Beneficial Effects of cART Initiated during Primary and Chronic HIV-1 Infection on Immunoglobulin-Expression of Memory B-Cell Subsets

<div><p>Introduction</p><p>During HIV-1 infection the B-cell compartment undergoes profound changes towards terminal differentiation, which are only partially restored by antiretroviral therapy (cART).</p><p>Materials and Methods</p><p>To investigate the impact of infection as early as during primary HIV-1 infection (PHI) we assessed distribution of B-cell subsets in 19 PHI and 25 chronic HIV-1-infected (CHI) individuals before and during 48 weeks of cART as compared to healthy controls (n = 23). We also analysed Immunoglobulin-expression of memory B-cell subsets to identify alterations in Immunoglobulin-maturation.</p><p>Results</p><p>Determination of B-cell subsets at baseline showed that total and Naive B-cells were decreased whereas Activated Memory (AM), Tissue-like Memory (TLM) B-cells and Plasma cells were increased in both PHI and CHI patients. After 4 weeks of cART total B-cells increased, while AM, TLM B-cells and Plasma cells decreased, although without reaching normal levels in either group of individuals. This trend was maintained until week 48, though only total B-cells normalized in both PHI and CHI. Resting Memory (RM) B-cells were preserved since baseline. This subset remained stable in CHI, while was expanded by an early initiation of cART during PHI. Untreated CHI patients showed IgM-overexpression at the expenses of switched (IgM-IgD-) phenotypes of the memory subsets. Interestingly, in PHI patients a significant alteration of Immunoglobulin-expression was evident at BL in TLM cells, and after 4 weeks, despite treatment, in AM and RM subsets. After 48 weeks of therapy, Immunoglobulin-expression of AM and RM almost normalized, but remained perturbed in TLM cells in both groups.</p><p>Conclusions</p><p>In conclusion, aberrant activated and exhausted B-cell phenotypes rose already during PHI, while most of the alterations in Ig-expression seen in CHI appeared later, despite 4 weeks of effective cART. After 48 weeks of cART B-cell subsets distribution improved although without full normalization, while Immunoglobulin-expression normalized among AM and RM, remaining perturbed in TLM B-cells of PHI and CHI.</p></div

Viro-immunological follow-up of study groups.

<p>CD4+ T-cell count (cells/mm³) and plasma viremia (log copies/ml) for PHI and CHI patients are here represented. Left panels show medians with IQR of each parameter; right panels show median values of each time-point to highlight the trend over time. P values are * = 0.01–0.05, ** = 0.001–0.01, *** = <0.001, obtained with Mann-Whitney test for left panels and with Two-sample paired sign test for right panels.</p

Schematic representation of the chemokine receptors CCR5 and CXCR4 and the chimeric CCR5/CXCR4 receptors.

<p>Chimeric receptors FC-1, FC-2, FC-4b, FC-5, FC-6 and FC-7 were obtained by replacing successively larger parts of CCR5 with corresponding regions of CXCR4.</p

Baseline characteristics of study participants.

<p>PHI are individuals recruited at Fiebig stage III to V of HIV-1 infection before donating baseline samples. CHI are chronically HIV-1 infected individuals having been infected for at least 0.8 years (median 2.9, IQR 0.8–5.3 years,) and not receiving cART at recruitment. MSM means Men having sex with men; IVDU means intravenous drug user; WBC means White Blood Cells; AST means aspartate aminotransferase; ALT means alanine aminotransferase. Statistical analysis was performed using (a) Mann-Whitney test and (b) Chi-square test. P values <0.05 were considered significant differences.</p

B-cell subsets distribution and trend of study groups.

<p>Indicated are frequencies of B-cells subsets in PHI and CHI at baseline and after 4 and 48 weeks of cART, as well as values of healthy controls (CS). Column A shows medians with IQR of each parameter; Column B shows median values of each time-point to highlight the trend over time. Dotted horizontal line represents the mean of the CS individuals for a given cell population. P values are * = 0.01–0.05, ** = 0.001–0.01, *** = <0.001, obtained with Mann-Whitney test for results of column A and with Two-sample paired sign test for results of column B.</p

Comparison of viral phenotypes of 21 mother-child pairs.

(a)<p>Isolates from corresponding mother-child pairs are indicated by the same number preceded by a letter: A for mothers and B for children. Time of sampling is indicated in parenthesis as months before (-), after (+) or within 1 week from (0) delivery/birth. </p><p>Pairs were grouped according to the mother's virus phenotype, i.e. first those carrying an R5^narrow, than an R5^broad and last an R5X4 virus.</p><p>Samples were used to infect U87.CD4 cells expressing wild type CCR5 or CXCR4, or one of the chimeric receptors FC-1, FC-2, FC-4b, FC-5, FC-6 or FC-7. Experiments were repeated twice. –, means no chimeric receptor is used.</p

Clinical and viral characteristics of HIV-1 infected children^(a).

(a)<p>symbol - means that the event has not occurred. n.a. = not available. Mos means months of age. Age of appearance of the different conditions is always indicated in months.</p>(b)<p>Narrow and broad refer to viruses with R5 phenotype. Viruses able to exclusively use wild type CCR5 receptor are defined narrow, whereas those using chimeric receptors besides the wild type CCR5 are defined broad. In parenthesis is indicated the age in months of the virus phenotype determination.</p><p>Statistical analysis: Influence of virus R5^broad phenotype on disease progression including children of group 1 and 2, or children of group 1, 2 and 3: p = 0.0260 and p = 0.0218 (Pearson's chi Square), respectively.</p>(c)<p>Age of entry into clinical or immunological category. Categories are defined according to the Centers for Disease Controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003292#pone.0003292-Control2" target="_blank">[21]</a>: CDC 3 = severe immune suppression; CDC B = moderate clinical manifestations; CDC C = severe clinical manifestations.</p>(d)<p>Age of start of mono or dual antiretroviral therapy, not HAART.</p

Distribution of the viral phenotype of transmitting and non transmitting mothers.

<p>Distribution of R5 (black) <i>vs.</i> R5X4 (white) viruses within all virus phenotypes (n = 59 viruses; p = n.s., Fisher's Exact Test) and of R5^narrow (dark gray) <i>vs.</i> R5^broad (light gray) within the R5 phenotype (n = 49 viruses; p = n.s., Fisher's Exact Test).</p

Baseline characteristics of participants.

<p>Baseline characteristics of participants.</p

Discomfort from injections with HIV-DNA using ZetaJet ID, electroporation ID or HIV-MVA IM injections according to a Visual Analogue Scale 0–10; 0 = no pain.

<p>^aObservation after combination of needle-free ZetaJet delivery and electroporation</p><p>Discomfort from injections with HIV-DNA using ZetaJet ID, electroporation ID or HIV-MVA IM injections according to a Visual Analogue Scale 0–10; 0 = no pain.</p