ABSTRACT In order to cure human immunodeficiency virus (HIV), we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persist within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics, however, did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average of >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were heterogeneous in terms of age, with some potentially originating from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally enriched and genetically younger sequences that date to the period of untreated infection when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination. IMPORTANCE Characterizing the human immunodeficiency virus (HIV) reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. We observed that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) dated to nearer ART initiation. This helps explain why studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled replication-competent HIV found almost none. Together with our findings that intact proviruses were more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that (i) on-ART and rebound viremia can have distinct within-host origins, (ii) intact proviruses have shorter lifespans than grossly defective ones and thus depend more heavily on clonal expansion for persistence, and (iii) an HIV reservoir predominantly “dating” to near ART initiation will be substantially adapted to within-host pressures, complicating immune-based cure strategies.

HIV proviral burden, genetic diversity, and dynamics in viremic controllers who 1 subsequently initiated suppressive antiretroviral therapy 2 3 F. Harrison Omondi a,b , Hanwei Sudderuddin b , Aniqa Shahid a,b , Natalie N. Kinloch a,b , Bradley R. 4 Jones b,c , Rachel L. Miller b,c , Olivia Tsai a , Daniel MacMillan b , Alicja Trocha d , Mark A. 5 Brockman a,e , Chanson J. Brumme b,f , Jeffrey B. Joy b,c,f , Richard Liang b , Bruce D. Walker d , 6 Zabrina L. Brumme a,b# 7 8

HIV therapy is lifelong because integrated, replication-competent viral copies persist within long-lived cells. To cure HIV, we need to understand when these viral reservoirs form, how large and genetically diverse they are, and how long they endure.

HIV-1-specific CD8+ T cells are an important component of HIV-1 curative strategies. Viral variants in the HIV-1 reservoir may limit the capacity of T cells to detect and clear virus-infected cells. We investigated the patterns of T cell escape variants in the replication-competent reservoir of 25 persons living with HIV-1 (PLWH) durably suppressed on antiretroviral therapy (ART). We identified all reactive T cell epitopes in the HIV-1 proteome for each participant and sequenced HIV-1 outgrowth viruses from resting CD4+ T cells. All non-synonymous mutations in reactive T cell epitopes were tested for their effect on the size of the T cell response, with a≥50% loss defined as an escape mutation. The majority (68%) of T cell epitopes harbored no detectable escape mutations. These findings suggest that circulating T cells in PLWH on ART could contribute to control of rebound and could be targeted for boosting in curative strategies.

Volume 92, no. 19, e00811-18, 2018, [https://doi.org/10.1128/JVI.00811-18][1]. Page 15, Acknowledgments, line 15: The following sentence should be added after “KwaZulu-Natal”: “J.K.M. received additional funding from the Poliomyelitis Research Foundation of South Africa. Poliomyelitis

CD8+ T cell-mediated escape mutations in Gag can reduce HIV-1 replication capacity (RC) and alter disease progression, but less is known about immune-mediated attenuation in other HIV-1 proteins. We generated 487 recombinant viruses encoding RT-integrase from individuals with chronic (n = 406) and recent (n = 81) HIV-1 subtype C infection and measured their in vitro RC using a green fluorescent protein (GFP) reporter T cell assay. In recently infected individuals, reverse transcriptase (RT)-integrase-driven RC correlated significantly with viral load set point (r = 0.25; P = 0.03) and CD4+ T cell decline (P = 0.013). Moreover, significant associations between RT integrase-driven RC and viral load (r = 0.28; P < 0.0001) and CD4+ T cell count (r = -0.29; P < 0.0001) remained in chronic infection. In early HIV infection, host expression of the protective HLA-B*81 allele was associated with lower RC (P = 0.05), as was expression of HLA-B*07 (P = 0.02), suggesting early immune-driven attenuation of RT-integrase by these alleles. In chronic infection, HLA-A*30:09 (in linkage disequilibrium with HLA-B*81) was significantly associated with lower RC (P = 0.05), and all 6 HLA-B alleles with the lowest RC measurements represented protective alleles, consistent with long-term effects of host immune pressures on lowering RT-integrase RC. The polymorphisms V241I, I257V, P272K, and E297K in reverse transcriptase and I201V in integrase, all relatively uncommon polymorphisms occurring in or adjacent to optimally described HLA-restricted cytotoxic T-lymphocyte epitopes, were associated with reduced RC. Together, our data suggest that RT-integrase-driven RC is clinically relevant and provide evidence that immune-driven selection of mutations in RT-integrase can compromise RC.IMPORTANCE Identification of viral mutations that compromise HIV's ability to replicate may aid rational vaccine design. However, while certain escape mutations in Gag have been shown to reduce HIV replication and influence clinical progression, less is known about the consequences of mutations that naturally arise in other HIV proteins. Pol is a highly conserved protein, but the impact of Pol function on HIV disease progression is not well defined. Here, we generated recombinant viruses using the RT-integrase region of Pol derived from HIV-1C-infected individuals with recent and chronic infection and measured their ability to replicate in vitro We demonstrate that RT-integrase-driven replication ability significantly impacts HIV disease progression. We further show evidence of immune-mediated attenuation in RT-integrase and identify specific polymorphisms in RT-integrase that significantly decrease HIV-1 replication ability, suggesting which Pol epitopes could be explored in vaccine development.

ABSTRACT Human leukocyte antigen (HLA) class I-associated polymorphisms in HIV-1 that persist upon transmission to HLA-mismatched hosts may spread in the population as the epidemic progresses. Transmission of HIV-1 sequences containing such adaptations may undermine cellular immune responses to the incoming virus in future hosts. Building upon previous work, we investigated the extent of HLA-associated polymorphism accumulation in HIV-1 polymerase (Pol) through comparative analysis of linked HIV-1/HLA class I genotypes sampled during historic (1979 to 1989; n = 338) and modern (2001 to 2011; n = 278) eras from across North America (Vancouver, BC, Canada; Boston, MA; New York, NY; and San Francisco, CA). Phylogenies inferred from historic and modern HIV-1 Pol sequences were star-like in shape, with an inferred most recent common ancestor (epidemic founder virus) sequence nearly identical to the modern North American subtype B consensus sequence. Nevertheless, modern HIV-1 Pol sequences exhibited roughly 2-fold-higher patristic (tip-to-tip) genetic distances than historic sequences, with HLA pressures likely driving ongoing diversification. Moreover, the frequencies of published HLA-associated polymorphisms in individuals lacking the selecting HLA class I allele was on average ∼2.5-fold higher in the modern than in the historic era, supporting their spread in circulation, though some remained stable in frequency during this time. Notably, polymorphisms restricted by protective HLA alleles appear to be spreading to a greater relative extent than others, though these increases are generally of modest absolute magnitude. However, despite evidence of polymorphism spread, North American hosts generally remain at relatively low risk of acquiring an HIV-1 polymerase sequence substantially preadapted to their HLA profiles, even in the present era. IMPORTANCE HLA class I-restricted cytotoxic T-lymphocyte (CTL) escape mutations in HIV-1 that persist upon transmission may accumulate in circulation over time, potentially undermining host antiviral immunity to the transmitted viral strain. We studied >600 experimentally collected HIV-1 polymerase sequences linked to host HLA information dating back to 1979, along with phylogenetically reconstructed HIV-1 sequences dating back to the virus' introduction into North America. Overall, our results support the gradual spread of many—though not all—HIV-1 polymerase immune escape mutations in circulation over time. This is consistent with recent observations from other global regions, though the extent of polymorphism accumulation in North America appears to be lower than in populations with high seroprevalence, older epidemics, and/or limited HLA diversity. Importantly, the risk of acquiring an HIV-1 polymerase sequence at transmission that is substantially preadapted to one's HLA profile remains relatively low in North America, even in the present era.

AIDS Research and Human RetrovirusesVol. 30, No. S1 ImmunogeneticsSubtype-Specific HIV-1 Adaptation to Host HLAGuinevere Q. Lee, Jonathan Carlson, Chanson J. Brumme, Helen Byakwaga, Conrad Muzoora, Daniel MacMillan, Natalie Kinloch, Kyle Cobarrubias, Mark A. Brockman, Peter W. Hunt, Jeff N. Martin, Mary Carrington, David R. Bangsberg, P. Richard Harrigan, and Zabrina L. BrummeGuinevere Q. LeeBC Centre for Excellence in HIV/AIDS, Vancouver, BC, CanadaSearch for more papers by this author, Jonathan CarlsonMicrosoft Research, Seattle, WA, United StatesSearch for more papers by this author, Chanson J. BrummeBC Centre for Excellence in HIV/AIDS, Vancouver, BC, CanadaSearch for more papers by this author, Helen ByakwagaMbarara University of Science and Technology, Mbarara, UgandaUniversity of California San Francisco, San Francisco, CA, United StatesSearch for more papers by this author, Conrad MuzooraMbarara University of Science and Technology, Mbarara, UgandaSearch for more papers by this author, Daniel MacMillanSimon Fraser University, Faculty of Health Sciences, Burnaby, BC, CanadaSearch for more papers by this author, Natalie KinlochSimon Fraser University, Faculty of Health Sciences, Burnaby, BC, CanadaSearch for more papers by this author, Kyle CobarrubiasSimon Fraser University, Faculty of Health Sciences, Burnaby, BC, CanadaSearch for more papers by this author, Mark A. BrockmanBC Centre for Excellence in HIV/AIDS, Vancouver, BC, CanadaSimon Fraser University, Faculty of Health Sciences, Burnaby, BC, CanadaSearch for more papers by this author, Peter W. HuntUniversity of California San Francisco, San Francisco, CA, United StatesSearch for more papers by this author, Jeff N. MartinUniversity of California San Francisco, San Francisco, CA, United StatesSearch for more papers by this author, Mary CarringtonFrederick National Laboratory for Cancer Research, Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC Frederick, Inc., Frederick, MD, United StatesSearch for more papers by this author, David R. BangsbergMassachusetts General Hospital and Harvard University, Boston, MA, United StatesSearch for more papers by this author, P. Richard HarriganBC Centre for Excellence in HIV/AIDS, Vancouver, BC, CanadaUniversity of British Columbia, Department of Medicine, Vancouver, BC, CanadaSearch for more papers by this author, and Zabrina L. BrummeBC Centre for Excellence in HIV/AIDS, Vancouver, BC, CanadaSimon Fraser University, Faculty of Health Sciences, Burnaby, BC, CanadaSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5475.abstractAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Subtype-Specific HIV-1 Adaptation to Host HLA." AIDS Research and Human Retroviruses, 30(S1), p. A218FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Guinevere Q. Lee, Jonathan Carlson, Chanson J. Brumme, Helen Byakwaga, Conrad Muzoora, Daniel MacMillan, Natalie Kinloch, Kyle Cobarrubias, Mark A. Brockman, Peter W. Hunt, Jeff N. Martin, Mary Carrington, David R. Bangsberg, P. Richard Harrigan, and Zabrina L. Brumme.Subtype-Specific HIV-1 Adaptation to Host HLA.AIDS Research and Human Retroviruses.Oct 2014.A218-A218.http://doi.org/10.1089/aid.2014.5475.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download