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. 2013 Jun 21:10:63.
doi: 10.1186/1742-4690-10-63.

Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia

Barbara C Bachler  1 , Michael Humbert, Samir K Lakhashe, Robert A Rasmussen, Ruth M Ruprecht
Affiliations

Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia

Barbara C Bachler et al. Retrovirology. .

Abstract

Background: We addressed the question whether live-virus challenges could alter vaccine-induced antibody (Ab) responses in vaccinated rhesus macaques (RMs) that completely resisted repeated exposures to R5-tropic simian-human immunodeficiency viruses encoding heterologous HIV clade C envelopes (SHIV-Cs).

Results: We examined the Ab responses in aviremic RMs that had been immunized with a multi-component protein vaccine (multimeric HIV-1 gp160, HIV-1 Tat and SIV Gag-Pol particles) and compared anti-Env plasma Ab titers before and after repeated live-virus exposures. Although no viremia was ever detected in these animals, they showed significant increases in anti-gp140 Ab titers after they had encountered live SHIVs. When we investigated the dynamics of anti-Env Ab titers during the immunization and challenge phases further, we detected the expected, vaccine-induced increases of Ab responses about two weeks after the last protein immunization. Remarkably, these titers kept rising during the repeated virus challenges, although no viremia resulted. In contrast, in vaccinated RMs that were not exposed to virus, anti-gp140 Ab titers declined after the peak seen two weeks after the last immunization. These data suggest boosting of pre-existing, vaccine-induced Ab responses as a consequence of repeated live-virus exposures. Next, we screened polyclonal plasma samples from two of the completely protected vaccinees by peptide phage display and designed a strategy that selects for recombinant phages recognized only by Abs present after - but not before - any SHIV challenge. With this "subtractive biopanning" approach, we isolated V3 mimotopes that were only recognized after the animals had been exposed to live virus. By detailed epitope mapping of such anti-V3 Ab responses, we showed that the challenges not only boosted pre-existing binding and neutralizing Ab titers, but also induced Abs targeting neo-antigens presented by the heterologous challenge virus.

Conclusions: Anti-Env Ab responses induced by recombinant protein vaccination were altered by the multiple, live SHIV challenges in vaccinees that had no detectable viral loads. These data may have implications for the interpretation of "vaccine only" responses in clinical vaccine trials.

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Figures

Figure 1
Figure 1
Anti-Env Ab responses in vaccinated RMs before and after virus challenge. A. Time line of vaccine/challenge study [12]. In red, immunization phase; in blue, challenge phase. Plasma samples were collected at weeks −1, 0, 1, 2 and 6. B. Quantitative ELISA to determine Env-specific Ab titers in two vaccine-protected RMs. In red, time points post 3rd immunization and pre-challenge; in blue, time points post challenge. C. Time line of immunogenicity study (unpublished). The last protein immunization was designated as week −2, so that the subsequent weeks would correspond to the study in (A). D. Quantitative ELISA to determine Env-specific Ab titers in eight vaccinated, but not challenged RMs. In red, 1 or 2 weeks post 3rd immunization; in green, subsequent time points. E. The gp140CN54-specific Ab titers were compared before and after live-virus challenges in five completely protected vaccinees. The challenge viruses used are indicated. Height of each bar, average titer from three independent assays; error bars, standard error of the mean (SEM). P values are shown (P < 0.05 was considered significant). F. Subtractive biopanning. Three rounds of selection were performed to identify Ab epitopes linked to live-virus exposure. Each round of selection consists of (1) positive selection, (2) negative selection and (3) amplification of the selected phages. Light gray, the Fc portion of all Abs. Dark gray, Fab portion of anti-RM IgG immobilized onto paramagnetic beads via the Fc. Positive selection used week 7 plasma from a protected animal. In dark blue, Fab portions of the live-virus induced Abs and the corresponding phages. Positively selected recombinant phages were counter-selected with plasma from the same vaccinee but collected at week 0 (containing vaccine-induced Abs only). In red, Fab portions of negative selector Abs and the corresponding bound phages. Purple or yellow phages, unspecific phages bound to anti-RM Ab or beads, respectively.
Figure 2
Figure 2
Mapping of anti-V3 binding Abs. A. Sequences of recombinant phages were assigned to V3 crown of HIV1084i or SHIV-1157ipEL-p. Gray shading, linear homologies; blue shading/white letters, amino acid difference between the two HIV-C envelopes (M307I). V3 mimotopes isolated using week 7 plasma (wk7-V3 mimotopes) were tested for plasma Ab binding using 14 time points of RRi-11 and RTr-11. Negative control, pre-immune plasma; weeks −1 and 0, vaccine-induced Ab responses; weeks 1–7, also include Ab responses induced during low-dose virus challenges; weeks 8–30, Ab responses after all challenges. Data illustrate results from two independent assays. Binding patterns are shown in form of a heat-map. OD signals 3x higher than signals detected with the wildtype phage control were considered positive. B. V3 amino-acid sequences of HIV1084i, SHIV-1157ipEL-p and SHIVSF162P4. The two HIV clade C envelopes of SHIV-1157ipEL-p and HIV1084i differ in only one residue in the V3 crown (M307I; HXB2 numbering scheme [17]; highlighted in blue). SHIVSF162P4 shares the same gp120-sequence as HIVSF162[18] and has three mutations compared with the 1084i immunogen (M307I (blue), and R308P and Q315K (gray)). C. Sequences of synthetic peptide sets used for plasma Ab titration and peptide absorption analysis. Peptides corresponding to consensus clade C sequence differed in two amino-acid residues compared with immunogen-related peptides (HIV1084i): M307I (blue) and A316T (gray). D. Plasma samples of five vaccinees [12] were assessed for binding Ab specificities at weeks 0 and 7 using two different V3 peptide sets (red bars, 1084i; blue bars, consensus clade C). Height of each bar, average titer calculated from three independent assays; error bars, standard error of the mean (SEM). Statistically significant differences between weeks 0 and 7 are indicated (if P < 0.05).
Figure 3
Figure 3
Induction and boosting of cross-neutralizing anti-V3 Abs in the absence of viremia. Plasma samples (weeks 0 and 7) were tested for neutralizing activity against the challenge virus, SHIV-1157ipEL-p (A) and the heterologous clade B SHIVSF162P4 (B) by TZM-bl assay [20]. Neutralization titers are expressed as the reciprocal plasma dilution inhibiting 50% of virus infection (IC50). Each bar represents the average titer of at least two independent assays using triplicates of each sample and error bars show the SEM. Plasma nAb titers were compared between weeks 0 (before live-virus challenges) and 7 (after 5x multiple low-dose challenges). Plasma samples were incubated with either medium (plasma only, white bars) or one of the two peptide sets (red bars, 1084i V3; blue bars, consensus clade C V3) and IC50 values were calculated. In case of a decreased IC50 (depletion of neutralizing activity), percentages illustrate the degree of inhibition reached with each peptide set. P-values are shown (significance after Bonferroni correction, P < 0.025). Gray box and asterisks indicate neoantigen reactivity.
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