.