Lynch, Rebecca

Shakhtour, Leyn

Wilson, Andrew

English

George Washington University: Health Sciences Research Commons (HSRC)

Viral RNA cDNARole of HIV-1 Diversity in Sensitivity to Treatment with Monoclonal Antibodies Leyn Shakhtour1, Andrew Wilson1,2, Rebecca Lynch11The George Washington University School of Medicine and Health Sciences  2The Institute for Biomedical Sciences, George Washington UniversityIntroductionMethodsPeripheral blood mononuclear cells were collected from 8 well-characterizedHIV-1+ males on ART with varied lengths of active infection. Resting CD4 T cellswere plated at multiple dilutions in replicate and maximally stimulated to inducelatent viruses to grow in the supernatants. Cultures were screened for thepresence of virus antigen and those that were positive were collected foranalysis. ln order to genetically define each person’s viral reservoir, the HIV-1envelope gene (env) was amplified and sequenced from each culture by singlegenome sequencing (SGS). At least 3 sequences were obtained from each culturewell and 3-5 wells were sequenced from each individual. All sequences from anindividual were aligned and used to generate a maximum-likelihood tree. If weconfirmed a single virus was contained in the well, all well sequences were thenused to generate a well consensus sequence, which were aligned to produce amaximum-likelihood tree. Finally, cultures confirmed by sequencing to containsingle viruses were then titered and measured for sensitivity to bNAbs in thestandard HIV-1 TZM-bl neutralization assay.ConclusionsWe would like to thank and acknowledge the BELIEVE collaboratory and the DC CFAR forfunding this project.Acknowledgements• SGS revealed differences in reservoir diversity between participants, with someindividuals harboring unique viruses while others harbor a few clonal strains.• Our data suggests that average pairwise distance (APD) is associated with greaterresistance to bNAbs; however, individuals who start ART early in infection anddevelop limited virus variation are more likely to be sensitive to bNAb treatment.Figure 1: Amplifying env genesAntiretroviral therapy (ART) can successfully suppress HIV-1 replication, however,patients on ART must maintain consistent therapy in order to avoid rebound froma latent viral reservoir. Strategies are urgently needed to clear or limit thisreservoir. Individuals infected for longer periods with replicating virus prior to ARTharbor greater viral genetic diversity in their reservoir. Broadly neutralizingantibodies (bNAbs) have been identified and are capable of neutralizing highlydiverse viruses. More studies are needed to explore the role viral diversity plays inthe potential efficacy of treatment with bNAbs. To do so, we have characterizedHIV-1 genetic diversity in individuals with varying lengths of infection, as well asthe sensitivity of their inducible virus reservoir to bNAbs.Adapted from Arbeitskreis But, Transfus Med Hemother 2016 and  Mens, Coffin et.al, J Vis Exp. 2011CIRC0196 OM5001OM5346OM5365OM5267 Consensus B OM5334OM5162Consensus AG OM5148Maximum likelihood tree of gapstripped amino acidconsensuses from each outgrowth virus based on treesfrom Figure 2. Wells containing multiple viruses wereexcluded from the tree. Participant OM5346 was co-infected with subtypes B and AG viruses. For individualswith higher levels of viral diversity, no wells had identicalviruses and branched separately; however, low-diversityindividuals harbored essentially 1-2 different viruses.APD vs bNAb geomean IC50(multiply-infected individual removed)(A) Average pairwise distance (APD) was calculated as a measure of reservoir diversityfor each participant in the study using DIVEIN (Deng et al., Biotechniques, 2010).(B) Each virus was tested for neutralization sensitivity against a panel of 10 HIV-1bNAbs: CD4bs targeting antibodies VRC01, VRC07-523, 3BNC117, and N6, V1V2targeting antibodies PGDM1400, CAP256.VRC26.25, and PG09, V3 targeting antibodiesPGT121 and 10-1074, and MPER targeting antibody 10E8. Geometric mean IC50s werecalculated against this panel as a measure of overall bNAb sensitivity for eachparticipant. Spearman’s correlations were performed to determine the relationshipbetween APD and bNAb sensitivity. Interestingly, while these factors are not statisticallysignificant in our data set, there is a trend towards greater resistance to bNAbs inindividuals who have greater diversity. APD could not be calculated for participantOM5148 due to QVOA wells containing multiple viruses as determined by SGS.Figure 4: Participants with greater reservoir diversity display greater bNAb resistancer = 0.6377p = 0.2ParticipantAverage pairwise distanceAverage Geomean IC50 for bNAbsOM5346 19.20% 4.40OM5334 0.28% 4.22OM5365 0.00% 1.42OM5267 0.37% 3.90OM5001 1.59% 1.42OM5162 8.26% 13.78CIRC0196 6.64% 13.95(A) Structure of the HIV-1 genome. (B) Viral RNA was extracted from each wells’supernatants to produce cDNA, using reverse genetics. The HIV-1 envelope gene(env) was amplified using SGS and Sanger sequencing from these reservoir-derived outgrowth viruses.AFigure 2: Viral Reservoir Diversity using Maximum Likelihood Trees Maximum likelihood trees for each participants’ sequencing. Trees are rooted at midpointfor visualization. Each dot represents a single sequence and is colored based on the well itoriginated from. Bootstraps over 90% are indicated. Trees revealed 80% of wells harboreda single virus. Despite utilizing a very small sample size in the analysis, we observed aclear trend toward lower diversity in individuals with short active infections.BFigure 3: Maximum likelihood tree of participant reservoir virusesTable 1: Clinical Characteristics of Study ParticipantsRen et al., Journal of Virology, 2018OM5162 CIRC0196 OM514875.5 months infected****OM534625.5 months infectedOM5334*OM5001*3 months infected*10.5 months infectedOM5267 OM5365*multiple viruses >4 AA81.5 months infectedBAConsensus B Consensus AG ***

Role of HIV-1 Diversity in Sensitivity to Treatment with Monoclonal Antibodies

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Role of HIV-1 Diversity in Sensitivity to Treatment with Monoclonal Antibodies

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