CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

stained for the down-modulation of CD4 and T cell activation by CD69 expression. The expression levels for MHC and CD4 were compared to primary CD4 T cells that went through the experiment without virus infection. Results: At 24 and 48 hours post-infection we detected two HIV-infected cell populations: GFP+p24- and GFP+p24+. CD4 was partially down-modulated in the GFP+p24- population and completely down-modulated in the GFP+p24+ population. The down-modulation of MHC molecules was more complex. Down-modulation of all 5 HLA molecules was only observed in the GFP+p24+ population at the 48hr time point. We also observed a significant increase in HLA expression in the GFP+p24- population at 24 hr. Finally, we also observed a significant increase in HLA expression in cells that up-regulated CD69. Conclusion: Our results show that down-modulation of MHC molecules occurred late in the virus replicative cycle in contrast to CD4 that began earlier. These data suggest that MHCs presenting virus peptides in HIV-infected cells could be expressed for most of the virus replicative cycle. Thus, our data offer an explanation for how CD8 T cells exert selective pressure on HIV-1 in vivo even with the expression of Nef. 176 HLA-B27–RESTRICTED CTL-ESCAPE MUTATIONS INCREASE SENSITIVITY OF HIV-1 TO INTERFERON Α Philipp Schommers 1 , Christopher Ford 2 , Angelique Hölzemer 2 , Gerd Fätkenheuer 1 , Marcus Atlfeld 2 1 Univ of Cologne, Cologne, Germany, 2 Heinrich Pette Inst, Hamburg, Germany Background: Type1 interferons (IFNs), including IFNα, upregulate cellular innate sensors acting as restriction factors in CD4+ T cells to suppress HIV-1 replication. HIV-1 strains can differ in their sensitivity to IFNα-mediated restriction. HLA-B27+ “Elite Controllers” have been suggested to control HIV-1 through specific cytotoxic T-cells (CTL), which exert immune selection pressure on HIV resulting in CTL-escape mutations. Our aimwas to determine whether the HLA-B27-restricted CTL escape mutation R264K (RK) in p24 Gag and the compensatory mutations R264K+L268M (RK+LM) and S173A+L268M+R264K (SA+RK+LM) influence the sensitivity of HIV-1 to IFNα-induced restriction. Methods: Site-directed mutagenesis of an HIV1 NL4-3Δenv strain expressing GFP was performed to introduce the respective p24 Gag mutations. Infection was assessed in Jurkat T-cells and Jurkat T-cells with a CypA knockout (Jurkat CypA-/-). For IFNα-sensitivity assays, HIV-1 GFP-infected Jurkats and Jurkat CypA-/- T-cells were pre-incubated with increasing doses of IFNα. Infectivity of Jurkat cells was measured using flow cytometry. Results: Wild-type HIV-1 NL4-3 (WT) infection rates of Jurkat cells were significantly higher than those of Jurkat CypA-/- cells, suggesting dependence of WT replication on CypA. In IFNα-stimulated Jurkat cells, WT infection rates were reduced by 19%, while WT infection rates were reduced by 50% in IFNα-stimulated Jurkat CypA -/-, indicating that CypA/ capsid interactions enable WT virus to evade IFNα-induced restriction factors. In contrast, HIV-1 containing the RK and RK+LMmutations showed higher infection rates of Jurkat CypA-/- cells compared to Jurkat cells, and viral replication in Jurkat cells was strongly inhibited by IFNα-exposure (RK: 41%; RK+LM: 35%). Addition of the compensatory SA mutation (SA+RK+LM) reconstituted infection rates to levels similar as WT, including increased susceptibility to IFNα-mediated inhibition in Jurkats CypA -/- cells. Conclusion: Our data suggest that HIV containing the RK and RK+LMmutation are less dependent on CypA than WT. However, weak CypA/capsid interactions might expose these mutated strains to IFNα-induced restriction factors that compete with CypA for their binding site, thereby rendering these strains more sensitive to IFNa in Jurkats. This highlights the role of CypA/capsid interactions in protecting HIV-1 from antiviral restriction factors, and provide a mechanism by which HLA-B27-restricted CTL escape mutations in capsid can result in a loss of viral fitness.

Poster and Themed Discussion Abstracts

177 CXCR4 VARIANTS ARE NEUTRALIZATION-RESISTANT COMPARED TO CCR5-USING VIRUSES IN HIV-1B

Ludy Registre 1 , Yvetane Moreau 2 , Sila Ataca 1 , Manish Sagar 1 1 Boston Univ, Boston, MA, USA, 2 Boston Med Cntr, Boston, MA, USA Background: Emergence of CXCR4 tropic variants during HIV-1 infection is associated with faster progression towards AIDS. The mechanism for emergence remains poorly understood. We hypothesized that X4-utilizing variants emerge as neutralization escape variants from selective pressure on the HIV envelope (env). Methods: Full-length envelopes (envs) were isolated from 4 individuals from ACTG study A5095 using single genome amplification. Envs were incorporated into an NL4-3 backbone to generate replication competent recombinant viruses. Viruses were examined for co-receptor usage and neutralization sensitivity using TZM-bl cells. Neutralization was assessed by measure of area under the curve (AUC), which assesses average neutralization within the range of concentrations. Structural modeling using Rosetta Structure Predication Server was done to visualize structural differences between X4 and R5 V3 loops. Comparisons were done using a paired t test or Wilcoxon rank sum test. Results: A median of 4.5 CCR5-using (R5) (range 1-5) and 3 co-circulating CXCR4-using (X4) envs (range 1-5) were amplified from 4 subjects. All samples were confirmed as subtype B by sequence analysis. All X4 variants (n=12) had a 2 – 3 amino acid insertion either prior to the V3 crown or towards the end of the V3 loop. Structural models of an X4 and R5 variant predicted an additional loop structure within the V3 loop of the X4 variant and not the R5 variant. In all 4 subjects, X4 envs were less neutralization sensitive to autologous contemporaneous plasma compared to the R5 envs (p=0.04). X4 and R5 envs demonstrated equivalent susceptibility to CD4 binding antibody, VRC01, suggesting the envs do not have significant differences in the CD4-binding site. Comparison of all X4 (n=12) to all R5 (n=10) envs demonstrated that X4 envs were less neutralization sensitive to anti-V3 loop directed antibody, PGT121 (p=0.03). Conclusion: Some subtype B env X4 variants possess a unique V3 loop genotype leading to a predicted novel loop structure compared to the R5 strains. In some individuals, X4 variants are less sensitive to autologous and V3 directed antibodies, potentially suggesting that CXCR4 usage emerges as a consequence of host generated humoral immune response. Our studies imply that treatment with some broadly neutralizing antibodies, such as those directed at the V3 loop, may select for CXCR4-using strains.

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CROI 2017