TAG HIV Basic Science, Vaccines, and Cure Project Blog

Sex and the Single Microbicide

The levels and distribution of an anti-HIV microbicide in the genital tract are likely to be critical factors in determining potential efficacy. Up until now, research studies have typically assessed microbicide levels in sexually abstinent women, which neglects to consider the potential impact of sexual activity. A new study in PLoS One looks at whether the physical act of sex and the introduction of semen into the genital tract affects the microbcide candidate 0.5% PRO 2000 gel. The study was conducted and completed before the recent announcement that 0.5% PRO 2000 gel had failed to show efficacy in preventing HIV infection in a large randomized clinical trial.

The results showed that 0.5% PRO 2000 gel levels were significantly lower after sex, and this correlated with a reduced ability of cervicovaginal lavage (CVL) from gel-treated women to inhibit HIV and HSV-2 in vitro. It was noted, however, that lower gel concentrations did not fully explain the reduction in antiretroviral activity; additional experiments revealed that seminal plasma also had an independent effect.

The researchers acknowledge that their study has limitations, including a small sample size and a single-dose approach that may underestimate microbicide levels compared to repeat dosing. Nevertheless, they suggest that “the current paradigm of microbicide development should be modified to include postcoital sampling following single and repeated dosing with both active and placebo products and should be expanded to include both CVL and biopsies to more fully define the pharmacokinetics and pharmacodynamics of lead candidates prior to embarking on large-scale efficacy trials.”

PLoS ONE 5(1): e8781. doi:10.1371/journal.pone.0008781

Postcoital Bioavailability and Antiviral Activity of 0.5% PRO 2000 Gel: Implications for Future Microbicide Clinical Trials

Marla J. Keller1,2, Pedro M. M. Mesquita3, N. Merna Torres3, Sylvia Cho3, Gail Shust3, Rebecca P. Madan3, Hillel W. Cohen4, Julie Petrie1, Tara Ford1, Lydia Soto-Torres6, Albert T. Profy7, Betsy C. Herold2,3,5*

1 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Obstetrics and Gynecology and Women's Health, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 4 Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York, United States of America, 5 Department of and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 6 Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America, 7 Endo Pharmaceuticals, Chadds Ford, Pennsylvania, United States of America

Background

The pharmacokinetics and pharmacodynamics of vaginal microbicides are typically assessed among sexually abstinent women. However, the physical act of sex may modulate gel distribution, and preclinical studies demonstrate seminal plasma interferes with the antiviral activity of several microbicides. This study compared the biological activity and concentration of PRO 2000 in cervicovaginal lavage (CVL) collected in the absence or following coitus.

Methods

CVL samples were collected from ten heterosexual couples at baseline, after sex, after a single dose of 0.5% PRO 2000 gel and sex, and after gel application without sex. The impact of CVL on HIV-1 infection of TZM-bl cells and HSV-2 infection of CaSki cells was monitored by luciferase and plaque assay, respectively. PRO 2000 concentrations were measured by fluorescence.

Results

CVL collected after PRO 2000 application significantly inhibited HIV-1 and HSV-2 (p = 0.01). However, the antiviral activity was reduced following sex and no significant protective effect was observed in postcoital CVL obtained in the presence compared to the absence of PRO 2000 for HIV (p = 0.45) or HSV-2 (p = 0.56). Less PRO 2000 was recovered in postcoital CVL, which, in conjunction with interference by seminal plasma, may have contributed to lower antiviral activity.

Conclusions

Postcoital responses to PRO 2000 differ from precoital measures and the results obtained may provide insights into the clinical trial findings in which there was no significant protection against HIV-1 or HSV-2. Postcoital studies should be incorporated into clinical studies before embarking on large-scale efficacy trials.

CMV-Specific T Cells in HIV: A Harbinger of Immunosenescence?

A large body of data points to CMV infection as the strongest risk factor for the development of the “immune risk profile” (IRP) in elderly individuals without HIV infection, a profile that is associated with ill health (including neurocognitive decline) and relatively earlier mortality. The association between the IRP and CMV infection is linked to large accumulations of CMV-specific CD4 and CD8 T cells displaying a senescent phenotype (lacking expression of the co-stimulatory molecule CD28). A new paper in PLoS One reports results of a comprehensive evaluation of CMV-specific T cell responses in individuals at varying stages of HIV infection, both on and off antiretroviral therapy, in comparison to matched HIV-negative controls. The goal of the study was to gain insight into whether CMV-specific CD4 and CD8 T cell expansions occur at an earlier age in people with HIV and might therefore contribute to the accelerated onset of immunosenescence.

The researchers found that CD8 T cells responses to two major CMV antigens (the pp65 and IE proteins) were consistently higher in people with HIV compared to age-matched controls, even at the earliest stages of infection. The same pattern held true for CD4 T cells although the number of CMV-specific cells was lower compared to CD8 T cells. The highest levels were seen among individuals on antiretroviral therapy; on average 6% of their peripheral blood CD8 T cells were specific for pp65 or IE antigens. Because CMV encodes many other proteins, the researchers estimate that an average of around 20% of CD8 T cells were CMV-specific in this group, a strikingly high number. The authors conclude that “longitudinal studies to determine whether CMV drives early immunosenescence during HIV disease are warranted.”

PLoS ONE 5(1): e8886. doi:10.1371/journal.pone.0008886

Cytomegalovirus-Specific T Cells Persist at Very High Levels during Long-Term Antiretroviral Treatment of HIV Disease

David M. Naeger1, Jeffrey N. Martin1,2, Elizabeth Sinclair1, Peter W. Hunt1, David R. Bangsberg3, Frederick Hecht1, Priscilla Hsue1, Joseph M. McCune1, Steven G. Deeks1

1 Department of Medicine, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California, United States of America, 3 Massachusetts General Hospital, Harvard Medical School, Harvard Initiative for Global Health, Boston, Massachusetts, United States of America

Background

In healthy, HIV seronegative, CMV seropositive adults, a large proportion of T cells are CMV-specific. High-level CMV-specific T cell responses are associated with accelerated immunologic aging (“immunosenesence”) in the elderly population. The impact of untreated and treated HIV infection on the frequency of these cells remains undefined.

Methodology/Principal Findings

We measured the proportion of CD4+ and CD8+ T cells responding to CMV pp65 and IE proteins was measured using flow cytometry in 685 unique HIV seronegative and seropositive individuals. The proportion of CMV-specific CD8+ T cells was consistently higher in the HIV-seropositive subjects compared to the HIV-seronegative subjects. This HIV effect was observed even in patients who lacked measurable immunodeficiency. Among the HIV-seropositive subjects, CMV-specific CD8+ T cell responses were proportionately lower during recent infection, higher during chronic untreated infection and higher still during long-term antiretroviral treated infection. The CD8+ T cell response to just two CMV proteins (pp65 and IE) was approximately 6% during long-term therapy, which was over twice that seen in HIV-seronegative persons. CMV-specific CD4+ T cell responses followed the same trends, but the magnitude of the effect was smaller.

Conclusions/Significance

Long-term successfully treated HIV infected patients have remarkably high levels of CMV-specific effector cells. These levels are similar to that observed in the elderly, but occur at much younger ages. Future studies should focus on defining the potential role of the CMV-specific inflammatory response in non-AIDS morbidity and mortality, including immunosenescence.

Adapting Yellow Fever Vaccine for HIV

The attenuated vaccine against yellow fever (YFV) is extremely efficacious and has been used by more than 400 million people over the past 70 years. Because of its impressive track record, researchers are now investigating whether YFV can be modified to work against other diseases, including Japanese encephalitis, dengue, West Nile virus and malaria.  The latest addition to this list is HIV; in a new study published recently by the Journal of Virology, Myrna C. Bonaldo and colleagues report preliminary data showing that YFV can be modified to encode the Gag protein from SIV and is a potent inducer of T cell immune responses in macaques. The levels of SIV-specific CD8 T cells induced by the YFV/SIV construct were equivalent to those induced live-attenuated SIV vaccines (the most successful vaccines studied to date in the macaque model). Challenge studies have not yet been conducted but the researchers suggest YFV is an excellent candidate for further development as an HIV vaccine vector.

JVI Accepts, published online ahead of print on 20 January 2010

J. Virol. doi:10.1128/JVI.02255-09

Recombinant Yellow Fever Vaccine Virus 17D Expressing SIVmac239 Gag Induces SIV-Specific CD8+ T Cell Responses in Rhesus Macaques

Myrna C. Bonaldo, Mauricio A. Martins, Richard Rudersdorf, Philip A. Mudd, Jonah B. Sacha, Shari M. Piaskowski, Patrícia C. Costa Neves, Marlon G. Veloso de Santana, Lara Vojnov, Saverio Capuano III, Eva G. Rakasz, Nancy A. Wilson, John Fulkerson, Jerald C. Sadoff, David I. Watkins, and Ricardo Galler

Laboratorio de Biologia Molecular de Flavivírus, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, Brazil; Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin; Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin; Aeras Global TB Vaccine Foundation, Rockville, MD; Instituto de Tecnologia em Imunobiologicos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

Abstract

Here we describe a novel vaccine vector for expressing HIV antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8+ T cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8+ T cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8+ T cells secreted several cytokines, were largely effector memory and suppressed viral replication in CD4+ T cells.

Immune Responses Out of the Frame

The translation of an RNA or DNA template into proteins is a complex process that can produce a wide variety of functional and non-functional protein products. Translation involves “reading” the genetic code, and the nature of the protein produced depends on where the reading of the code starts and stops (called the “reading frame”) and also which direction the code is read in (forwards is called “sense” while backwards is called “antisense”). HIV’s RNA genome contains nine reading frames encoding functional proteins, but it is also known that alternate reading frames exist which can be translated into proteins that are non-functional (or have unknown functions). Antisense translation can also lead to the production of such proteins. The extent to which these proteins are targeted by the immune system in HIV-infected people has not been well characterized.

Two new papers in the Journal of Experimental Medicine address this knowledge gap by evaluating CD8 T cell response to “cryptic” epitopes derived from HIV proteins produced from both alternate reading frames and antisense transcription. The reported results indicate that targeting of cryptic HIV epitopes by CD8 T cells is surprisingly common. Both papers also present evidence that CD8 T cell responses against cryptic epitopes can result in mutations in the RNA sequences encoding these epitopes that abrogate CD8 T cell recognition (immune escape).

The implication of these findings is that there is an unexplored plethora of potential targets for HIV-specific T cell responses that could conceivably be exploited to improve vaccine design. Anju Bansal and colleagues note that the process of codon optimization, which is commonly used to try and improve expression of HIV proteins by vaccine candidates, alters the proteins produced from alternate reading frames so that they differ (by as much as 80%) from those generated by natural infection. So counter-intuitively, it is possible that codon optimization – which was used in the Merck Ad5 vaccine – actually reduces the breadth of the resultant HIV-specific CD8 T cell response. Bansal et al also point out that that the ALVAC-HIV vaccine used in the recently reported RV-144 vaccine trial is not codon optimized and could potentially induce responses to cryptic epitopes (CE); they conclude that "it may be important to take into account the contribution of CE-induced responses in the overall CD8 T cell repertoire of future vaccine constructs."

Published online January 11, 2010

doi:10.1084/jem.20092060

The Journal of Experimental Medicine

Brief Definitive Report

CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription

Anju Bansal1, Jonathan Carlson4, Jiyu Yan1, Olusimidele T. Akinsiku2, Malinda Schaefer5,6, Steffanie Sabbaj1, Anne Bet2, David N. Levy7, Sonya Heath1, Jianming Tang1,2, Richard A. Kaslow3, Bruce D. Walker8,9, Thumbi Ndung’u8,9, Philip J. Goulder8,9,10, David Heckerman4, Eric Hunter5,6, and Paul A. Goepfert1,2

1 Department of Medicine, 2 Department of Microbiology, and 3 Department of Epidemiology, University of Alabama at Birmingham, Birmingham, AL 35294. 4 Microsoft Research, Redmond, WA 98052. 5 Emory Vaccine Center at Yerkes National Primate Research Center and 6 Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30329. 7 Department of Basic Science, New York University College of Dentistry, New York, NY 10010. 8 Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Boston, MA 02129. 9 HIV Pathogenesis Program, Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban 4013, South Africa. 10 Department of Pediatrics, University of Oxford, Oxford OX1 3SY, England, UK

Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.

Published online January 11, 2010

doi:10.1084/jem.20091808

The Journal of Experimental Medicine

Article

Viral adaptation to immune selection pressure by HLA class I–restricted CTL responses targeting epitopes in HIV frameshift sequences

Christoph T. Berger1, Jonathan M. Carlson2, Chanson J. Brumme1, Kari L. Hartman1, Zabrina L. Brumme1,3,4, Leah M. Henry1, Pamela C. Rosato1, Alicja Piechocka-Trocha1, Mark A. Brockman1,3,4, P. Richard Harrigan3,5, David Heckerman2, Daniel E. Kaufmann1, and Christian Brander1,6,7

1 Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Boston, MA 02129. 2 Microsoft Research, Seattle, WA 98033. 3 British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC V6B 5S8, Canada. 4 Simon Fraser University, Burnaby, BC V5A 4Y7, Canada. 5 Division of AIDS, University of British Columbia, Vancouver, BC V6Z 1Y6, Canada. 6 Institucio Catalana de Recerca i Estudis Avancats, 08010 Barcelona, Spain. 7 Irsicaixa HIV Research Institute–HIVACAT, Hospital Germans Trias i Pujol, Badalona, 08916 Barcelona, Spain

CD8+ cytotoxic T lymphocyte (CTL)–mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q < 0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03–restricted +2 frame–encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

Early Predictors of Disease Progression

Recent research involving SIV-infected macaques has suggested that the early loss of a particular type of memory CD4 T cell (known as a “central memory” T cell or Tcm) may be a key predictor of the subsequent pace of disease progression. Tcm are a long-lived subset of memory T cells that can proliferate robustly in response to antigen. Tcm proliferation generates a fleet of T cells belonging to a shorter-lived subset called “effector memory” (Tem) cells. Tem are generally viewed as first-responders that can rapidly execute anti-pathogen functions, while Tcm provide a stem-cell like renewal source for new Tem if their numbers need to be bolstered. Studies in HIV-infected people have consistently shown a loss of Tcm and increase in Tem (which equates to a decrease in long-lived resting T cells and an increase in short-lived activated T cells), but whether changes in the numbers of different T cell subsets during early infection can predict disease progression has not been thoroughly evaluated.

A new study published in the Journal of Infectious Diseases set out to answer the question of whether quantifying Tcm in early infection provides prognostic information. To provide sufficient statistical power to ensure confidence in the findings, a total of 466 individuals were studied, among whom 101 progression events occurred.

It turned out that the proportion or absolute number of Tcm did not correlate with subsequent disease progression (defined as the time to AIDS or death), but several other parameters did. These included the proportion of naïve CD8 T cells, with a greater proportion being strongly associated with slower disease progression (p<0.001); this correlation remained significant after adjustment for CD4 T cell count. The numbers of CD8 T cells expressing the IL-7 receptor (CD127) were also linked to the rate of progression; having fewer of these cells correlated with a faster disease course.

Immune activation was assessed by measuring the proportion of CD4 and CD8 T cells expressing the proliferation marker Ki67. In both subsets, higher proportions of Ki67-expressing cells equated to faster progression, and for CD8 T cells this relationship held up after adjustment for baseline CD4 T cell count, age, and viral load. The median time to AIDS or death among subjects with the highest levels of Ki67-expressing CD8 T cells (based on dividing participants into quartiles) was 4 years for those in the top quartile compared to 10 years for those in the lowest.

Finally, measures of cell-associated viral load (CAVL: the proportion of CD4 T cells containing HIV DNA) were correlated significantly with progression in those participants sampled within 225 days of their estimated date of seroconversion (225 days was the median time after the estimated date of seroconversion that samples were obtained). Among participants sampled later, CAVL was not significantly correlated with rate of progression, suggesting an important impact of the early spread of HIV among CD4 T cells on subsequent disease course. The researchers also evaluated CAVL in different CD4 T cell subsets: naïve, central memory, transitional memory and effector memory. To their surprise, naïve CD4 T cells showed relatively high rates of infection, albeit around 10-fold lower than the memory subsets. Because resting naïve CD4 T cells are known to be very resistant to HIV, the researchers speculate that the infected naïve cells may have been rendered susceptible by immune activation (naïve CD4 T cells have been shown to become susceptible to R5-using HIV after they receive activation signals).

The authors conclude by stating: “we find that quantification of Tcm cells in early infection does not provide predictive power for progression. However, measures of homeostasis and activation, including CD127 expression and Ki-67, do provide such information and should be studied further to determine their role in clinical monitoring of HIV-1 progression…Future efforts to identify markers of subsequent progression should focus on measures of activation and homeostasis during the earliest stages of infection.”

The Journal of Infectious Diseases 2010;201:272–284

This article is in the public domain, and no copyright is claimed.

DOI: 10.1086/649430

MAJOR ARTICLE

Immunologic and Virologic Events in Early HIV Infection Predict Subsequent Rate of Progression

Anuradha Ganesan,1,a Pratip K. Chattopadhyay,2,a Tess M. Brodie,2 Jing Qin,3 Wenjuan Gu,4 John R. Mascola,2 Nelson L. Michael,5 Dean A. Follmann,3 and Mario Roederer,2 for the Infectious Disease Clinical Research Program HIV Working Groupb

1National Naval Medical Center, Infectious Disease Clinical Research Program, Uniformed Services University, 2Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and 3Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, Bethesda, 4Biostatistics Research Branch, Scientific Application International Corporation–Frederick, Frederick, and 5United States Military HIV Research Program, Walter Reed Army Institute of Research, Rockville, Maryland

Background. Variability in human immunodeficiency virus (HIV) disease progression cannot be fully predicted by CD4+ T cell counts or viral load (VL). Because central memory T (TCM) cells play a critical role in the pathogenesis of simian immunodeficiency virus disease, we hypothesized that quantifying these cells in early HIV infection could provide prognostic information.

Methods. We measured expression of CD45RO, chemokine (C‐C motif) receptor (CCR) 5, CCR7, CD27, and CD28 to enumerate naive and memory subsets in samples from recently infected individuals. We also quantified proliferation, CD127 expression, and cell‐associated VL. Disease progression was compared across subgroups defined by these measurements, using Kaplan‐Meier survival curves and multivariate Cox proportional hazards regression.

Results. Four hundred sixty‐six subjects contributed 101 events. The proportion or absolute count of TCM cells did not correlate with disease progression, defined as the time to AIDS or death. However, significant associations were observed for proliferation within CD4+ or CD8+ T cells, loss of naive or CD127+ memory CD8+ T cells, and CD4+ T cell–associated VL.

Conclusions. Our results demonstrate that the extent of the immunopathogenesis established early in HIV infection predicts the course of future disease. Because antiretroviral drug treatment reverses such defects in part, our study provides mechanistic clues to why early use of antiretrovirals may prove beneficial.

Reservoir Macaques

In the online-ahead-of-print section of the Journal of Virology, a group of researchers led by Thomas North describe a new macaque model of viral persistence during antiretroviral therapy. Because non-nucleoside reverse transcriptase inhibitor (NNRTI) antiretroviral drugs are not active against the reverse transcriptase (RT) of simian immunodeficiency virus (SIV), the study employs a SIV-HIV hybrid encoding the RT from HIV (RT-SHIV). A group of nine macaques were treated with a combination of the NNRTI efavirenz (Sustiva) and the nucleotide RT inhibitors emtricitabine and tenofovir (Truvada), while three served as untreated controls. All treated animals showed peripheral blood viral load levels of less than 50 copies/mL after 18 weeks of treatment. After a total of 26 weeks of ART, five treated animals and the three untreated controls were euthanized and comprehensive analyses of different tissues were conducted to assess for the presence of viral DNA and RNA.

The results showed that, as expected, viral DNA and RNA levels were much higher in untreated versus treated macaques. The highest levels of DNA and RNA in the treated group were in lymphoid tissues, particularly spleen and lymph nodes, while lower levels were found in GI tract tissues. All the treated animals had detectable levels of viral RNA in their spleen and all lymph nodes. Some also showed RNA in thymus (3 of 5) and tonsils (4 of 5), but none had detectable viral RNA in bone marrow. All lymphoid tissues from the treated macaques had detectable viral DNA except bone marrow from two of the animals. The authors of the study state: “these findings are consistent with a widespread distribution of latent viral reservoirs in RT-SHIV-infected macaques.” They go on to explain that the goal of developing this model is to explore strategies for eradicating viral reservoirs, in the hopes of eventually discovering effective interventions that can be evaluated in humans.

JVI Accepts, published online ahead of print on 23 December 2009

J. Virol. doi:10.1128/JVI.02356-09

VIRAL SANCTUARIES DURING HIGHLY ACTIVE ANTIRETROVIRAL THERAPY IN A NONHUMAN PRIMATE MODEL FOR AIDS

Thomas W. North*, Joanne Higgins, Jesse D. Deere, Timothy L. Hayes,Andradi Villalobos, Lourdes A. Adamson, Barbara L. Shacklett,Raymond F. Schinazi, and Paul A. Luciw

Center for Comparative Medicine, Department of Veterinary Molecular Biosciences, Department of Medical Microbiology and Immunology, and Department of Pathology, School of Medicine, University of California, Davis, CA 95616; and Emory University School of Medicine, Veterans Affairs Medical Center, Decatur, GA 30033

Abstract

Highly active antiretroviral therapy (HAART) enables long-term suppression of plasma HIV-1 loads in infected persons, but low level virus persists and rebounds following cessation of therapy. During HAART, this virus resides in latently-infected cells, such as resting CD4+ T cells, and in other cell types that may support residual virus replication. Therapeutic eradication will require elimination of virus from all reservoirs. We report here a comprehensive analysis of these reservoirs in fluids, cells and tissues in a rhesus macaque model that mimics HAART in HIV-infected humans. This nonhuman primate model uses RT-SHIV, a chimera of simian immunodeficiency virus containing the HIV-1 reverse transcriptase (RT). Methods were developed for extraction, pre-amplification and real-time PCR analyses of viral DNA (vDNA) and viral RNA (vRNA) in tissues from RT-SHIV-infected macaques. These methods were used to identify viral reservoirs in RT-SHIV-infected macaques treated with a potent HAART regimen consisting of efavirenz, emtricitabine and tenofovir. Plasma virus loads at necropsy ranged from 11-28 copies of vRNA per ml. Viral RNA and DNA were detected during HAART in tissues from numerous anatomical locations. Additional analysis provided evidence for full-length viral RNA in tissues of animals with virus suppressed by HAART. The highest levels of vDNA and vRNA in HAART-treated macaques were in lymphoid tissues, particularly spleen, lymph nodes, and gastrointestinal tract tissues. This study is the first comprehensive analysis of tissue and organ distribution of a primate AIDS virus during HAART. These data demonstrate widespread persistence of residual virus in tissues during HAART.