Anti-HIV Antibody Responses and the HIV Reservoir Size during Antiretroviral Therapy

<div><p>Background</p><p>A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the “reservoir”). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART).</p><p>Methods</p><p>We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (<i>Alu</i> PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables.</p><p>Results</p><p>Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results.</p><p>Conclusions</p><p>Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.</p></div

Association between measures of cell-associated HIV-1 DNA and RNA from gut-associated lymphoid tissue (GALT) and anti-HIV antibody responses^a.

<p>Association between measures of cell-associated HIV-1 DNA and RNA from gut-associated lymphoid tissue (GALT) and anti-HIV antibody responses<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160192#t003fn002" target="_blank">^a</a>.</p

Estimated effects of measures of peripheral blood total or integrated HIV-1 DNA on anti-HIV antibody responses^a.

<p>Estimated effects of measures of peripheral blood total or integrated HIV-1 DNA on anti-HIV antibody responses<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160192#t002fn002" target="_blank">^a</a>.</p

Antibody profiles against seven different HIV-1 antigens in the HIV cohort used for reservoir measurements.

<p>The antibody levels against each of the seven HIV proteins were determined in HIV-uninfected (N = 8) and HIV-infected ART-suppressed (N = 51) participants. The antibody levels are plotted on the Y-axis using a log₁₀scale, and the geometric mean with 95% CI are shown. Comparison between the two groups for all the antigens revealed highly statistically significant higher antibody responses in HIV-infected participants (p<0.0001), as expected. Abbreviations: GP120 = envelope glycoprotein 120; GP41 = envelope glycoprotein 41; RT = reverse transcriptase; INT = integrase; PR = protease; MA = matrix; CA = capsid; NV = HIV-uninfected; HIV = HIV-infected.</p

Integrated HIV DNA is preferentially cleared over 2-LTR circles after coculture recreating the EC phenotype in vitro in the absence of spinoculation.

<p>EC resting CD4+T cells were inoculated without spinoculation, cultured for 3 days and cocultured with autologous CD8+T cells or cultured alone for 16 hours. DNA was then isolated. The fold changeis calculated by dividing the copies of the specific HIV DNA intermediate per resting CD4+T cell (i.e. integrated or 2-LTRs) cultured alone by the copies from the same samples cultured with autologous CD8+T cells. For cocultured samples the level measured was multiplied by 10, which is the dilution factor of effectors added to targets. The line represents the median and p value was calculated using the Student's t-test.</p

Association between peripheral blood HIV-1 DNA or RNA measures of transcription and anti-HIV antibody responses^a.

<p>Association between peripheral blood HIV-1 DNA or RNA measures of transcription and anti-HIV antibody responses<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160192#t004fn002" target="_blank">^a</a>.</p

Spearman rank correlations of log₂-transformed predictors (HIV reservoir measures in rows) versus log₂-transformed outcomes (HIV antibody levels in columns).

<p>Correlation coefficients shown in each box with p-values below. Abbreviations: GP120 = envelope glycoprotein 120; GP41 = envelope glycoprotein 41; RT = reverse transcriptase; INT = integrase; PR = protease; MA = matrix; CA = capsid; rtPCR = reverse transcriptase polymerase chain reaction (PCR); ddPCR = droplet digital PCR; <i>Alu</i> PCR = PCR using a primer in an <i>Alu</i> element to detect integrated HIV-1 DNA; LTR = long terminal repeat; Gag = HIV-1 Gag protein; rCD4 = resting CD4+ T cells; PBMC = peripheral blood mononuclear cells; SCA = single copy assay.</p

Superinfected resting CD4+T cells express HIV proteins without producing detectable viral spread.

<p>(A) Resting CD4+T cells were isolated from frozen PBMC by negative bead depletion and spinoculated with HIV-1_NL4-3. Cells were cultured for 3 days without stimulation in the presence saquinavir (SQV). Resting conditions refer to culture in RPMI+ 10% FCS. After 3 days cells were collected, labeled with a viability dye and activation markers, then fixed and permeabilized and stained for intracellular HIV Gag. (B) Resting CD4+T cells were gated by excluding HLA-DR, CD25 and CD69 and analyzed for the presence of intracellular Gag with or without addition of Raltegravir at the time of infection, using a reverse-transcriptase inhibitor-treated control to set the Gag positive gates. At time 0 and day 3 resting CD4+T were determined to be 99% negative for activation markers. (C) The frequency of cells expressing HIV Gag after <i>in vitro</i> infection and 3 day culture was compared among normal donors (ND, n = 6) and elite controllers (EC, n = 6). (D) One representative experiment in an EC. Total HIV DNA was measured in superinfected resting or activated cells and compared between +SQV and -SQV fractions. (E) Summary data from 4 different EC donors as in C, showing the ratio of total HIV DNA in the -SQV divided by the +SQV fraction. (Lines represent the median and p values were generated using the Students t-test.</p

Characteristics of HIV-infected antiretroviral therapy (ART)-suppressed study participants.

<p>Characteristics of HIV-infected antiretroviral therapy (ART)-suppressed study participants.</p

HIV Gag is expressed <i>in vivo</i> in resting CD4+T cells.

<p>The percentage of integrated HIV expressing HIV Gag was calculated by dividing the Gag positive cells per sorted cells (column 3) by the correction for live cells (column 4). We then divided this number by the integrated copies per PBMC (column 5) and multiplied by the RU5 per cell of the sorted Gag + resting CD4+T cells (and then multiplied by 100) to get the percent of integrated expressing Gag. This calculation assumes that all of the integrated HIV DNA is in the resting CD4+T cell compartment of the PBMC, which makes the percent expressed an underestimate.</p