Contrasting antibody responses to intrasubtype superinfection with CRF02_AG

<div><p>HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for <i>pol</i> and <i>env</i> regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.</p></div

Timeline and clinical parameters of the two cases of intrasubtype CRF02_AG superinfection.

<p><b>A)</b> Plasma samples were collected from 2002 to 2014 for patients NYU6501 and NYU6564. Samples are shown in green along the timeline. Red indicates the time span when superinfection occurred. Blue indicates antiretroviral treatment (ART). <b>B)</b> Collection dates, viral load, CD4 cell counts, and the time post diagnosis for each sample used in the study (mths abbreviates for months when listed). Red shades indicate the first time point collected after superinfection occurred. Blue shades highlight samples taken when the patient was on ART. Time points after superinfection are in bold.</p

Heterologous neutralization responses in two cases of intrasubtype CRF02_AG superinfection.

<p><b>A</b>, <b>B)</b> Table of IC50 values that represent the plasma dilutions (<b>A</b>) or IgG concentrations (<b>B</b>) needed for 50% neutralization of the respective pseudovirus. IC50 values were calculated using nonlinear regression fits of the neutralization curves in GraphPad Prism and are illustrated in a color-coded scheme. Resistance to neutralization was assumed if the plasma or IgG sample could not reach 50% neutralization at the lowest plasma dilution (<1) or highest IgG concentration (>500 μg/mL), respectively, and is indicated in the table with a green shade. Breadth and potency values were calculated as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.ref010" target="_blank">10</a>]. Pseudoviruses are all tier 2 covering subtypes A, B, C, G, and CRF02_AG, with the exception of lab strain BaL.26, tier 1, subtype B. MLV was tested as negative control and to ensure absence of ART (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.s009" target="_blank">S9 Fig</a>)</b>. Time points filled in with pale red are after superinfection, pale blue after initiation of ART. IC50, breadth, and potency values are calculated using the averages of 2 or more experiments. <b>C)</b> Neutralization curves from plasma samples for NYU6501 & NYU6564 against pseudoviruses T250-4 and Q23.17, shown as the percent neutralization at the reciprocal plasma dilution. <b>D)</b> Neutralization curves from IgG samples for NYU6501 and NYU6564 against pseudoviruses T250-4, shown as the percent neutralization at the given IgG concentration.</p

Multiple amino acid alignments of V1V2 and V3 regions with NYU6501 and NYU6564 envelope consensus sequences pre and post superinfection.

<p>Alignments were made with consensus sequences generated from all functional Env clones per time point of NYU6501 and NYU6564 (according to phylogenetic analyses in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.g002" target="_blank">Fig 2</a></b>); two consensus sequences per time point were created when distinct populations were detected (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.g002" target="_blank">Fig 2</a></b>; see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.s002" target="_blank">S2 Fig</a></b>). Patient Env sequences were aligned with V1V2 and V3 antigens (≥10 fold change in EC50, <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.g004" target="_blank">Fig 4B</a></b>; <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.s014" target="_blank">S2 Table</a></b>) and reference strains of subtypes CRF02_AG, A, C and B, also used as pseudoviruses for neutralization. Red residues indicate a nonsynonymous substitution and blue residues indicate isofunctional mutations compared to the time point 1 consensus sequence before SI, thereby indicating changes post-SI. <b>A)</b> Patient V1V2 consensus sequences aligned with the V1V2 ZM109 antigen used for binding experiments and reference sequences. Green and yellow boxes indicate the residues that make up the glycan V2 region and the integrin binding site, respectively, located within the immunodominant V1V2 region. V1 and V2 loops are indicated with brackets. <b>B)</b> Patient V3 sequences compared with the V3 ZM109 antigen and reference sequences. The V3 crown residues are denoted underneath.</p

Differential binding patterns to <i>env</i> antigens after intrasubtype superinfection.

<p><b>A)</b> Plasma samples diluted 1:100 were used in ELISA to observe longitudinal plasma antibody binding to envelope antigens V1V2 sc (scaffolded), V3, MPER gp41, gp120 core, and a SOSIP gp140 trimer. Green colors indicate samples tested pre SI and red colors indicate samples post-SI. One-way ANOVA with repeated measures and a multiple comparisons test was used to determine if the binding changes observed to the time point immediately before superinfection (dark green) were significant. <b>B)</b> Binding curves with plasma purified IgG from one time point before [6501-(3); 6564-(1)] and after SI [6501-(5); 6564-(4)] against selected antigens with >5 fold affinity change observed in at least one individual. Analyzed IgG concentrations range from 500 μg/mL to 0.1 μg/mL. Fold change in relative apparent affinities (EC50) after superinfection are indicated as arrows in the binding curves; highest fold change is indicated with the value (see also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173705#pone.0173705.s014" target="_blank">S2 Table</a></b>). Nonlinear regression curves and EC50 values were calculated in GraphPad Prism.</p

Phylogenetic diversity before and after superinfection in two genomic regions.

<p><b>Top:</b> Envelope gene analysis for the ~1.6 kb portion (HXB2 6225–7817) for patients NYU6501 and NYU6564. Over 20 clones for at least 3 time points spanning 10 years were analyzed. Red indicates time points before SI. For NYU6501, we obtained both functional and non-functional (nf) <i>env</i> populations at the first time point post-SI (5), of which only the functional sequences evolved to closely cluster with subsequent lineages of time point 6. At the later time point NYU6564-(5), when the patient has undergone ART, we were only able to amplify non-functional (nf) <i>env</i> sequences out of the plasma. <b>Bottom:</b> The <i>pol</i> region was analyzed on the MiSeq platform generating over 40,000 sequences for each time point. Shown here are consensus sequences made from these data for ≥3 time points. Red and orange indicate time points before SI. Phylogenetic trees were generated using MEGA and FigTree software and were created using the indicated reference sequences downloaded from the Los Alamos Database (black). CRF02_AG reference sequences are marked with an asterisk.</p