Michael Palm Basic Science, Vaccines and Prevention Weblog

One of the most efficient means by which HIV spreads between cells is by promoting cell to cell contacts. The virus is then transferred from infected to uninfected cell via what has been dubbed a “virological synapse.” Some prior studies have raised the concern that this process may be invulnerable to approaches designed to inhibit viral entry into cells, including neutralizing antibodies and entry inhibitor drugs. A new study published in the Journal of Virology offers some reassurance that this is not in fact the case. The researchers show that a variety of entry inhibitors both big (in the form of the monoclonal antibody b12) and small (the CCR5-blocking entry inhibitor compound TAK-779) remain capable of inhibiting infection in the setting of CD4 T cell to CD4 T cell transfer. They suggest prior results may have been misleading due to problems with the readout used to assess infection, and state: “these results are encouraging for use of prophylactic vaccines designed to elicit neutralizing antibodies and for entry inhibitors applied in a prophylactic or therapeutic setting.” There is, however, a caveat in that the results may not apply to other cell types such as macrophages, and the researchers note that some relatively recent data has indicated that transfer of HIV between dendritic cells and CD4 T cells may be resistant to inhibition by neutralizing antibodies. The paper is accompanied online by supplemental videos showing contacts between infected (red) and uninfected (green) cells (video S1) and visualizing the synapse in the context of both X4-using (video S2) and R5-using (video S3) HIV isolates.

Journal of Virology, April 2010, p. 3516-3527, Vol. 84, No. 7

doi:10.1128/JVI.02651-09

Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

Nicola Martin,1 Sonja Welsch,2,4 Clare Jolly,3 John A. G. Briggs,4 David Vaux,1 and Quentin J. Sattentau1

The Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom,1 Structural Biology Unit, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom,2 Wohl Virion Centre and MRC/UCL Centre for Medical Molecular Virology, University College London, London W1T 4JF, United Kingdom,3 Structural and Computational Biology Unit, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany4

Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4+ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.

The loss of naïveté that comes with getting older is familiar to just about everyone. Somewhat less familiar is the fact that this is also true immunologically; our repertoire of naïve T cells and B cells – vital to responding to pathogens that have not previously been encountered, and keeping up with evolving chronic infections – steadily diminishes. For T cells, the loss is associated with declining output from the thymus, which also shrinks in size over time. It has been appreciated for many years that chronic infections can accelerate the pace of naïve T cell decline, but the effects of HIV far exceed those of any other chronic virus; progression to AIDS is typically associated with an almost complete loss of naïve CD4 and CD8 T cells.

Recent studies of long-term immune reconstitution on antiretroviral therapy (ART) have identified the ratio of naïve T cells to memory T cells as an important factor predicting the extent of CD4 T cell repopulation after 7+ years of treatment. A study from Timothy Schacker at the University of Minnesota now addresses the question of whether baseline measures of naïve CD4 T cell numbers can predict the potential for immune reconstitution on ART. The results of an analysis of 348 participants in AIDS Clinical Trials Group (ACTG) trials show that, indeed, baseline naive but not total CD4 T cell counts strongly predicted the magnitude of CD4 T cell increases after ART initiation. Lower naïve CD4 T cell levels at baseline were also associated with greater time spent with low CD4 T cell counts on ART, which is known to be associated with a greater risk of clinical events. The study findings suggest that measurements of naïve CD4 T cells could help optimize timing of ART initiation and lessen the incidence of poor immune reconstitution despite HIV suppression.

J Acquir Immune Defic Syndr. 2010 Feb 24. [Epub ahead of print]

Measurement of Naive CD4 Cells Reliably Predicts Potential for Immune Reconstitution in HIV.

Schacker TW, Bosch RJ, Bennett K, Pollard R, Robbins GK, Collier AC, Gulick RM, Spritzler J, Mildvan D; for the AIDS Clinical Trials Group (ACTG).

From the *Department of Medicine, University of Minnesota, Minneapolis, MN; daggerDepartment of Biostatistics, Harvard School of Public Health, Boston, MA; double daggerDivision of Infectious Diseases, University of California, Sacramento, CA; section signDivision of Infectious Diseases, Massachusetts General Hospital, Boston, MA; parallelDepartment of Medicine, University of Washington, Seattle, WA; paragraph signDivision of Infectious Diseases, Weill-Cornell Medical College, New York, NY; and #Division of Infectious Diseases, Beth Israel Medical Center, New York, NY.

BACKGROUND:: Pathogenesis studies show that naive CD4 cells are preferentially depleted in lymphoid tissues during HIV infection, and studies of advanced patients suggest levels of naive CD4 cells in blood correlate to total CD4 cells after starting antiretroviral therapy (ARV). We hypothesized that measuring naive CD4 cells in blood in people at earlier stages of disease would identify those at highest risk for poor CD4 reconstitution who may benefit from earlier initiation of ARV. METHODS AND FINDINGS:: We identified 348 patients from multiple AIDS Clinical Trials Group studies who were ARV naive, had a CD4 count between 200 and 500 cells per microliter, a measure of pretreatment-naive CD4 percent, and serial follow-up measures of CD4 count and plasma HIV RNA after starting ARV. We used logistic regression to model the ability of naive CD4 percent to predict 100 and 200 CD4 cell increases after 24 months of therapy. After controlling for baseline viral load and demographic variables, baseline naive but not total CD4 cell count strongly predicted CD4 cell increases. Lower baseline naive CD4 percent was associated with greater time spent at lower CD4 T-cell counts after initiating ARV. CONCLUSIONS:: Measurement of naive CD4 percent in patients can identify those least likely to reconstitute immunity, who may benefit from earlier ARV treatment.

The 17th annual CROI took place last week in San Francisco. For the first time this year, poster discussion sessions are also included in the conference webcasts, meaning that essentially the entire content of the meeting can be accessed online. Several media outlets also provided wide-ranging coverage and interviews during CROI, including AIDSMeds.com, The Body and AIDSMap.  

There was no single study that dominated the headlines, but several emerging themes in HIV research were reinforced by new data. Chief among them, the idea that increased uptake of antiretroviral therapy (ART) and a greater degree of viral suppression at the community level might reduce new HIV diagnoses received preliminary support in an analysis by Dr. Moupali Das-Douglas from the San Francisco Department of Health. The presentation is the first in the Testing and Transmission session at 9:30am on Wednesday. More direct evidence came from the partners in prevention trial, which enrolled 3,408 discordant couples in order to assess the impact of suppressing HSV-2 with acyclovir on HIV transmission. While acyclovir had no effect, an analysis presented at CROI (paper #136) revealed that among HIV positive partners on ART, there was one transmission of HIV to the uninfected partner, which occurred within the first 18 days of ART initiation. Among couples where the HIV positive partner was not on ART, there were 102 HIV transmissions.

In terms of the intersection of HIV pathogenesis and clinical care, a group of researchers at UCSF presented a suite of studies showing that markers of inflammation, immune activation and immunosenescence correlate with a wide spectrum of adverse phenomena that clinician scientist Steve Deeks groups under the umbrella term “badness.” Examples include measures of arterial health such as the presence of plaques and reduced arterial distensibility. Priscilla Hsue summarized some of these findings in an insightful talk on HIV and cardiovascular health (the webcast is the third presentation in the Symposium on Pathogenesis and Clinical Management of Complications at 4pm on Wednesday 2/17).

After many years of often contentious debate, the idea that HIV replication is completely stopped in the majority of people on suppressive ART therapy finally seemed to gain acceptance at the 2010 CROI. The use of an assay developed by Sarah Palmer that can measure down to 0.2 copies of HIV RNA in peripheral blood has played a key role; many studies – including several presented at CROI - have now shown that intensifying ART has no effect on the residual HIV RNA that is below the range of standard viral load tests (which can’t measure less than 50 copies per ml of blood), but detectable using Palmer’s technique. These findings have led to a general consensus that, in most cases, the low-level HIV RNA (<50 copies) that can sometimes be detected in people on ART is being produced by long-lived cells containing integrated HIV DNA. These long-lived reservoirs can occasionally pump out new viruses, but ART prevents them from going on to infect new cells. As described in a talk by Frank Malderelli from the National Cancer Institute, this new understanding of HIV persistence is reinvigorating efforts to extinguish the remaining embers of infection and fully cure HIV. Malderelli’s talk is the first in the The Future of HIV Therapeutic Research Symposium on Friday 2/19, the last session listed on the Friday webcast page.

Other selected presentations that may be of interest, with brief comments on each:

Plenary (webcast): Pathogenic vs Nonpathogenic Retrovirus Infections

Guido Silvestri

Univ of Pennsylvania, Philadelphia, US

- Excellent review of the state of the science and particularly on the crucial role of persistent immune activation in distinguishing pathogenic from non-pathogenic immunodeficiency virus infections. Also offers this quote (from Guido Silvestri & Steve Deeks), which may turn out to be a harbinger of where HIV pathogenesis research is headed now that fully suppressive ART regimens have become available: “In the absence of HIV replication, residual inflammation rather than drug toxicity may be the most important obstacle towards reaching a normal healthy human lifespan.”

Paper # 82

Broad HIV Neutralizing Antibodies Can Be Elicited by the GBV-C glycoprotein E2 and Neutralize HIV via a 2F5-like Mechanism

S Jung, H Wend, N Donhauser, K Eissmann, B Fleckenstein, and Heide Reil*

Univ Hosp Erlangen, Germany

Webcast, Thursday 2/18 (last presentation in session)

- Reports that some antibodies against the E2 protein of GB virus C (GBV-C) can broadly neutralize HIV isolates, due to a mechanism involving interactions with phospholipids in the lipid bilayer of the virus (a coating acquired from the host cell during virus budding). A similar mechanism has been reported for the broadly neutralizing monoclonal antibody 2F5 and these findings may open novel routes toward an effectve neutralizing antibody-based HIV vaccine. This presentation is also cited on JBM's PS blog which is focused on developments in this field of study.

Paper # 140

Monospecific Expansion of SIVmac251 during Acute Infection May Mask Multiple Transmitted Virus Variants Revealed during the Chronic Phase

Barbara Felber*1, E-Y Kim2, R Pal3, R Desrosiers4, S Wolinsky2, and G Pavlakis1

1NCI-Frederick, MD, US; 2Northwestern Univ, Chicago, IL, US; 3Advanced Biosci Labs, Inc, Kensington, MD, US; and 4New England Primate Res Ctr, Boston, MA, US

Webcast, Friday 2/19 (last but one presentation in session)

- Uses challenge with a diverse SIVmac251 stock and finds that some variants present in the stock remain undetectable until chronic infection. This calls into question the interpretation that a single dominant virus detectable during acute HIV infection means that only a single variant was transmitted.

Paper # 285

Maraviroc Intensification for Suboptimal CD4+ Cell Response Despite Sustained Virologic Suppression: ACTG 5256

Timothy Wilkin1, C Lalama2, A Tenorio3, A Landay3, H Ribaudo2, J McKinnon4, R Gandhi5, J Mellors4, J Currier6, and R Gulick1

1Weill Cornell Med Coll, New York, NY, US; 2Harvard Sch of Publ Hlth, Boston, MA, US; 3Rush Univ Med Ctr, Chicago, IL, US; 4Univ of Pittsburgh Sch of Med, PA, US; 5Massachusetts Gen Hosp, Boston, US; and 6Univ of California, Los Angeles, US

Poster PDF: http://www.retroconference.org/2010/PDFs/285.pdf

- Adding Maraviroc did not increase CD4 T cell counts in people with poor CD4 T cell recovery despite sustained viral load suppression. There was, however, a statistically significant reduction in measures of immune activation; one interpretation of these findings is that – at least in some cases – persistent immune activation may be a consequence of poor CD4 T cell recovery rather than the primary cause. These results may note bode well for the four other ongoing clinical trials of maraviroc with similar designs.

Paper # 714

Incomplete Immune Recovery on HAART Is Associated with Significant More Cardiovascular Events and a Trend Towards More Non-AIDS Related Malignancies in Dutch ATHENA Cohort

Steven van Lelyveld*1, L Gras2, A Kesselring2, S Zhang2, F de Wolf2, A Wensing1, and A Hoepelman1

1Univ Med Ctr Utrecht, The Netherlands and 2Stichting HIV Monitoring, Amsterdam, The Netherlands

Poster PDF: http://www.retroconference.org/2010/PDFs/714.pdf

- A study showing that poor immune recovery despite suppression of viral load is a risk factor for clinical illness, emphasizing the need to research and develop new therapies to promote immune reconstitution for people in this situation.

The primary mechanism by which CD8 T cells contribute to controlling pathogens is by killing infected cells. The afflicted cells display pathogen epitopes on their surface via class I HLA molecules that constantly shuttle disused protein fragments to the cell surface in a manner akin to taking out the garbage. CD8 T cells that recognize the pathogen epitope in the context of an HLA molecule that matches the HLA molecule expressed by the CD8 T cell initiate a cascade of events that results in the infected cell being killed. Examples of the process have been captured on video (the Howard Hughes Medical Institute website has an excellent excerpt from a lecture by Bruce Walker showing such a video and explaining it). However, although this is the canonical mechanism of CD8 T cell activity (and the reason for their alternate moniker of cytotoxic T cell lymphocyte or CTL), CD8 T cells can also suppress pathogens by other means such as release of chemokines and cytokines and, in the case of HIV, an as-yet-unidentified antiviral factor that goes by the acronym of CAF. While there is a vast amount of evidence demonstrating the importance of CD8 T cells in suppressing replication of HIV (and other similar viruses such as SIV), the relative contributions of direct killing and indirect suppression have not been clearly delineated. Attempting to do so is a complex task, as the contribution of these activities may vary based on factors such as the extent of immunologic control and disease stage.

Two new studies in PLoS Pathogens make a valiant first attempt to shed light on this issue by evaluating whether artificial depletion of CD8 T cells impacts the speed of SIV suppression by antiretroviral therapy (ART) in macaques. The rationale of both studies is that the kinetics of ART-mediated viral suppression should be slower in the absence of CD8 T cells because infected cells will have a longer lifespan. In both experiments, no such effect is demonstrated, leading the researchers to argue that CD8 T cells do not impact the lifespan of productively infected cells and that therefore their primary mechanism of action in chronic SIV infection is indirect suppression as opposed to direct killing. In an accompanying commentary, Miles P. Davenport and Janka Petravic invoke the tortured language of former US Secretary of Defense Donald Rumsfeld and suggest that CD8 T cell activity against HIV may qualify as a “known unknown.”

But there are a number of issues that make interpreting these data complicated. Both studies involve chronically SIV-infected macaques, and CD8 T cell dysfunction and exhaustion in the setting of chronic infection is well documented. It could be that in a milieu in which viral control is only partial and both functional and dysfunctional SIV-specific CD8 T cells co-exist, indirect suppression plays a larger role than in the setting of robust immunological control (e.g. elite controllers). The selection pressure imposed upon SIV and HIV by HLA-restricted CD8 T cell responses is also extremely well documented, and although the authors of these new papers argue that indirect suppression could also exert selective pressure this scenario is somewhat difficult to envision. It is straightforward to grasp how a virus with an immune escape mutation could survive as a result of CD8 T cells being unable to kill the cell it is occupying. But in the case of indirect suppression, the escape mutation would have to abrogate localized release of suppressive substances by virus-specific CD8 T cells; it seems unlikely that the epitope specificities of CD8 T cells in a localized environment would be so uniform that a single virus carrying an escape mutation would switch off the suppression to a sufficient degree to obtain a survival advantage (although this question may be amenable to study).

Both papers note the possibility that CD8 T cells might kill virus-infected cells prior to the release of new virions, therefore making their activity essentially invisible in this particularly experimental system. The data cited in support of this possibility comes from Jonah Sacha, who has shown that epitopes from incoming virions can be processed and presented to CD8 T cells prior to virus integration, leading to killing of infected cells before the establishment of productive infection. Another possibility is that CD8 T cell-mediated killing reduces the average number of virions produced by each infected cell while having only a minor impact on the average lifespan (I think the mathematical model used in these papers assumes the same average virion production – or “burst size” – for every infected cell). Due to considerable variation from animal to animal, relatively subtle differences in infected cell lifespan may not be easy to capture.

Despite all the complexity and caveats, the papers are provocative and highlight the need to better understand the mechanism of action of CD8 T cells in SIV, and by extension, HIV infection.

In a related development, a group of researchers headed by members of the Human Immunology Laboratory at the International AIDS Vaccine Initiative (IAVI) have just published details of an assay that measures HIV inhibition by CD8 T cells in vitro. Although the numbers of people studied is small, the paper reports that inhibition measured by the assay correlated with viral load control, i.e. CD8 T cell-mediated inhibition was strong among untreated individuals with viral loads <10,000 copies/mL but weak in those above that threshold. The researchers also use the assay to measure the inhibitory capacity of CD8 T cells from seven uninfected individuals immunized with a DNA/Ad5 HIV vaccine regimen, reporting that significant inhibition could be documented but only after receipt of the Ad5 boost. Importantly, there was no correlation between the degree of in vitro virus inhibition and the numbers of vaccine-induced CD8 T cell responses measured by ELISpot, which up until now has been the standard way to measure the immunogenicity of T cell-based vaccine candidates. The new assay takes three weeks to run and involves less than 2 million cells, making it more practical than those developed previously. The study authors write: “We believe the viral inhibition assay will be a useful tool in the study of HIV‐1 pathogenesis and vaccine development, complementing existing methods used to prioritize candidates for further trials.”

PLoS Pathog 6(1): e1000747. doi:10.1371/journal.ppat.1000747

CD8+ Lymphocytes Control Viral Replication in SIVmac239-Infected Rhesus Macaques without Decreasing the Lifespan of Productively Infected Cells

Nichole R. Klatt1,2, Emi Shudo3, Alex M. Ortiz1, Jessica C. Engram1, Mirko Paiardini1, Benton Lawson2, Michael D. Miller4, James Else2, Ivona Pandrea5, Jacob D. Estes6, Cristian Apetrei5, Joern E. Schmitz7, Ruy M. Ribeiro3, Alan S. Perelson3, Guido Silvestri1,2

1 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, United States of America, 3 Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America, 4 Gilead Sciences, Inc., Foster City, California, United States of America, 5 Tulane National Primate Research Center and Tulane Health Sciences Center, Tulane University, New Orleans, Louisiana, United States of America, 6 AIDS and Cancer Virus Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, Frederick, Maryland, United States of America, 7 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication.

PLoS Pathog 6(1): e1000748. doi:10.1371/journal.ppat.1000748

In Vivo CD8+ T-Cell Suppression of SIV Viremia Is Not Mediated by CTL Clearance of Productively Infected Cells

Joseph K. Wong1,2#*, Matthew C. Strain2, Rodin Porrata3, Elizabeth Reay4, Sumathi Sankaran-Walters4, Caroline C. Ignacio2, Theresa Russell2, Satish K. Pillai1,2, David J. Looney2, Satya Dandekar4#

1 Department of Medicine, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Medicine, VA San Diego Healthcare System, University of California San Diego, La Jolla, California, United States of America, 3 Department of Physics, University of California Berkeley, Berkeley, California, United States of America, 4 Department of Medical Microbiology and Immunology, University of California Davis, Davis, California, United States of America

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

PLoS Pathog 6(1): e1000728. doi:10.1371/journal.ppat.1000728

CD8+ T Cell Control of HIV—A Known Unknown

Miles P. Davenport*, Janka Petravic

Complex Systems in Biology Group, Centre for Vascular Research, University of New South Wales, Sydney, New South Wales, Australia

The Journal of Infectious Diseases 2010;201:720–729

DOI: 10.1086/650492

MAJOR ARTICLE

Viral Inhibition Assay: A CD8 T Cell Neutralization Assay for Use in Clinical Trials of HIV‐1 Vaccine Candidates

Aggeliki Spentzou,1,a Philip Bergin,1,a Dilbinder Gill,1 Hannah Cheeseman,1 Ambreen Ashraf,1 Harry Kaltsidis,1 Michelle Cashin‐Cox,1 Insiyah Anjarwalla,1 Alan Steel,2 Christopher Higgs,2 Anton Pozniak,2 Alicja Piechocka‐Trocha,3 Johnson Wong,4 Omu Anzala,6 Etienne Karita,7 Len Dally,5 Frances Gotch,1 Bruce Walker,3 Jill Gilmour,1 and Peter Hayes1

1Human Immunology Laboratory, International AIDS Vaccine Initiative, Imperial College London, and 2Chelsea and Westminster Hospital, London, United Kingdom; 3Ragon Institute of MGH, MIT and Harvard and 4Massachusetts General Hospital, Boston; 5Emmes Corporation, Rockville, Maryland; 6Kenya AIDS Vaccine Initiative, University of Nairobi, Nairobi, Kenya; 7Projet San Francisco, Kigali, Rwanda

We have characterized an assay measuring CD8 T cell–mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV‐1 isolates. The assay uses relatively simple autologous cell culture and enzyme‐linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell–mediated cross‐clade inhibition of HIV‐1 replication in vitro was demonstrated in antiretroviral therapy–naive HIV‐1–infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV‐1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV‐1 in a HLA‐I–dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.

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.