Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

HIV-1-infected individuals harbor a latent reservoir of infected CD4⁺ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones

Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies

A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs

Effective Treatment of SIVcpz-Induced Immunodeficiency in a Captive Western Chimpanzee

Abstract Background Simian immunodeficiency virus of chimpanzees (SIVcpz), the progenitor of human immunodeficiency virus type 1 (HIV-1), is associated with increased mortality and AIDS-like immunopathology in wild-living chimpanzees (Pan troglodytes). Surprisingly, however, similar findings have not been reported for chimpanzees experimentally infected with SIVcpz in captivity, raising questions about the intrinsic pathogenicity of this lentivirus. Findings Here, we report progressive immunodeficiency and clinical disease in a captive western chimpanzee (P. t. verus) infected twenty years ago by intrarectal inoculation with an SIVcpz strain (ANT) from a wild-caught eastern chimpanzee (P. t. schweinfurthii). With sustained plasma viral loads of 105 to 106 RNA copies/ml for the past 15 years, this chimpanzee developed CD4+ T cell depletion (220 cells/μl), thrombocytopenia (90,000 platelets/μl), and persistent soft tissue infections refractory to antibacterial therapy. Combination antiretroviral therapy consisting of emtricitabine (FTC), tenofovir disoproxil fumarate (TDF), and dolutegravir (DTG) decreased plasma viremia to undetectable levels (<200 copies/ml), improved CD4+ T cell counts (509 cell/μl), and resulted in the rapid resolution of all soft tissue infections. However, initial lack of adherence and/or differences in pharmacokinetics led to low plasma drug concentrations, which resulted in transient rebound viremia and the emergence of FTC resistance mutations (M184V/I) identical to those observed in HIV-1 infected humans. Conclusions These data demonstrate that SIVcpz can cause immunodeficiency and other hallmarks of AIDS in captive chimpanzees, including P. t. verus apes that are not naturally infected with this virus. Moreover, SIVcpz-associated immunodeficiency can be effectively treated with antiretroviral therapy, although sufficiently high plasma concentrations must be maintained to prevent the emergence of drug resistance. These findings extend a growing body of evidence documenting the immunopathogenicity of SIVcpz and suggest that experimentally infected chimpanzees may benefit from clinical monitoring and therapeutic intervention

Effect of Rev1-Vpu expression on HIV-1 replication in PBMCs and tonsillar explant cultures.

<p>(A) PHA-stimulated PBMCs were infected with adjusted amounts of the indicated viruses. Virus replication was monitored by analyzing RT-activity in the supernatant. The means of three independent experiments +/- SEM are shown. (B, C) Surface expression levels of (B) tetherin and (C) CD4 were determined by flow cytometry at day 3 post infection. Infected cells were identified by intracellular p24 staining after surface staining of CD4 or tetherin. Dot plots indicating the gating strategy are shown in the right panels. Bar diagrams summarizing four to five independent experiments +/- SD are shown on the left. (D) Sequence analyses of viral mixtures. Wt and fs mutant virus stocks were normalized for infectivity, mixed at the indicated ratios, and the <i>rev1-vpu</i> region was sequenced after bulk amplification of cDNA. The lower panels show respective standard curves. The peak fluorescence of the T residue at position 1 (ZM246 wt) and the A residue at position 3 (ZM247 wt) is expressed as a fraction of the total fluorescence (relative peak height). (E) Sequence chromatograms of 1:1 input mixtures and viral cultures 10 days post infection (dpi). Percentages of wt and fs sequences displayed in the right panels were calculated from the standard curves shown in (D). (F) Human tonsil explant cultures were infected and analyzed as described in (A). One representative experiment for one of three independent donors is shown (***p<0.001; **p<0.01; *p<0.05; n.s. not significant).</p

Splice sites generating Rev1-Vpu encoding mRNA.

<p>The HIV-1 genome (black) and nine ORFs encoding structural, regulatory and accessory proteins are depicted on top. Splice donor (D1-6) and acceptor (A1-8) sites are indicated by dotted lines. mRNAs encoding Rev1-Vpu, Vpu and Env are shown in grey. Depending on the cell type and time point post infection, 75–90% of Vpu and Env encoding mRNA species fail to express Rev1-Vpu since the usage of splice acceptor site A5 removes an intron containing the initiation codon of <i>rev1</i>. Only 10–25% of the Vpu encoding mRNAs have the potential to be translated into Rev1-Vpu since splice acceptor sites A4a, A4b and A4c retain the complete first exon of <i>rev</i>.</p

Frequency of the <i>rev1-vpu</i> polymorphism in HIV-1 quasispecies of infected individuals over time.

<p>^a single genome sequences (SGS) per sample visit</p><p>Frequency of the <i>rev1-vpu</i> polymorphism in HIV-1 quasispecies of infected individuals over time.</p

Functional activity of Rev1-Vpu expressed from pCG expression plasmids.

<p>(A) CMV-IE promoter-based pCG expression vectors containing <i>vpu</i> (left panel) or the <i>rev1-vpu</i> fusion gene (right panel). An enhanced version of the green fluorescent protein (eGFP) is co-expressed via an IRES. (B) Expression of Rev1-Vpu and Vpu in HEK293T cells transfected with the indicated pCG vectors. A Vpu-specific antiserum was used for detection. eGFP was detected to check transfection efficiencies. (C-F) FACS analysis of (C) CD4, (D) tetherin, (E) CD1d or (F) NTB-A receptor modulation by ZM247 Vpu and Rev1-Vpu. HEK293T cells were transfected with expression vectors for the respective surface receptor and Vpu or Rev1-Vpu. 40 h post transfection, surface receptor levels were monitored by two-color flow cytometry. Dot plots indicating the gating strategy are shown in the right panels. Bar diagrams summarizing three to five independent experiments +/- SD are shown on the left (***p<0.001; **p<0.01; *p<0.05; n.s. not significant).</p

Expression of the Rev1-Vpu fusion protein.

<p>(A) Western blot analysis of HEK293T cells co-transfected with the proviral clones described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142118#pone.0142118.g001" target="_blank">Fig 1A</a> or a <i>vpu</i>-deficient mutant thereof. Expression vectors containing <i>rev1</i>-<i>vpu</i> or <i>vpu</i> cassettes served as size controls. Vpu and Rev1-Vpu were detected with an antiserum raised against ZM247 Vpu. (B, C) Expression of Vpu and Rev1-Vpu in ZM247-infected PBMCs or SupD1 cells. Bands representing the Rev1-Vpu fusion protein are highlighted by red arrows. Detection of p55, p24 and actin served as internal controls.</p

Rev function in isogenic viruses differing only in their ability to express Rev1-Vpu.

<p>(A) Gene arrangement of a reporter construct expressing GFP in a Rev-dependent manner. The <i>GFP</i> ORF is flanked by splice donor site 4 (D4) and splice acceptor site 7 (A7). Rev mediates the export of intron-containing GFP expressing mRNA via binding to the RRE. (B) Rev-dependent gene expression was determined by co-transfection of HEK293T cells with increasing amounts of the indicated molecular clones of HIV-1, the GFP reporter construct and a BFP expressing control plasmid. 40 h post transfection, GFP expression levels of BFP positive cells were analyzed by flow cytometry. Examples of primary FACS data are shown in the lower panel. (C) Western blot, showing Rev-dependent expression of p24-capsid and Rev-independent expression of Nef in HEK293T cells transfected with the indicated proviral constructs.</p

Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) Identifies Immune-Selected HIV Variants

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations of mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus “hot-spots” under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. With well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent “cocktail” vaccines