Michael Palm Basic Science, Vaccines and Prevention Weblog

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.”

Notably, significantly lower drug was detected in CVL obtained following coitus (median (IQR) 14 (3, 27)) compared to CVL obtained in the absence of coitus (28 (22, 110) (p = 0.035; Table 1) and the antiviral activity correlated with the concentration of PRO 2000 (r = 0.48, p = 0.03 for both HIV and HSV-2).

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.

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.

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.

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.

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.