Different Pattern of Immunoglobulin Gene Usage by HIV-1 Compared to Non-HIV-1 Antibodies Derived from the Same Infected Subject

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis

Biased usage of VH family genes by anti-HIV-1 mAbs produced from one infected individual.

1<p>VH family 2, 6 and 7 were not detected; predominantly used VH family genes by mAbs are in bold type.</p

Anti-HIV-1 envelope mAbs produced from single IgG⁺ memory B cells selected using JRFL expressing virus-like particles.

1<p>A standard ELISA was used to determine the binding activity of mAbs to antigens. Monoclonal Abs were tested at a concentration of 10 µg/ml against gp120 and gp41 coated onto ELISA plate at 1 µg/ml; cardiolipin at a concentration of 45 µg/ml in ethanol was coated by evaporation at 4°C overnight. The numbers are O.D. values; bold numbers indicate specific reactivity based on value above cutoff which was defined as the mean binding of mAb 1418 (specific to parvovirus B19) +3 standard deviations. ²Flow cytometry was used to measure the mAb binding to JRFL, SF162 Env-transfected 293T cells and native 293T cells as control. The bold numbers indicate percent of cells specifically reactive with mAbs based on values above cutoff determined with irrelevant mAb 1418 as described above. C(+) – positive control; C(-) – negative control; nt – not tested.</p

Cell sorting of B cells stained with VLPs, anti-CD27, and anti-IgG

<p>. The top panel (Gag-VLP) and bottom panel (JRFL-VLP) indicate the gating of non-HIV-1 and anti-HIV-1 Env Abs expressing B cells, respectively. (1A and 1D) FSC and SSC show forward scatter and side scatter, measures of cell size and granularity. The selected area shows the gated single live cells from CD19 magnetic beads enriched B cells. (1B and 1E) The dot plots show the percentages of CD27⁺ memory B cells. Numbers indicate the percentage of gated cells stained with anti-CD27-APC. The percentages of CD27⁺ memory B cells are similar prior to VLPs⁺ selection in both non-HIV-1 and anti-HIV Env Abs expressing cells. (1C and 1F) The dot plots show the gating of IgG⁺ and Gag-VLP⁺ cells (1C) or IgG⁺ and JRFL-VLP⁺ cells (1F) on the CD27⁺ memory B cells. The selected area shows percent of total B cells stained for Gag-VLP (1C) and JRFL-VLP (1F).</p

Anti-HIV-1 envelope mAbs produced from single IgG⁺ memory B cells selected using JRFL expressing virus-like particles.

<p>The sequences have been submitted to GenBank (accession numbers: JQ301900– JQ301919).</p

The usage of VH family genes by human anti-HIV-1 and non-HIV-1 mAbs.

<p>These mAbs were produced from single B cells derived from one HIV infected individual and are compared to antibodies with undefined specificities produced from single B cells of four healthy control subjects <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039534#pone.0039534-Tiller2" target="_blank">[21]</a>. The preferential usage of the VH3 versus VH1 family genes by non-HIV-1 mAbs compared controls and significantly increased usage of VH1 family genes of anti-HIV-1 versus non-HIV-1 mAbs was determined by the Chi-Squared test. NS – not significant.</p