Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques

Abstract

Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig) purified from previously infected animals (SHIVIG) or control Ig prior to intra-rectal challenge with SHIVSF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased de novo Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2-3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host's ability to eliminate HIV.

Fig 1. Effect of passively transferred neutralizing IgG on plasma viral load and CD4⁺ T cells in SHIV_SF162P4 infected macaques.

Male rhesus macaques were treated with NIgG (n = 4) or SHIVIG (n = 5) and blood samples collected at regular intervals after viral exposure. (A) RNA was isolated from plasma, and viral SHIV RNA was quantified by RT-PCR. (B) CD4⁺ T cell count was determined by flow cytometry and normalized to percentage of baseline value. Baseline value was defined as the average value of the -1 w.p.i and either -1 day p.i. (NIgG group) or 0 day p.i. (SHIVIG group). Symbols represent group mean±SEM. * indicates significant difference (p<0.05) between groups at indicated time point as determined by two-tailed t-test.

Fig 2. SHIVIG treatment mitigates gross B cell dysregulation.

The absolute peripheral blood total CD20+ B cell count (A) and frequency of CD19+CD20+ IgD/IgG B cell subsets (B-E) was determined by flow cytometry. Symbols represent group mean+SEM. * indicates significant difference (p<0.05) between groups at indicated time point as determined by two-tailed t-test.

Fig 3. High-resolution longitudinal B cell phenotypic profile.

Heatmap shows data corresponding to the change (log2) from baseline value for each subset frequency at each time point. Paired two tailed t-test for all post-baseline time points vs. baseline was computed independently for NIgG and SHIVIG groups; populations shown had at least one significant time point p<0.01 compared to baseline. Subsets were clustered hierarchically based on Euclidean distance and complete linkage. Magenta subset label indicates a significant difference of at least one time point for the log2 change from baseline between NIgG and SHIVIG groups at p<0.01 as determined by two-tailed unpaired t-test. [X] indicates subset is measured as the frequency of X parent population, otherwise parent population is total CD19+CD20+ B cells. Rectangle outline is used to emphasize select subsets.

Fig 4. SHIVIG treatment enhances plasma HIV-1 SF162 envelope binding antibody response.

(A) Binding antibody (BAb) titer was determined by IgG ELISA against HIV-1 SF162 gp140 envelope protein. (B) Neutralization antibody (NAb) titer against HIV_SF162 pseudovirus was determined by TZM-bl neutralization assay. Symbols represent group mean±SEM. * indicates significant difference (p<0.05) between groups at indicated time point as determined by two-tailed t-test.

Fig 5. Kinetic analysis of binding antibody association with peripheral B cell subsets.

(A) Correlation of the eigenvalues of the kinetic profile of the B cell subsets (n-128) and plasma Env-specific binding Ab (BAb). Red indicates significant overall (group independent) correlation (p<0.05) of B cell subset and BAb eigenvalues. Blue indicates significant difference (p<0.05) between groups and their correlation with BAb. Magenta indicates both significant overall correlation with BAb and significant difference between groups. (B) Individual kinetic profile of de novo BAb, and B cell subsets. Each line represents an individual animal. Black indicates NIgG, red indicates SHIVIG. (C) Eigenvalues of BAb and B cell subsets.

Fig 6. Detailed phenotype of CD21⁺ and CD5⁺ B cell subsets.

B cell flow cytometry gating strategy and marker expression profile of a representative baseline PBMC sample.