9G4 autoreactivity is increased in HIV-infected patients and correlates with HIV broadly neutralizing serum activity

Abstract

The induction of a broadly neutralizing antibody (BNAb) response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs) have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE) patients, both of which positively correlated with HIV viral load. Compared to the global 9G4-IgD--memory B cell population, the 9G4+IgD--memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward "IgM only" memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated (r = 0.403, p = 0.0019) with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE (anti-dsDNA, ANA, anti-CL) did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in tolerance, but rather a more intricate expansion of selective autoreactive B cells and antibodies.

Figure 1. 9G4+ serum antibodies and 9G4+ B cells are increased in HIV patients, correlating with CD4 and viral load.

Peripheral blood was collected from ART-negative HIV patients and healthy control (HC) subjects, and serum and PBMC were isolated. A. 9G4+ serum IgG was determined by ELISA. B. The frequency of 9G4+ B cells was determined by flow cytometry. C–F. Spearmann correlation of 9G4+ serum IgG and 9G4+ B cells with CD4 and VL was determined for HIV patients. Each symbol represents a unique patient. * p<0.05 (Mann Whitney test).

Figure 2. Phenotype of 9G4+ B cells in HIV patients.

PBMC were incubated at 4°C or 37°C for 30 minutes prior to incubation at 4°C with antibodies for flow cytometric analysis. A. Representative B cell 9G4 expression profiles. Inset numbers represent frequency of 9G4+ B cells among total CD19+ B cells. B. 9G4+ B cell frequency for multiple samples; -A, -B, -C, indicate consecutive longitudinal samples from the same HIV patient. C–E. 9G4+ and 9G4− B cells incubated at 37°C for 30 minutes and then stained for surface markers and phenotypic analysis peformed using indicated gating strategy (C) to determine composition of total CD19+ total B cell population (D) and IgD−CD19+ memory B cell population (E) determined for 9 samples from unique HIV patients. * p<0.05 (two-tailed paired t-test).

Figure 3. Detection of 9G4+ gp140-reactive serum antibodies.

A. Serum was diluted 1∶100 and 9G4+ YU2 gp140-reactive antibody detected by ELISA. Each symbol represents a unique patient. *p<0.05. B–C. Serum from 3 HIV patients and 1 HC subject was serially diluted and 9G4+ gp140-reactive (B) and total IgG gp140-reactive (C) antibodies determined, and mean +/− SEM for assay triplicates presented.

Figure 4. Identification of 9G4+ gp140-reactive B cells.

PBMC were incubated at 37°C for 30 minutes, then stained at 4°C with fluorescently conjugated gp140 and antibodies. A. Representative flow profile of a HC and HIV sample (HIV040). Plots are gated on live, CD14−CD3−CD19+IgD−IgM−p24− B cells. B. The frequency of 9G4+ B cells within the total IgD− IgM− B cell subset or gp140+ IgD−IgM− B cell subset is indicated.

Figure 5. 9G4+ serum antibodies and 9G4+ B cells correlate with HIV serum broadly neutralizing activity.

HIV neutralizing activity of serum against a panel of five Tier II isolates was determined by TZMbl assay and geometric mean ID50 presented. Serum IgG reactive to 9G4 (A), ANA (B), dsDNA (C) and cardiolipin (D) was determined by ELISA. E. The frequency of 9G4+ B cells was determined by flow cytometry. Spearmann correlation indicated. Each symbol represents a unique patient.