TAG HIV Basic Science, Vaccines, and Cure Project Blog

More Bad News For Proposed HIV Vaccine Trial

At the Keystone Symposia on HIV Prevention that took place last week, Dan Barouch presented some new and disturbing data from a study exploring DNA and adenovirus vector immunizations in the macaque SIV challenge model. The vaccine regimen used in the study is similar to  - but not exactly the same as - the Vaccine Research Center’s DNA/Ad5 combination, which is to be evaluated for efficacy in the next proposed human HIV vaccine trial, HVTN 505. Barouch’s results have provoked controversy regarding whether HVTN 505 should proceed, not only due to the findings themselves but because they have highlighted the lack of evidence that the VRC’s DNA/Ad5 regimen is superior to Ad5 alone. Ad5 alone, of course, failed to show any efficacy in the recent STEP study.

Barouch’s study involved 30 rhesus macaques, split into five different groups. Animals with MHC genes known to be associated with enhanced control of SIV replication (Mamu A*01, B*08 and B*17) were excluded. The five groups in the study were:

1) DNA prime including Gag, Pol, Nef & Env. Booster immunization with an adenovirus vector called Ad5HVR48 encoding the same antigens. This vector is almost entirely the Ad5 serotype, but with the hexon protein swapped with the same protein from the less common Ad48 serotype.

2) DNA prime including Gag, Pol, Nef & Env, given with DNA adjuvants encoding the chemokine MIP-1 alpha and flt-3 ligand. Booster immunization with Ad5HVR48 encoding the same antigens.

3) Ad5HVR48 encoding Gag, Pol, Nef & Env.

4) Ad5HVR48 encoding Gag, Pol & Nef.

5) Sham (placebo) vaccination.

Six months after the last immunization, all animals were given a high dose intravenous challenge with SIVmac251 (the same virus from which the vaccine antigens were derived, which is called a homologous challenge).

Prior to the challenge, Barouch noted that vaccine-induced T cell responses were five-fold higher in the macaques that received the DNA prime versus those that received Ad5HVR48 alone. However, post-challenge outcomes did not mirror the immunogenicity data. Set point viral loads were lowest in the animals that received Ad5HVR48, averaging 4.4 logs, compared to 5.8 logs in recipients of the DNA prime plus adjuvants (group 2), 5.2 logs in the DNA prime arm (group 1) and 5.5 logs in the placebo arm. In an exploratory analysis comparing the Ad5HVR48 and DNA/ Ad5HVR48 groups, viral loads were significantly higher (by around 0.75 logs) in the latter. In terms of survival, after 500 days of follow up, only 4/12 animals in the DNA groups were alive, compared to 10/12 in the Ad5HVR48 groups. Among the placebo recipients, 1/6 animals survived, comparable with the DNA groups.

Barouch speculated that the results might be explained by the preponderance of Env-specific CD4 T cell responses induced by the DNA prime; animals in these groups showed 4-5 fold higher responses to Gag, Pol & Nef compared to the Ad5HVR48 recipients, but 10-fold higher responses to Env. Barouch cited the possibility that Env-specific CD4 responses might provide additional targets for SIV and thereby enhance viral replication (as was seen in a prior monkey vaccine study that induced only Env-specific CD4 T cell responses using a VZV vector). However, Dr. Barouch took pains to emphasize that the negative impact of DNA priming in his study was only statistically significant in the exploratory combined analysis, and thus the results must be viewed as hypothesis-generating rather than conclusive.

It’s also important to stress that there are differences between the vaccines used in this study and the VRC constructs. The VRC adenovirus vector is entirely Ad5, while Barouch’s has a substituted hexon protein. The VRC DNA vaccine being used in humans consists of six different plasmids, one for each antigen (Gag, Pol, Nef and Envs from clades A, B & C), and splitting the vaccine in this way has been shown to reduce the bias toward Env-specific CD4 T cell responses by enhancing Gag-specific and Nef-specific CD4 T cell responses. However, in terms of the published and presented data on the performance of VRC constructs in the macaque challenge model, it is perhaps surprising to note that there is essentially no evidence supporting the superiority of the DNA/Ad5 approach compared to Ad5 alone. In the most-cited macaque study, published in Science in 2006, the authors write that:

“The vaccination regimens elicited high-frequency SIV-specific cellular immune responses, with greater responses detected in the monkeys that received the DNA prime/rAd boost than those that received only rAd immunizations (Fig. 1). After virus challenge, there were no statistically significant differences in any parameters of clinical outcome between the various groups of experimentally vaccinated monkeys. Therefore, all 24 experimentally vaccinated monkeys were subsequently treated as a single group and compared to the 6 sham-vaccinated control monkeys."

The macaques in this experiment all received the same high-dose intravenous  SIVmac251 challenge that was used in Dan Barouch’s study. The vaccine groups in (six animals in each) were as follows:

1) DNA-SIVmac239 gag/pol/nef prime, rAd5-SIVmac239 gag/pol boost

2) DNA-SIVmac239 env prime, rAd5-SIVmac239 env boost

3) DNA-SIVmac239 gag/pol/nef + DNA-SIVmac239 env prime, rAd-SIVmac239 gag/pol + rAd-SIVmac239 env boost

4) rAd5-SIVmac239  gag/pol + rAd5-SIVmac239 env only

5) sham DNA + empty Ad5

One other challenge study presented at the Keystone meeting by Diane Bolton also included a comparison between the VRC’s DNA/Ad5 and Ad5 alone; while both performed significantly better than placebo in terms of viral load reductions, no significant difference between the vaccine strategies could be discerned.

The implications for the HVTN 505 trial are troubling, to say the least. It appears that there is little – if any – evidence suggesting that the VRC’s DNA/Ad5 approach has a better chance of success than Merck’s Ad5 vaccine. In addition to the safety issues with Ad5 raised by the Merck vaccine results, there is also cause for concern that Env-specific CD4 responses induced by the DNA priming could lead to increased target cells for HIV infection. TAG has previously opposed proceeding with a larger antecedent of HVTN 505 called PAVE100A, and takes the position that HVTN 505 should not go ahead.

Keystone abstracts:

Keystone Symposia: Prevention of HIV/AIDS (X3), Keystone, Colorado, March 22 - 27, 2009. Abstract #017.

Novel Adenovirus Vector-Based Vaccines for HIV-1

Dan H. Barouch

Beth Israel Deaconess Medical Center, Boston, MA, USA

We have recently shown that heterologous rAd prime-boost regimens utilizing two serologically distinct rAd vectors can elicit potent and polyfunctional cellular immune responses and can afford superior protective efficacy as compared with a homologous rAd5 regimen against SIVmac251 challenges in rhesus monkeys.  We will present an update regarding our preclinical and clinical studies involving rare serotype rAd vectors.  We will also describe our efforts to develop novel HIV-1 immunogens aimed at optimizing cellular immune breadth.  Finally, we will address important safety considerations regarding the use of rare serotype rAd vectors in humans.

Keystone Symposia: HIV Immunobiology: From Infection to Immune Control (X4), Keystone, Colorado, March 22 - 27, 2009. Abstract #125.

Aerosol Adenovirus Immunization Controls Early Viremia

Diane L. Bolton(1), Kamei Song(1), Pam Kozlowski(2), Srinivas Rao(3), and Mario Roederer(1).

1) Immunotechnology Section, Vaccine Research Center (VRC), NIAID, NIH, Bethesda, MD 20892; 2) Gene Therapy Program, LSU Health Sciences Center, New Orleans, LA 70012; 3) Laboratory Animal Medicine, VRC.

Objective: Immunization in the lung may prevent diseases caused by airborne pathogens as well as confer immunity at distal mucosal sites, where HIV and most sexually transmitted pathogens initially replicate. We induced strong immune responses in the respiratory tract of rhesus macaques (RM) using fine aerosol droplets of recombinant adenovirus (serotype 5, rAd5) generated by the PARI eFlow nebulizer. Indian-origin RM were immunized twice either intramuscularly (i.m.) or by aerosol with rAd5 encoding SIV gag-pol and env (5x1010 pfu each). Two additional groups were first primed with three doses of gag-pol-nef and env DNA (4mg each), while a sham group received only the empty aerosolized empty rAd5. SIVmac251 challenge was intravenous.

Results: The aerosol route induced much stronger cellular immune responses in the lung (represented by the bronchoalveolar lavage, BAL) than i.m. (p<0.05), with up to 20% of CD4 and 45% of CD8 T lymphocytes specific for the SIV immunogens. By contrast, PBMC T cell responses were greater following i.m. immunization and even undetectable in the aerosol group at the time of challenge (9 weeks post-rAd). Equivalent systemic humoral IgG and IgA responses were elicited to SIV Env, despite much lower anti-vector antibody responses generated by aerosol. Aerosol rAd5 induced mucosal IgA in the BAL and nasal washes at titers greater than in serum, suggesting local (secretory) IgA production. It also primed for an anamnestic CD8 response upon SIV challenge, albeit weaker than systemic vaccination. Strikingly, all immunization groups significantly controlled  acute viremia by 1-2 logs; control was maintained for ~5 months. Lung CD4 T cells were also preserved in immunized animals compared to controls.

Conclusions: Aerosolized adenovirus vaccines generate strong and polyfunctional lung cellular immune responses and persistent systemic and mucosal humoral responses. In the RM SIV model, aerosol immunization affords the same protection against viremia and pathogenicity as i.m. delivery despite the lack of peripheral T cell responses and neutralizing antibodies at the time of challenge. This modality represents a powerful immunization vehicle for respiratory pathogens such as influenza respiratory syncytial virus and tuberculosis, and it may complement vaccine strategies against HIV and other pathogens by inducing excellent mucosal humoral immunity.

IL-7 Clinical Trial Results Published

Coincidentally, results from one of the IL-7 clinical trials mentioned in the prior post have just been published in the Journal of Clinical Investigation. Access to the full text of the paper is free of charge. The study reports that a short course of IL-7 treatment led to sustained increases in naïve and central memory CD4 and CD8 T cell levels out to 48 weeks of follow up. The primary mechanism of the increase appears to be increased proliferation of the cells; the researchers could not discern a contribution of increased thymic output, although they note that this could have been due to technical considerations. Limited data is presented on the functionality of the T cells; future studies will need to address this issue in more detail, perhaps by studying the impact on immune responses to routine immunizations (which was done previously with IL-2, showing no effect).

IL-7 treatment was well tolerated and clearly lacks the severe side effects associated with IL-2 administration. However, one individual in the higher dose group with a history of liver disease developed a grade 3 elevation in liver enzymes, necessitating cessation of treatment. Four out of seven individuals in the higher dose group also experienced transient viral load “blips,” which may relate to the reported ability of IL-7 to increase HIV replication in vitro. The number of infected cells – as assessed by measuring HIV proviral DNA – did not change as a result of IL-7 administration. Immune activation levels were also stable out to 12 weeks of follow up (48 week data is not reported). The researchers did not evaluate whether the CD4 T cell count changes induced by IL-7 had any impact on microbial translocation.

Overall, the data offer support for further evaluation of IL-7, particularly in individuals with low CD4 T cell counts despite viral suppression.

J Clin Invest. 2009 Mar 16. pii: 38052. doi: 10.1172/JCI38052. [Epub ahead of print]

Enhanced T cell recovery in HIV-1-infected adults through IL-7 treatment.

Levy Y, Lacabaratz C, Weiss L, Viard JP, Goujard C, Lelièvre JD, Boué F, Molina JM, Rouzioux C, Avettand-Fénoêl V, Croughs T, Beq S, Thiébaut R, Chêne G, Morre M, Delfraissy JF.

HIV infection results in CD4+ T cell deficiency, but efficient combination antiretroviral therapy (c-ART) restores T cells and decreases morbidity and mortality. However, immune restoration by c-ART remains variable, and prolonged T cell deficiency remains in a substantial proportion of patients. In a prospective open-label phase I/IIa trial, we evaluated the safety and efficacy of administration of the T cell regulator IL-7. The trial included 13 c-ART-treated HIV-infected patients whose CD4+ cell counts were between 100 and 400 cells/mul and plasma HIV RNA levels were less than 50 copies/ml. Patients received a total of 8 subcutaneous injections of 2 different doses of recombinant human IL-7 (rhIL-7; 3 or 10 mug/kg) 3 times per week over a 16-day period. rhIL-7 was well tolerated and induced a sustained increase of naive and central memory CD4+ and CD8+ T cells. In the highest dose group, 4 patients experienced transient increases in viral replication. However, functional assays showed that the expanded T cells responded to HIV antigen by producing IFN-gamma and/or IL-2. In conclusion, in lymphopenic HIV-infected patients, rhIL-7 therapy induced substantial functional and quantitative changes in T cells for 48 weeks. Therefore, patients may benefit from intermittent therapy with IL-7 in combination with c-ART.

IL-7 Trial Begins Recruiting

After a prolonged planning phase, the phase I/IIa trial of the immune-based therapy interleukin-7 (IL-7) has begun enrolling at multiple sites in the US, Canada and France (see the updated clinicaltrials.gov entry for details, including inclusion/exclusion criteria). IL-7 is a cytokine (immune system messenger protein) that plays a key role in maintaining naive and memory T cell numbers. The study is evaluating a new form of IL-7 designed to allow less frequent (once-a-week) dosing. Two prior phase I trials of IL-7 have been completed in people with HIV, results were presented at the CROI conference in 2007 and the presentations can be viewed via webcast. A follow-up poster from one of the trials was also presented at CROI in 2008. Overall, the results indicated that IL-7 was safe - in the relatively short-term - and led to significant increases in both naive and memory CD4 T cell numbers. The 2008 poster also reported some unexpected evidence of improved functionality of HIV-specific CD4 T cell responses. Because the new trial is primarily evaluating safety, people with low CD4 T cells despite viral suppression - the population most in need of immunological interventions - are not being included. 

Recent results from two large trials of a different cytokine, IL-2, showed that the CD4 T cell count increases produced by the approach did not confer benefit in terms of reducing the incidence of illness compared to people taking only antiretroviral therapy (ART). IL-7 works by a different mechanism than IL-2, but it remains unknown if CD4 T cell increased prompted by IL-7 treatment can benefit health. If the new form of IL-7 increases CD4 T cell counts to the same extent as seen in the prior phase I trials, and is found to be safe, additional studies will be required to evaluate the clinical impact of the approach. 

Viral Resistance to a Vaginal Microbicide in Macaques

Several years ago it was shown that very high doses of an entry inhibitor-based microbicide could protect macaques from infection with SHIV162-p3. A paper in J Virology now raises some important caveats about this approach, including the first reported example of a microbicide selecting for drug-resistant virus. The microbicide in question is a CCR5 inhibitor called PSC-RANTES. The challenge virus used in the studies is called SHIV162-p3, a lab-created SIV/HIV hybrid in which an HIV envelope that uses CCR5 to gain entry into CD4 T cells is inserted into an SIV genome instead of the SIV envelope.

The new paper, authored by Dawn Dudley and colleagues from Case Western Reserve University and the Tulane Primate Research Center, is based on an analysis of macaques given lower doses of PSC-RANTES that were not protective against SHIV162-p3 infection. In most cases, viruses in these animals showed mutations that were deemed unrelated to PSC-RANTES because similar mutations were seen in controls that were challenged with the same virus in the absence of the microbicide. But the virus in one macaque contained two mutations, K315R in gp120 and N640D in gp41, which were very rare in the challenge virus stock (after infection, compared to their frequency in the challenge virus, these mutations were increased at least 25-fold and 75-fold). Importantly, PSC-RANTES failed to completely inhibit this mutant SHIV, even at high concentrations that inhibited the parent SHIV162-p3 and all other R5-using HIV isolates tested.

Dudley and colleagues acknowledge that this apparent example of resistance was only seen in 1/25 macaques studied, and there is an outlying possibility that the mutations represent a random adaptation of the virus to better replicate in macaque cells. However, they argue that the weight of evidence supports their conclusion that the mutations arose from drug selection pressure, and that this possibility is particularly concerning given that SHIV162-p3 is a very homogenous virus stock from which the emergence of resistance would have been considered unlikely.

Discussing the implications for microbicides generally, they note that the positive results reported to date with entry inhibitor-based microbicides (PSC-RANTES, BMS-378806, CMPD167, and C52L) have all involved administering relatively high doses and the SHIV162-p3 challenge virus (which “is exquisitely sensitive to many entry inhibitors”).  As a result, they believe that “the barrier for selecting resistance to an anti-HIV microbicide (such as PSC-RANTES) has been set very high and possibly beyond physiological relevance.” To address these concerns, the authors recommend the development of macaque models that use multiple R5-SHIVs as a challenge in order to better duplicate the diversity of viruses to which individuals are typically exposed. Because the resistance described in their study was associated with a dose of PSC-RANTES lower than that which conferred full protection, they also caution that waning microbicide levels after application could conceivably provide the conditions necessary for the emergence of drug resistance. 

JVI Accepts, published online ahead of print on 11 March 2009

J. Virol. doi:10.1128/JVI.00055-09

Selection of SHIV resistant to a vaginal microbicide in macaques

Dawn M. Dudley, Jennifer L. Wentzel, Matthew S. Lalonde, Ronald S. Veazey, and Eric J. Arts

Department of Molecular Biology and Microbiology, Division of Infectious Diseases, Department of Medicine, Department of Biochemistry, Case Western Reserve University, Cleveland, OH, 44106; Department of Pathology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, LA

Abstract

PSC-RANTES binds to CCR5, inhibits HIV-1 entry, and has been shown to protect rhesus macaques as a vaginal microbicide from SHIVSF162-p3 infection in a dose-dependent manner. In this study, env gene sequences from SHIVSF162-p3-infected rhesus macaques treated with PSC-RANTES were analyzed for possible drug escape variants. Two specific mutations located in the V3 region of gp120 (K315R) and C-helical domain of gp41 (N640D) were identified in a macaque (m584) pre-treated with a 100 µM dose of PSC-RANTES. These two env mutations were found throughout infection (through week 77) but were only found at low frequencies in the inoculating SHIVSF162-p3 stock and in the other SHIVSF162-p3-infected macaques. HIV-1 env genes from the m584 macaque and from inoculating SHIVSF162-p3 were cloned into an HIV-1 backbone. Increases in IC50 values to PSC-RANTES with envm584 was modest (7-fold) and most pronounced in cells expressing rhesus macaque as compared to human CCR5. Nonetheless, virus harboring the envm584, unlike inoculating envp3, could replicate even at the highest tissue culture PSC-RANTES concentrations (100 nM). Dual virus competitions revealed a dramatic increase in fitness of chimeric virus containing the envm584 (K315R/N640D) over that containing the envp3, but again, only in rhesus CCR5-expressing cells. This study is the first to describe the immediate selection and infection of a drug-resistant SHIV variant in the face of a protective vaginal microbicide, PSC-RANTES. This rhesus CCR5-specific/PSC-RANTES-resistance selection is particularly alarming given the relative homogeneity of the SHIVSF162-p3 stock as compared to the potential exposure to a heterogeneous HIV-1 population in human transmission.

Regulation Gone Awry? Elevated Levels of Regulatory T Cells Linked To Disease Progression

Over the past decade it has become accepted that some T cells play a role in dampening down immune responses. These cells are dubbed regulatory T cells or Treg for short. In lab studies, Treg have been shown to suppress the activation of other T cells, and there is considerable evidence that this regulatory function is also performed in vivo. One problem, however, is that there are no definitive markers for Tregs. Initially, expression of a molecule called CD25 was used to define Treg, but CD25 can also be upregulated by other activated T cells. More recently, a transcription factor, Foxp3, has emerged as a more specific marker for Treg. Although several studies have explored the role of Treg in HIV infection, results have been inconsistent and hard to interpret because approaches to define the cells have varied. A new study in the journal AIDS Research & Human Retroviruses uses the combination of CD25 and Foxp3 to explore whether Treg levels differ in individuals with varying rates of disease progression.

Weiwei Cao and colleagues from UCLA employed a case-control approach to compare twenty fast progressors from the Multicenter AIDS Cohort Study (MACS) to forty matched slow progressors and nine uninfected individuals. Fast progression was defined as an AIDS diagnosis within 4 years after enrollment into MACS, whereas slow progressors were categorized based on the absence for an AIDS diagnosis for at least 8 years after entry into the cohort. Treg constituted 4.6% of CD4 T cells in uninfected study participants and this proportion was only slightly increased to 5.6% in slow progressors. Among fast progressors, however, the proportion of Treg was significantly higher at 11.3%.  Further analysis showed that Treg levels correlated with CD4 T cell activation; the proportion of CD4 T cells expressing the activation marker CD38 was 1.5% in uninfected individuals, 7.2% in slow progressors and 16.3% in fast progressors.

In the discussion section of the paper, Cao and colleagues suggest that these two phenomena may be linked, because other studies have shown that T cell activation can lead to the generation of Foxp3-expressing Treg from resting T cells that previously did not express the marker. Therefore it is possible that the persistently elevated levels of T cell activation in HIV infection leads to the observed accumulation of Tregs. Some researchers have suggested that immune activation in HIV infection is caused by a lack of Treg, but Cao and colleagues note that their data appears incompatible with this possibility. Rather, their data argues that Treg either have a negative effect – perhaps by interfering with HIV-specific immune responses, as some prior studies have posited – or alternatively the relative expansion of Tregs is just a by-product of immune activation which has no specific role in HIV disease progression.

AIDS Research and Human Retroviruses. February 2009, 25(2): 183-191. doi:10.1089/aid.2008.0140.

Regulatory T Cell Expansion and Immune Activation during Untreated HIV Type 1 Infection Are Associated with Disease Progression (free access to full text PDF)

Weiwei Cao,1 Beth D. Jamieson,2 Lance E. Hultin,1 Patricia M. Hultin,1 and Roger Detels1

1Department of Epidemiology, School of Public Health, University of California, Los Angeles, California 90095.

2Department of Medicine, Division of Hematology and Oncology, David Geffen School of Medicine, University of California, Los Angeles, California 90095.

Abstract

Regulatory T cells (Tregs) may play an important role in the immunopathology of chronic HIV-1 infection due to their potent suppressive activity of both T cell activation and effector function. To investigate the correlation between Tregs and immune activation during untreated chronic HIV-1 infection, we conducted a nested case–control study within the Multicenter AIDS Cohort Study (MACS). Twenty HIV-1-infected fast progressors (FP) and 40 slow progressors (SP) were included in our study using risk-set sampling. Nine age-matched HIV-1-uninfected men (UI) were also included. Cryopreserved peripheral blood mononuclear cells (PMBCs) were tested using flow cytometry analyses. We identified Tregs as Foxp3+CD25+CD4+ T cells and assessed the activation of CD4+ and CD8+ T cells by the expression of CD38, HLADR, or both markers simultaneously. There is a relative expansion of Tregs during HIV-1 infection, which is associated with disease progression. The increased CD38 expression on both CD4+ and CD8+ T cells expressed as either percentage or median fluorescence intensity (MFI) and the elevated proportion of CD8+ T cells that is HLADR+CD38+ were all associated with rapid HIV-1 progression. Counter to the assumed role of Tregs as the suppressors of activation, the expansion of Tregs was positively correlated with CD4+ T cell activation among HIV-1-infected fast progressors. The high level of Tregs associated with rapid HIV progression may suggest a detrimental role of these cells in the immune control of HIV-1 infection.

Gut Bacteria Breach the Barrier: Further Confirmation of Microbial Translocation in HIV Infection

Last October, Guila Marchetti and colleagues reported that DNA from gut bacteria can be detected in the bloodstream of people with HIV, and that elevated bacterial DNA levels correlate with poor immune reconstitution in people on antiretroviral therapy (ART). The data were concordant with the hypothesis proposed by Daniel Douek, David Price and Jason Brenchley that leaking of gut bacteria into systemic circulation – a phenomenon called microbial translocation - contributes to HIV pathogenesis. The Marchetti study was small, however, involving just 47 participants. A paper just published online in the Journal of Infectious Diseases confirms and significantly extends these findings using several larger cohorts of HIV-infected individuals.

The researchers - led by Wei Jiang and Michael Lederman from Case Western Reserve University - first compared levels of bacterial DNA in a group of uninfected individuals to those in people with untreated HIV infection, or treated HIV infection. Study participants on ART were further subdivided into two groups based on whether their HIV viral load was above or below the limit of detection (400 copies/mL). None of the 15 HIV-negative individuals showed detectable levels of bacterial DNA (the lowest level the test used in the study can reliably detect is 5 DNA copies). In contrast, bacterial DNA was found in 18 out of 19 untreated individuals with HIV infection, with median levels of 132.5 copies per microliter. Study participants on ART fell between these two extremes: the median number of bacterial DNA copies was 8.6 among individuals with undetectable viral loads and 22.8 for those with viral loads above the detection limit (this difference was not statistically significant). The researchers also found that there was a significant association between viral load and bacterial DNA levels in the untreated individuals, but there was no such association with total CD4 counts in peripheral blood (naïve and memory CD4 T cells were not analyzed separately).

Next, a separate cohort of 114 individuals on long-term ART were studied, in order to gain further insight into the impact of microbial translocation on the response to treatment. Higher levels of bacterial DNA were associated with lower CD4 count gains on ART; for every 100-copy increase in bacterial DNA levels, 11 fewer CD4 T cells were gained (although this could also be looked at the other way around, i.e. larger increases in CD4 T cells were associated with lower levels of bacterial DNA). Bacterial DNA levels also correlated with levels of lipopolysaccharide (LPS), another indicator of microbial translocation, and – more weakly - with CD8 T cell activation.

Finally, the researchers used samples from a longitudinal, 54-person study of ART treatment (ACTG 5014) to evaluate changes in bacterial DNA levels over time. Samples were taken before and at 1, 8, and 48 weeks after starting ART. Levels of bacterial DNA declined progressively, and were significantly lower than baseline at the week 8 and 48 timepoints. These changes were independent of changes in HIV viral load, but correlated inversely with CD4 counts after 48 weeks of treatment (the higher the CD4 count, the lower the bacterial DNA level). The study authors note that median bacterial DNA levels were still higher at week 48 (75 copies per microliter) than had been seen in the first cohort analyzed (8.6 copies). Participants in the first cohort had been on ART for a median of over 6 years, suggesting that bacterial DNA levels decline slowly over a protracted period after starting treatment.

In discussing their data, the authors suggest that microbial translocation in HIV infection is driven by viral replication, either via direct effects on mucosal cells that would normally maintain the integrity of the gut wall, or by indirect effects on immune responses that help contain commensal bacteria within the GI tract. Unlike Marchetti and colleagues, these researchers do not cite the basic immunology literature indicating that T cell depletion can contribute to microbial translocation, although they do consider the possibility that in some cases poor CD4 T cell reconstitution might be a cause as well as a consequence of microbial translocation. In arguing against a primary causative role of CD4 T cell depletion, they note the lack of a correlation between CD4 T cell counts and bacterial DNA levels in untreated individuals, and also cite the fact that microbial translocation occurs in conditions such as inflammatory bowel disease that aren’t associated with loss of CD4 T cells.

While this type of research can seem dauntingly complex, it’s worth emphasizing that understanding and addressing the causes of poor CD4 T cell reconstitution on ART is critical for improving the care of individuals with HIV infection.  As the authors of this paper point out in their introduction, “persistently low CD4 T cell counts are associated with significant increases in non–AIDS-associated morbidities, including cardiovascular disease, liver disease, cancer, and, perhaps, renal disease.” Consequently, there is an urgent need to develop and study therapies that might have the potential to improve CD4 T cell recovery in individuals who remain at risk for clinical illness despite ART-mediated viral suppression. The recent failure of interleukin-2 to provide benefit should not discourage development of approaches with different mechanisms of action, such as enhancement of T cell production by the thymus. Other possible avenues of intervention may include attempting to restore the integrity of the gut wall and/or evaluating anti-inflammatory approaches that aim to reduce the immune activation caused by microbial translocation.

The Journal of Infectious Diseases 2009;199:000–000

MAJOR ARTICLE

Plasma Levels of Bacterial DNA Correlate with Immune Activation and the Magnitude of Immune Restoration in Persons with Antiretroviral‐Treated HIV Infection

Wei Jiang,1 Michael M. Lederman,1 Peter Hunt,5 Scott F. Sieg,1 Kathryn Haley,1 Benigno Rodriguez,1 Alan Landay,2 Jeffrey Martin,5 Elizabeth Sinclair,5 Ava I. Asher,3 Steven G. Deeks,5 Daniel C. Douek,3 and Jason M. Brenchley4

1Case Western Reserve University, Cleveland, Ohio; 2Rush Medical School, Chicago, Illinois; 3Human Immunology Section, Vaccine Research Center, and 4Immunopathogenesis Unit, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; 5University of San Francisco, San Francisco General Hospital, San Francisco, California

The significance of elevated plasma levels of bacterial lipopolysaccharide (LPS) in persons with chronic HIV infection remains undefined. We measured LPS levels by use of limulus lysate assay, and DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) were assessed by quantitative polymerase chain reactions in plasma samples obtained from 242 donors. Plasma levels of 16S rDNA were significantly higher in human immunodeficiency virus (HIV)–infected subjects than in uninfected subjects, and they correlated with LPS levels. Higher levels of 16S rDNA were associated with higher levels of T cell activation and with lower levels of CD4 T cell restoration during antiretroviral therapy. Antiretroviral therapy reduces but does not fully normalize plasma levels of bacterial 16S rDNA, an index of microbial translocation from the gastrointestinal tract. High levels of 16S rDNA during therapy are strongly associated with reduced increases in the CD4+ T lymphocyte count, irrespective of plasma HIV RNA levels. These findings are consistent with the importance of microbial translocation in immunodeficiency and T cell homeostasis in chronic HIV infection.