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1.  Clinicopathologic correlates of hepatitis C virus in brain: A pilot study 
Journal of neurovirology  2008;14(1):17-27.
Hepatitis C virus (HCV) has been detected in the brain tissues of 10 individuals reported to date; it is unclear what clinical factors are associated with this, and with what frequency it occurs. Accordingly, a pilot analysis utilizing reverse transcriptase–polymerase chain reaction (RT- PCR) to detect and sequence HCV in premortem plasma and postmortem brain and liver from 20 human immunodeficiency virus (HIV)-infected and 10 HIV-naïve individuals was undertaken. RNA encoding the first 126 amino acids of the HCV E1 envelope protein and the majority of the E1 signal sequence was analyzed in parallel with an 80-base-long segment of the 5′ untranslated region (UTR). Liver HCV was detected only in subjects with premortem HCV viremia (10 HIV-infected and 3 HIV-naïve). Brain HCV was detected in 6/10 HCV/HIV-coinfected and 1/3 HCV-monoinfected subjects. In the setting of HIV, the magnitude of plasma HCV load did not correlate with the presence of brain HCV. However, coinfected patients with brain HCV were more often off antiretroviral therapy and tended to have higher plasma HIV loads than those with HCV restricted to liver. Furthermore, premortem cerebrospinal fluid (CSF) analysis revealed that HCV/HIV-coinfected patients with brain HCV had detectable CSF HIV, whereas those without brain HCV had undetectable CSF HIV loads (P = .0205). Neuropsychologic tests showed a trend for hierarchical impairment of abstraction/executive functioning in HIV/HCV coinfection, with mean T scores for HIV monoinfected patients 43.2 (7.3), for liver-only HCV 39.5 (9.0), and for those with HCV in brain and liver 33.2 (5.1) (P = .0927). Predominant brain HCV sequences did not match those of the plasma or liver in 4 of the 6 coinfected patients analyzed. We conclude that in the setting of HIV/HCV coinfection, brain HCV is a common phenomenon unrelated to the magnitude of HCV viremia, but related to active HIV disease and detectable CSF HIV. Furthermore, there is sequence evidence of brain compartmentalization. Differences in abstraction/executive function of HCV/HIV coinfected patients compared to HIV monoinfected warrant further studies to determine if neuropsychiatric effects are predicated upon brain infection.
doi:10.1080/13550280701708427
PMCID: PMC2729451  PMID: 18300072
brain; cognition; hepatitis C virus; HIV
2.  Kidney and liver organ transplantation in persons with human immunodeficiency virus 
Executive Summary
Objective
The objective of this analysis is to determine the effectiveness of solid organ transplantation in persons with end stage organ failure (ESOF) and human immunodeficiency virus (HIV+)
Clinical Need: Condition and Target Population
Patients with end stage organ failure who have been unresponsive to other forms of treatment eventually require solid organ transplantation. Similar to persons who are HIV negative (HIV−), persons living with HIV infection (HIV+) are at risk for ESOF from viral (e.g. hepatitis B and C) and non-viral aetiologies (e.g. coronary artery disease, diabetes, hepatocellular carcinoma). Additionally, HIV+ persons also incur risks of ESOF from HIV-associated nephropathy (HIVAN), accelerated liver damage from hepatitis C virus (HCV+), with which an estimated 30% of HIV positive (HIV+) persons are co-infected, and coronary artery disease secondary to antiretroviral therapy. Concerns that the need for post transplant immunosuppression and/or the interaction of immunosuppressive drugs with antiretroviral agents may accelerate the progression of HIV disease, as well as the risk of opportunistic infections post transplantation, have led to uncertainty regarding the overall benefit of transplantation among HIV+ patients. Moreover, the scarcity of donor organs and their use in a population where the clinical benefit of transplantation is uncertain has limited the availability of organ transplantation to persons living with ESOF and HIV.
With the development of highly active anti retroviral therapy (HAART), which has been available in Canada since 1997, there has been improved survival and health-related quality of life for persons living with HIV. HAART can suppress HIV replication, enhance immune function, and slow disease progression. HAART managed persons can now be expected to live longer than those in the pre-HAART era and as a result many will now experience ESOF well before they experience life-threatening conditions related to HIV infection. Given their improved prognosis and the burden of illness they may experience from ESOF, the benefit of solid organ transplantation for HIV+ patients needs to be reassessed.
Evidence-Based Analysis Methods
Research Questions
What are the effectiveness and cost effectiveness of solid organ transplantation in HIV+ persons with ESOF?
Literature Search
A literature search was performed on September 22, 2009 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 1, 1996 to September 22, 2009.
Inclusion Criteria
Systematic review with or without a Meta analysis, RCT, Non-RCT with controls
HIV+ population undergoing solid organ transplantation
HIV+ population managed with HAART therapy
Controls include persons undergoing solid organ transplantation who are i) HIV− ii) HCV+ mono-infected, and iii) HIV+ persons with ESOF not transplanted.
Studies that completed and reported results of a Kaplan-Meier Survival Curve analysis.
Studies with a minimum (mean or medium) follow up of 1-year.
English language citations
Exclusion Criteria
Case reports and case series were excluded form this review.
Outcomes of Interest
i) Risk of Death after transplantation
ii) Death censored graft survival (DCGS)
iii) HIV disease progression defined as the post transplant incidence of:
- opportunistic infections or neoplasms,
- CD4+ T-cell count < 200mm3, and
- any detectable level of plasma HIV viral load.
iv) Acute graft rejection,
v) Return to dialysis,
vi) Recurrence of HCV infection
Summary of Findings
No direct evidence comparing an HIV+ cohort undergoing transplantation with the same not undergoing transplantation (wait list) was found in the literature search.
The results of this review are reported for the following comparison cohorts undergoing transplantation:
i) Kidney Transplantation: HIV+ cohort compared with HIV− cohort
ii) Liver Transplantation: HIV+ cohort compared with HIV− negative cohort
iii) Liver Transplantation: HIV+ HCV+ (co-infected) cohort compared with HCV+ (mono-infected) cohort
Kidney Transplantation: HIV+ vs. HIV−
Based on a pooled HIV+ cohort sample size of 285 patients across four studies, the risk of death after kidney transplantation in an HIV+ cohort does not differ to that of an HIV− cohort [hazard ratio (HR): 0.90; 95% CI: 0.36, 2.23]. The quality of evidence supporting this outcome is very low.
Death censored graft survival was reported in one study with an HIV+ cohort sample size of 100, and was statistically significantly different (p=.03) to that in the HIV− cohort (n=36,492). However, the quality of evidence supporting this outcome was determined to be very low. There was also uncertainty in the rate of return to dialysis after kidney transplantation in both the HIV+ and HIV− groups and the effect, if any, this may have on patient survival. Because of the very low quality evidence rating, the effect of kidney transplantation on HIV-disease progression is uncertain.
The rate of acute graft rejection was determined using the data from one study. There was a nonsignificant difference between the HIV+ and HIV− cohorts (OR 0.13; 95% CI: 0.01, 2.64), although again, because of very low quality evidence there is uncertainty in this estimate of effect.
Liver Transplantation: HIV+ vs. HIV−
Based on a combined HIV+ cohort sample size of 198 patient across five studies, the risk of death after liver transplantation in an HIV+ cohort (with at least 50% of the cohort co-infected with HCV+) is statistically significantly 64% greater compared with an HIV− cohort (HR: 1.64; 95% CI: 1.32, 2.02). The quality of evidence supporting this outcome is very low.
Death censored graft survival was reported for an HIV+ cohort in one study (n=11) however the DCGS rate of the contemporaneous control HIV− cohort was not reported. Because of sparse data the quality of evidence supporting this outcome is very low indicating death censored graft survival is uncertain.
Both the CD4+ T-cell count and HIV viral load appear controlled post transplant with an incidence of opportunistic infection of 20.5%. However, the quality of this evidence for these outcomes is very low indicating uncertainty in these effects. Similarly, because of very low quality evidence there is uncertainty in the rate of acute graft rejection among both the HIV+ and HIV− groups
Liver Transplantation: HIV+/HCV+ vs. HCV+
Based on a combined HIV+/HCV+ cohort sample size of 156 from seven studies, the risk of death after liver transplantation is significantly greater (2.8 fold) in a co-infected cohort compared with an HCV+ mono-infected cohort (HR: 2.81; 95% CI: 1.47, 5.37). The quality of evidence supporting this outcome is very low. Death censored graft survival evidence was not available.
Regarding disease progression, based on a combined sample size of 71 persons in the co-infected cohort, the CD4+ T-cell count and HIV viral load appear controlled post transplant; however, again the quality of evidence supporting this outcome is very low. The rate of opportunistic infection in the co-infected cohort was 7.2%. The quality of evidence supporting this estimate is very low, indicating uncertainty in these estimates of effect.
Based on a combined HIV+/HCV+ cohort (n=57) the rate of acute graft rejection does not differ to that of an HCV+ mono-infected cohort (OR: 0.88; 95% CI: 0.44, 1.76). Also based on a combined HIV+/HCV+ cohort (n=83), the rate of HCV+ recurrence does not differ to that of an HCV+ mono-infected cohort (OR: 0.66; 95% CI: 0.27, 1.59). In both cases, the quality of the supporting evidence was very low.
Overall, because of very low quality evidence there is uncertainty in the effect of kidney or liver transplantation in HIV+ persons with end stage organ failure compared with those not infected with HIV. Examining the economics of this issue, the cost of kidney and liver transplants in an HIV+ patient population are, on average, 56K and 147K per case, based on both Canadian and American experiences.
PMCID: PMC3377507  PMID: 23074407
3.  Supervised and Unsupervised Self-Testing for HIV in High- and Low-Risk Populations: A Systematic Review 
PLoS Medicine  2013;10(4):e1001414.
By systematically reviewing the literature, Nitika Pant Pai and colleagues assess the evidence base for HIV self tests both with and without supervision.
Background
Stigma, discrimination, lack of privacy, and long waiting times partly explain why six out of ten individuals living with HIV do not access facility-based testing. By circumventing these barriers, self-testing offers potential for more people to know their sero-status. Recent approval of an in-home HIV self test in the US has sparked self-testing initiatives, yet data on acceptability, feasibility, and linkages to care are limited. We systematically reviewed evidence on supervised (self-testing and counselling aided by a health care professional) and unsupervised (performed by self-tester with access to phone/internet counselling) self-testing strategies.
Methods and Findings
Seven databases (Medline [via PubMed], Biosis, PsycINFO, Cinahl, African Medicus, LILACS, and EMBASE) and conference abstracts of six major HIV/sexually transmitted infections conferences were searched from 1st January 2000–30th October 2012. 1,221 citations were identified and 21 studies included for review. Seven studies evaluated an unsupervised strategy and 14 evaluated a supervised strategy. For both strategies, data on acceptability (range: 74%–96%), preference (range: 61%–91%), and partner self-testing (range: 80%–97%) were high. A high specificity (range: 99.8%–100%) was observed for both strategies, while a lower sensitivity was reported in the unsupervised (range: 92.9%–100%; one study) versus supervised (range: 97.4%–97.9%; three studies) strategy. Regarding feasibility of linkage to counselling and care, 96% (n = 102/106) of individuals testing positive for HIV stated they would seek post-test counselling (unsupervised strategy, one study). No extreme adverse events were noted. The majority of data (n = 11,019/12,402 individuals, 89%) were from high-income settings and 71% (n = 15/21) of studies were cross-sectional in design, thus limiting our analysis.
Conclusions
Both supervised and unsupervised testing strategies were highly acceptable, preferred, and more likely to result in partner self-testing. However, no studies evaluated post-test linkage with counselling and treatment outcomes and reporting quality was poor. Thus, controlled trials of high quality from diverse settings are warranted to confirm and extend these findings.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
About 34 million people (most living in resource-limited countries) are currently infected with HIV, the virus that causes AIDS, and about 2.5 million people become infected with HIV every year. HIV is usually transmitted through unprotected sex with an infected partner. HIV infection is usually diagnosed by looking for antibodies to HIV in blood or saliva. Early during infection, the immune system responds to HIV by beginning to make antibodies that recognize the virus and target it for destruction. “Seroconversion”—the presence of detectable amounts of antibody in the blood or saliva—usually takes 6–12 weeks. Rapid antibody-based tests, which do not require laboratory facilities, can provide a preliminary result about an individual's HIV status from a simple oral swab or finger stick sample within 20 minutes. However preliminary rapid positive results have to be confirmed in a laboratory, which may take a few days or weeks. If positive, HIV infection can be controlled but not cured by taking a daily cocktail of powerful antiretroviral drugs throughout life.
Why Was This Study Done?
To reduce the spread of HIV, it is essential that HIV-positive individuals get tested, change behaviors avoid transmitting the virus to other people by, for example, always using a condom during sex, and if positive get on to treatment that is available worldwide. Treatment also reduces transmission of virus to the partner and controls the virus in the community. However, only half the people currently living with HIV know their HIV status, a state of affairs that increases the possibility of further HIV transmission to their partners and children. HIV positive individuals are diagnosed late with advanced HIV infection that costs health care services. Although health care facility-based HIV testing has been available for decades, people worry about stigma, visibility, and social discrimination. They also dislike the lack of privacy and do not like having to wait for their test results. Self-testing (i.e., self-test conduct and interpretation) might alleviate some of these barriers to testing by allowing individuals to determine their HIV status in the privacy of their home and could, therefore, increase the number of individuals aware of their HIV status. This could possibly reduce transmission and, through seeking linkages to care, bring HIV under control in communities. In some communities and countries, stigma of HIV prevents people from taking action about their HIV status. Indeed, an oral (saliva-based) HIV self-test kit is now available in the US. But how acceptable, feasible, and accurate is self-testing by lay people, and will people who find themselves self-test positive seek counseling and treatment? In this systematic review (a study that uses pre-defined criteria to identify all the research on a given topic), the researchers examine these issues by analyzing data from studies that have evaluated supervised self-testing (self-testing and counseling aided by a health-care professional) and unsupervised self-testing (self-testing performed without any help but with counseling available by phone or internet).
What Did the Researchers Do and Find?
The researchers identified 21 eligible studies, two-thirds of which evaluated oral self-testing and a third of which evaluated blood-based self-testing. Seven studies evaluated an unsupervised self-testing strategy and 14 evaluated a supervised strategy. Most of the data (89%) came from studies undertaken in high-income settings. The study populations varied from those at high risk of HIV infection to low-risk general populations. Across the studies, acceptability (defined as the number of people who actually self-tested divided by the number who consented to self-test) ranged from 74% to 96%. With both strategies, the specificity of self-testing (the chance of an HIV-negative person receiving a negative test result is true negative) was high but the sensitivity of self-testing (the chance of an HIV-positive person receiving a positive test result is indeed a true positive) was higher for supervised than for unsupervised testing. The researchers also found evidence that people preferred self-testing to facility-based testing and oral self-testing to blood-based self testing and, in one study, 96% of participants who self-tested positive sought post-testing counseling.
What Do These Findings Mean?
These findings provide new but limited information about the feasibility, acceptability, and accuracy of HIV self-testing. They suggest that it is feasible to implement both supervised and unsupervised self-testing, that both strategies are preferred to facility-based testing, but that the accuracy of self-testing is variable. However, most of the evidence considered by the researchers came from high-income countries and from observational studies of varying quality, and data on whether people self-testing positive sought post-testing counseling (linkage to care) were only available from one evaluation of unsupervised self-testing in the US. Consequently, although these findings suggest that self-testing could engage individuals in finding our their HIV status and thereby help modify behavior thus, reduce HIV transmission in the community, by increasing the proportion of people living with HIV who know their HIV status. The researchers suggested that more data from diverse settings and preferably from controlled randomized trials must be collected before any initiatives for global scale-up of self-testing for HIV infection are implemented.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001414.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including information on HIV testing, and on HIV transmission and testing (in English and Spanish)
The UK National Health Service Choices website provides information about all aspects of HIV and AIDS; a “behind the headlines” article provides details about the 2012 US approval for an over-the-counter HIV home-use test
The 2012 World AIDS Day Report provides information about the percentage of people living with HIV who are aware of their HIV status in various African countries, as well as up-to-date information about the AIDS epidemic
Patient stories about living with HIV/AIDS are available through Avert; the nonprofit website Healthtalkonline also provides personal stories about living with HIV, including stories about getting a diagnosis
doi:10.1371/journal.pmed.1001414
PMCID: PMC3614510  PMID: 23565066
4.  Evolution of Envelope Sequences from the Genital Tract and Peripheral Blood of Women Infected with Clade A Human Immunodeficiency Virus Type 1 
Journal of Virology  1998;72(10):8240-8251.
The development of viral diversity during the course of human immunodeficiency virus type 1 (HIV-1) infection may significantly influence viral pathogenesis. The paradigm for HIV-1 evolution is based primarily on studies of male cohorts in which individuals were presumably infected with a single virus variant of subtype B HIV-1. In this study, we evaluated virus evolution based on sequence information of the V1, V2, and V3 portions of HIV-1 clade A envelope genes obtained from peripheral blood and cervical secretions of three women with genetically heterogeneous viral populations near seroconversion. At the first sample following seroconversion, the number of nonsynonymous substitutions per potential nonsynonymous site (dn) significantly exceeded substitutions at potential synonymous sites (ds) in plasma viral sequences from all individuals. Generally, values of dn remained higher than values of ds as sequences from blood or mucosa evolved. Mutations affected each of the three variable regions of the envelope gene differently; insertions and deletions dominated changes in V1, substitutions involving charged amino acids occurred in V2, and sequential replacement of amino acids over time at a small subset of positions distinguished V3. The relationship among envelope nucleotide sequences obtained from peripheral blood mononuclear cells, plasma, and cervical secretions was evaluated for each individual by both phylogenetic and phenetic analyses. In all subjects, sequences from within each tissue compartment were more closely related to each other than to sequences from other tissues (phylogenetic tissue compartmentalization). At time points after seroconversion in two individuals, there was also greater genetic identity among sequences from the same tissue compartment than among sequences from different tissue compartments (phenetic tissue compartmentalization). Over time, temporal phylogenetic and phenetic structure was detectable in mucosal and plasma viral samples from all three women, suggesting a continual process of migration of one or a few infected cells into each compartment followed by localized expansion and evolution of that population.
PMCID: PMC110179  PMID: 9733867
5.  Restriction of HIV-1 Genotypes in Breast Milk Does Not Account for the Population Transmission Genetic Bottleneck That Occurs following Transmission 
PLoS ONE  2010;5(4):e10213.
Background
Breast milk transmission of HIV-1 remains a major route of pediatric infection. Defining the characteristics of viral variants to which breastfeeding infants are exposed is important for understanding the genetic bottleneck that occurs in the majority of mother-to-child transmissions. The blood-milk epithelial barrier markedly restricts the quantity of HIV-1 in breast milk, even in the absence of antiretroviral drugs. The basis of this restriction and the genetic relationship between breast milk and blood variants are not well established.
Methodology/Principal Findings
We compared 356 HIV-1 subtype C gp160 envelope (env) gene sequences from the plasma and breast milk of 13 breastfeeding women. A trend towards lower viral population diversity and divergence in breast milk was observed, potentially indicative of clonal expansion within the breast. No differences in potential N-linked glycosylation site numbers or in gp160 variable loop amino acid lengths were identified. Genetic compartmentalization was evident in only one out of six subjects in whom contemporaneously obtained samples were studied. However, in samples that were collected 10 or more days apart, six of seven subjects were classified as having compartmentalized viral populations, highlighting the necessity of contemporaneous sampling for genetic compartmentalization studies. We found evidence of CXCR4 co-receptor using viruses in breast milk and blood in nine out of the thirteen subjects, but no evidence of preferential localization of these variants in either tissue.
Conclusions/Significance
Despite marked restriction of HIV-1 quantities in milk, our data indicate intermixing of virus between blood and breast milk. Thus, we found no evidence that a restriction in viral genotype diversity in breast milk accounts for the genetic bottleneck observed following transmission. In addition, our results highlight the rapidity of HIV-1 env evolution and the importance of sample timing in analyses of gene flow.
doi:10.1371/journal.pone.0010213
PMCID: PMC2857876  PMID: 20422033
6.  Compartmentalization of HIV-1 within the Female Genital Tract Is Due to Monotypic and Low-Diversity Variants Not Distinct Viral Populations 
PLoS ONE  2009;4(9):e7122.
Background
Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons.
Methodology/Principal Findings
Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by ≥2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage.
Conclusions/Significance
In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.
doi:10.1371/journal.pone.0007122
PMCID: PMC2741601  PMID: 19771165
7.  Fialuridine Induces Acute Liver Failure in Chimeric TK-NOG Mice: A Model for Detecting Hepatic Drug Toxicity Prior to Human Testing 
PLoS Medicine  2014;11(4):e1001628.
Gary Peltz, Jeffrey Glenn, and colleagues report that a pre-clinical mouse toxicology model can detect liver toxicity of a drug that caused liver failure in several early clinical trial participants in 1993.
Please see later in the article for the Editors' Summary
Background
Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers.
Methods and Findings
Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers.
Conclusions
FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Before new drugs are approved for clinical use, they undergo extensive preclinical (laboratory-based) and clinical testing. In the preclinical studies, scientists investigate the causes of diseases, identify potential new drugs, and test promising drug candidates in animals. Animal testing is performed to determine whether the new drug is likely to work, and to screen for drug-induced toxicity. In preclinical toxicology studies, new drugs are given to two or more animal species to find out whether the drug has any short- or long-term toxic effects such as damage to the liver (hepatotoxicity). Drugs that pass these animal tests enter clinical trials. Phase I clinical trials test new drugs in a handful of healthy volunteers or patients to evaluate their safety and to identify possible side effects. In phase II trials, a larger group of patients receives the new drug to evaluate its safety further and to get an initial idea of its effectiveness. Finally, in phase III trials, very large groups of patients are randomly assigned to receive the new drug or an established treatment for their disease. These randomized controlled trials provide detailed information about the effectiveness and safety of a candidate drug, and must be completed before a drug can be approved for clinical use.
Why Was This Study Done?
Since animals are not perfect models for people, candidate drugs can cause toxicities in clinical trials that were not predicted by preclinical toxicology testing performed using animal species. For example, in 1993, 15 participants in a phase II trial were given a nucleoside analogue called fialuridine to treat hepatitis B virus infection (nucleoside analogues often have antiviral activity). Seven participants developed liver failure and lactic acidosis (buildup of lactic acid in the blood). Analysis of liver tissue from the affected participants revealed steatosis (fatty degeneration), intracellular fat droplets, and swollen mitochondria (these organelles are the powerhouses of the cell). Five participants subsequently died, and two had to have a liver transplant. In preclinical toxicology testing in mice, rats, dogs, and primates, there had been no indications that fialuridine would be hepatotoxic in people. It now seems that the expression of a nucleoside transporter in the mitochondria of humans but not of other animals may underlie the human-specific mitochondrial toxicity and hepatotoxicity of fialuridine. With several other nucleoside analogues in development, a better screening tool for human-specific mitochondrial toxicity is needed. In this study, the researchers investigate whether fialuridine toxicity can be detected in TK-NOG mice with chimeric (humanized) livers. TK-NOG mice are immunodeficient mice that have been genetically engineered so that human liver cells (hepatocytes) transplanted into these animals establish a long-lived mature “human organ.”
What Did the Researchers Do and Find?
The researchers treated chimeric (with transplanted human liver cells) and control (without transplanted human liver cells) TK-NOG mice with several doses of fialuridine. After treatment with the highest dose (1,600-fold above the dose used in the phase II trial) for four days, the chimeric mice developed liver failure and lactic acidosis. Moreover, steatosis and lipid and mitochondrial abnormalities developed in the regions of their livers that contained human hepatocytes but not in regions that contained mouse hepatocytes. Notably, the control mice had not developed liver toxicity after 14 days of treatment with the highest dose of drug. Liver toxicity was also easily detectable in chimeric mice that had been treated for 14 days with a fialuridine dose only 10-fold above that used in the human trial. Treatment with another nucleoside analogue that does not cause liver toxicity in people did not cause liver toxicity in the chimeric mice.
What Do These Findings Mean?
These findings show that fialuridine-induced liver toxicity can be readily detected using TK-NOG mice that have humanized livers at drug doses only 10-fold higher than those that caused liver failure in the phase II trial. Although the liver toxicity developed much more quickly in these mice than in the human trial participants, the clinical features, laboratory abnormalities, and structural changes seen in the fialuridine-treated chimeric TK-NOG mice closely mirrored those seen in fialuridine-treated people. The use of TK-NOG mice containing humanized livers in toxicology testing will not reveal whether drugs have human-specific toxicities outside the liver. Since they are highly immunocompromised, chimeric TK-NOG mice cannot be used to detect immune-mediated drug toxicities. Nevertheless, these findings suggest that the use of chimeric mice in toxicology studies could help improve the safety of candidate drugs that are tested in humans.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001628.
The US Food and Drug Administration, the body that approves drugs for clinical use in the US, provides an overview for patients about the drug development process from the laboratory to the clinic
The UK Medicines and Healthcare Products Regulatory Agency (MHRA) provides more detailed information for patients and the public about the drug development process, including a section on preclinical research, which includes information on animal testing
The US National Institutes of Health provides information about clinical trials, including personal stories from people who have taken part in clinical trials
The UK National Health Service Choices website has information for patients about clinical trials and medical research, including personal stories about participation in clinical trials
Understanding Animal Research is a UK advocacy group that provides information about the importance of animal research to the public, teachers, scientists, journalists, and policy makers
Wikipedia has a page on animal testing (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001628
PMCID: PMC3988005  PMID: 24736310
8.  Prevention of SIV Rectal Transmission and Priming of T Cell Responses in Macaques after Local Pre-exposure Application of Tenofovir Gel 
PLoS Medicine  2008;5(8):e157.
Background
The rectum is particularly vulnerable to HIV transmission having only a single protective layer of columnar epithelium overlying tissue rich in activated lymphoid cells; thus, unprotected anal intercourse in both women and men carries a higher risk of infection than other sexual routes. In the absence of effective prophylactic vaccines, increasing attention is being given to the use of microbicides and preventative antiretroviral (ARV) drugs. To prevent mucosal transmission of HIV, a microbicide/ARV should ideally act locally at and near the virus portal of entry. As part of an integrated rectal microbicide development programme, we have evaluated rectal application of the nucleotide reverse transcriptase (RT) inhibitor tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy) propyl] adenine monohydrate), a drug licensed for therapeutic use, for protective efficacy against rectal challenge with simian immunodeficiency virus (SIV) in a well-established and standardised macaque model.
Methods and Findings
A total of 20 purpose-bred Indian rhesus macaques were used to evaluate the protective efficacy of topical tenofovir. Nine animals received 1% tenofovir gel per rectum up to 2 h prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given tenofovir gel 2 h after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a noncloned, virulent stock of SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from peripheral blood mononuclear cells (PBMC), quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive quantitative competitive (qc) RT-PCR, and presence of SIV-specific serum antibodies by ELISA. We report here a significant protective effect (p = 0.003; Fisher exact probability test) wherein eight of nine macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving tenofovir gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. We found a strong positive association between the concentration of tenofovir in the plasma 15 min after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged tenofovir-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of tenofovir. Intriguingly, in the absence of seroconversion, Gag-specific gamma interferon (IFN-γ)-secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/106 PBMC to 261 spot forming cells (SFC)/106 PBMC.
Conclusions
These results indicate that colorectal pretreatment with ARV drugs, such as tenofovir, has potential as a clinically relevant strategy for the prevention of HIV transmission. We conclude that plasma tenofovir concentration measured 15 min after rectal administration may serve as a surrogate indicator of protective efficacy. This may prove to be useful in the design of clinical studies. Furthermore, in vitro intestinal explants served as a model for drug distribution in vivo and susceptibility to virus infection. The finding of T cell priming following exposure to virus in the absence of overt infection is provocative. Further studies would reveal if a combined modality microbicide and vaccination strategy is feasible by determining the full extent of local immune responses induced and their protective potential.
Martin Cranage and colleagues find that topical tenofovir gel can protect against rectal challenge with SIV in a macaque model, and can permit the induction of SIV-specific T cell responses.
Editors' Summary
Background.
About 33 million people are now infected with the human immunodeficiency virus (HIV), which causes AIDS by killing immune system cells. As yet, there is no cure for AIDS, although HIV infections can be held in check with antiretroviral drugs. Also, despite years of research, there is no vaccine available that effectively protects people against HIV infection. So, to halt the AIDS epidemic, other ways of preventing the spread of HIV are being sought. For example, pre-exposure treatment (prophylaxis) with antiretroviral drugs is being investigated as a way to prevent HIV transmission. In addition, because HIV is often spread through heterosexual penile-to-vaginal sex with an infected partner, several vaginal microbicides (compounds that protect against HIV when applied inside the vagina) are being developed, some of which contain antiretroviral drugs.
Why Was This Study Done?
Because HIV can cross the membranes that line the mouth and the rectum (the lower end of the large intestine that connects to the anus) in addition to the membrane that lines the vagina, HIV transmission can also occur during oral and anal sex. The lining of the rectum in particular is extremely thin and overlies tissues rich in activated T cells (the immune system cells that HIV targets), so unprotected anal intercourse carries a high risk of HIV infection. Anal intercourse is common among men who have sex with men but is also more common in heterosexual populations than is generally thought. Tenofovir (an antiretroviral drug that counteracts HIV after it has entered human cells) given by mouth partly protects macaques against rectal infection with simian immunodeficiency virus (SIV; a virus that induces AIDS in monkeys and apes) so the researchers wanted to know whether this drug might be effective against rectal SIV infection if applied at the site where the virus enters the body.
What Did the Researchers Do and Find?
To answer this question, the researchers rectally infected several macaques with SIV up to 2 h after rectal application of a gel containing tenofovir, after rectal application of a gel not containing the drug, or after no treatment. In addition, a few animals were treated with the tenofovir gel after the viral challenge. Most of the animals given the tenofovir gel before the viral challenge were partly or totally protected from SIV infection, whereas all the untreated animals and most of those treated with the placebo gel or with the drug-containing gel after the viral challenge became infected with SIV. High blood levels of tenofovir 15 min after its rectal application correlated with protection from viral infection. The researchers also collected rectal and small intestine samples from tenofovir-treated macaques that had not been exposed to SIV and asked which samples were resistant to SIV infection in laboratory dishes. They found that only the rectal samples were resistant to infection and only rectal cells contained tenofovir. Finally, activated T cells that recognized an SIV protein were present in the blood of some of the animals that were protected from SIV infection by the tenofovir gel.
What Do These Findings Mean?
These findings, although based on experiments in only a few animals, suggest that rectal treatment with antiretroviral drugs before rectal exposure to HIV might prevent rectal HIV transmission in people. However, results from animal experiments do not always reflect what happens in people. Indeed, clinical trials of a potential vaginal microbicide that worked well in macaques were halted recently because women using the microbicide had higher rates of HIV infection than those using a control preparation. The finding that immune-system activation can occur in the absence of overt infection in animals treated with the tenofovir gel additionally suggests that a combination of a local antiretroviral/microbicide and vaccination might be a particularly effective way to prevent HIV transmission. However, because HIV targets activated T cells, viral rechallenge experiments must be done to check that the activated T cells induced by the virus in the presence of tenofovir do not increase the likelihood of infection upon re-exposure to HIV before this potential microbicide is tried in people.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050157.
Read the accompanying PLoS Medicine Perspective by Florian Hladik
An overview of HIV infection and AIDS is available from the US National Institute of Allergy and Infectious Diseases
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including an article on safer sex, which includes information on the risks associated with specific types of sex and on microbicides and other methods to prevent the sexual transmission of HIV
Information on all aspects of HIV/AIDS is available from Avert, an international AIDS charity, including information on HIV prevention and on microbicides
The World Health Organization has a fact sheet on microbicides
The UK charity NAM also provides detailed information on microbicides
PrEP Watch is a comprehensive information source on pre-exposure prophylaxis for HIV prevention
Global Campaign for Microbicides is an international coalition of organisations dedicated to accelerating access to new HIV prevention options
doi:10.1371/journal.pmed.0050157
PMCID: PMC2494562  PMID: 18684007
9.  Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41 
PLoS Medicine  2008;5(1):e9.
Background
The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env) variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes.
Methods and Findings
Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41) of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced neutralization sensitivity exceeded 1,000-fold in several cases.
Conclusions
Two amino acid mutations within gp41 were identified that expose multiple discontinuous neutralization epitopes on diverse HIV-1 Env proteins. These exposed epitopes were shielded on the unmodified viral Env proteins, and several of the exposed epitopes encompass desired target regions for protective antibodies. Env proteins containing these modifications could act as a scaffold for presentation of such conserved domains, and may aid in developing methods to target antibodies to such regions.
Julie Overbaugh and colleagues analyze an HIV strain with high susceptibility to antibody neutralization and identify two gp41 envelope mutations that confer this sensitivity by exposing multiple neutralization epitopes.
Editors' Summary
Background.
In 1984 when scientists identified human immunodeficiency virus (HIV)—the cause of acquired immunodeficiency syndrome (AIDS)—many experts believed that a vaccine against HIV infection would soon be developed. Nearly 25 years later, there is still no such vaccine and with about 2.5 million new HIV infections in 2007, an effective vaccine is urgently needed to contain the AIDS epidemic. Vaccines provide protection against infectious diseases by priming the immune system to deal quickly and effectively with viruses and other pathogens. Vaccines do this by exposing the immune system to an immunogen—a fragment or harmless version of the pathogen. The immune system mounts a response against the immunogen and also “learns” from this experience so that if it is ever challenged with a virulent version of the same pathogen, it can quickly contain the threat. Many vaccines work by stimulating an antibody response. Antibodies are proteins made by the immune system that bind to molecules called antigens on the surface of pathogens. Antibodies that inactivate the invader upon binding to it are called “neutralizing” antibodies.
Why Was This Study Done?
Several characteristics of HIV have hampered the development of an effective vaccine. An “envelope” protein consisting of two subunits called gp120 and gp41 covers the outside of HIV. Many regions of this protein change rapidly, so the antibody response stimulated by a vaccine containing the envelope protein of one HIV variant provides little protection against other variants. However, other regions of the protein rarely change, so a vaccine that stimulates the production of antibodies to these “conserved” regions is likely to provide protection against many HIV variants. That is, it will stimulate the production of broadly neutralizing antibodies. Unfortunately, it has been difficult to find HIV vaccines that do this, because these conserved regions are often hidden from the immune system by other parts of the envelope protein. In this study, the researchers investigate the envelope protein of an HIV-1 variant they have isolated that is highly susceptible to inactivation by antibodies specific for these conserved regions. Comparing the envelope protein of this sensitive virus to closely related envelope proteins that are resistant to neutralization could identify features that might, if included in an envelope protein immunogen, produce a vaccine capable of generating broadly neutralizing antibodies.
What Did the Researchers Do and Find?
The researchers isolated a subtype A HIV-1 variant from a newly infected woman in Kenya that was efficiently neutralized by monoclonal antibodies (antibodies made by cells that have been cloned in the laboratory). These antibodies were specific for several different conserved regions of gp41 and gp120. The isolate was also neutralized by antibodies in blood from HIV-1-infected people. The envelope protein of the sensitive variant was the same as that of a resistant variant isolated at the same time from the woman, except for four amino acid changes in conserved regions of gp41 (proteins are made from long strings of amino acids). Using a technique called site-directed mutagenesis, the researchers introduced these amino acid changes into envelope proteins made in the laboratory and determined that just two of these changes were responsible for the neutralization sensitivity of the HIV-1 variant. The introduction of these two changes into the neutralization resistant variant and into the unrelated envelope sequences of another subtype A (common in Africa) HIV-1 variant and a subtype B HIV-1 (common in Europe and the Western Hemisphere) variant increased the sensitivity of all these viruses to antibody neutralization.
What Do These Findings Mean?
These findings show that two amino acid changes in gp41 of a neutralization-sensitive HIV-1 variant are responsible for the sensitivity of this variant to several neutralizing antibodies. The finding that the inclusion of these changes in the envelope protein of neutralization-resistant HIV-1 variants greatly increases their sensitivity to neutralizing antibodies indicates that the normally shielded regions of the protein are somehow made accessible to antibody by these changes. One possibility is that the amino acid changes might modify the overall shape of the envelope protein, thus exposing multiple, normally hidden regions in the HIV-1 envelope protein to antibodies. Importantly, these findings open up the possibility that the inclusion of these modifications in envelope-based immunogens might improve the ability of vaccines to generate broadly neutralizing antibodies against HIV-1.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050009.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including links to resources dealing with HIV vaccine development
Information is available from Avert, an international AIDS charity, on all aspects of HIV and AIDS, including HIV vaccines
The US Centers for Disease Control and prevention provides information on HIV/AIDS including information on its HIV vaccine unit (in English and some information in Spanish)
The AIDS Vaccine Clearinghouse provides clear information about HIV vaccine science, research and product development
The International AIDS Vaccine Initiative also provides straightforward information about the development of HIV vaccines
doi:10.1371/journal.pmed.0050009
PMCID: PMC2174964  PMID: 18177204
10.  Adaptation to Different Human Populations by HIV-1 Revealed by Codon-Based Analyses 
PLoS Computational Biology  2006;2(6):e62.
Several codon-based methods are available for detecting adaptive evolution in protein-coding sequences, but to date none specifically identify sites that are selected differentially in two populations, although such comparisons between populations have been historically useful in identifying the action of natural selection. We have developed two fixed effects maximum likelihood methods: one for identifying codon positions showing selection patterns that persist in a population and another for detecting whether selection is operating differentially on individual codons of a gene sampled from two different populations. Applying these methods to two HIV populations infecting genetically distinct human hosts, we have found that few of the positively selected amino acid sites persist in the population; the other changes are detected only at the tips of the phylogenetic tree and appear deleterious in the long term. Additionally, we have identified seven amino acid sites in protease and reverse transcriptase that are selected differentially in the two samples, demonstrating specific population-level adaptation of HIV to human populations.
Synopsis
Despite the efforts devoted to surveying HIV genetic diversity and the development of an effective vaccine, there is still no consensus on the extent to which the former prejudices the latter. Experimental studies show that escape from cell-mediated immunity is selected for in HIV and SIV, and sometimes very quickly. Conversely, escape mutants may be selected against at transmission, so how much does this selection within individuals influence the genotype of the circulating HIV population overall? Kosakovsky Pond, Leigh Brown, and colleagues have developed a new statistical approach to address this question. Using sequences from the globally most abundant HIV subtype (subtype C), the authors directly compared virus of the same subtype infecting genetically different human populations. They show at least half of the amino acid sites selected within individuals are not selected at a population level, and they identify six amino acid sites in the RT gene that are selected differentially between populations. We can now partition molecular adaptation between individual and population components for whatever genes may be included in candidate vaccines, in the target populations themselves. The methods are also important beyond the HIV world, where analogous issues arise in the more general question of the evolution of virulence in pathogens.
doi:10.1371/journal.pcbi.0020062
PMCID: PMC1480537  PMID: 16789820
11.  Episodic Sexual Transmission of HIV Revealed by Molecular Phylodynamics 
PLoS Medicine  2008;5(3):e50.
Background
The structure of sexual contact networks plays a key role in the epidemiology of sexually transmitted infections, and their reconstruction from interview data has provided valuable insights into the spread of infection. For HIV, the long period of infectivity has made the interpretation of contact networks more difficult, and major discrepancies have been observed between the contact network and the transmission network revealed by viral phylogenetics. The high rate of HIV evolution in principle allows for detailed reconstruction of links between virus from different individuals, but often sampling has been too sparse to describe the structure of the transmission network. The aim of this study was to analyze a high-density sample of an HIV-infected population using recently developed techniques in phylogenetics to infer the short-term dynamics of the epidemic among men who have sex with men (MSM).
Methods and Findings
Sequences of the protease and reverse transcriptase coding regions from 2,126 patients, predominantly MSM, from London were compared: 402 of these showed a close match to at least one other subtype B sequence. Nine large clusters were identified on the basis of genetic distance; all were confirmed by Bayesian Monte Carlo Markov chain (MCMC) phylogenetic analysis. Overall, 25% of individuals with a close match with one sequence are linked to 10 or more others. Dated phylogenies of the clusters using a relaxed clock indicated that 65% of the transmissions within clusters took place between 1995 and 2000, and 25% occurred within 6 mo after infection. The likelihood that not all members of the clusters have been identified renders the latter observation conservative.
Conclusions
Reconstruction of the HIV transmission network using a dated phylogeny approach has revealed the HIV epidemic among MSM in London to have been episodic, with evidence of multiple clusters of transmissions dating to the late 1990s, a period when HIV prevalence is known to have doubled in this population. The quantitative description of the transmission dynamics among MSM will be important for parameterization of epidemiological models and in designing intervention strategies.
Using viral genotype data from HIV drug resistance testing at a London clinic, Andrew Leigh Brown and colleagues derive the structure of the transmission network through phylogenetic analysis.
Editors' Summary
Background.
Human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS), is mainly spread through unprotected sex with an infected partner. Like other sexually transmitted diseases, HIV/AIDS spreads through networks of sexual contacts. The characteristics of these complex networks (which include people who have serial sexual relationships with single partners and people who have concurrent sexual relationships with several partners) affect how quickly diseases spread in the short term and how common the disease is in the long term. For many sexually transmitted diseases, sexual contact networks can be reconstructed from interview data. The information gained in this way can be used for partner notification so that transmitters of the disease and people who may have been unknowingly infected can be identified, treated, and advised about disease prevention. It can also be used to develop effective community-based prevention strategies.
Why Was This Study Done?
Although sexual contact networks have provided valuable information about the spread of many sexually transmitted diseases, they cannot easily be used to understand HIV transmission patterns. This is because the period of infectivity with HIV is long and the risk of infection from a single sexual contact with an infected person is low. Another way to understand the spread of HIV is through phylogenetics, which examines the genetic relatedness of viruses obtained from different individuals. Frequent small changes in the genetic blueprint of HIV allow the virus to avoid the human immune response and to become resistant to antiretroviral drugs. In this study, the researchers use recently developed analytical methods, viral sequences from a large proportion of a specific HIV-infected population, and information on when each sample was taken, to learn about transmission of HIV/AIDS in London among men who have sex with men (MSM; a term that encompasses gay, bisexual, and transgendered men and heterosexual men who sometimes have sex with men). This new approach, which combines information on viral genetic variation and viral population dynamics, is called “molecular phylodynamics.”
What Did the Researchers Do and Find?
The researchers compared the sequences of the genes encoding the HIV-1 protease and reverse transcriptase from more than 2,000 patients, mainly MSM, attending a large London HIV clinic between 1997 and 2003. 402 of these sequences closely matched at least one other subtype B sequence (the HIV/AIDS epidemic among MSM in the UK primarily involves HIV subtype B). Further analysis showed that the patients from whom this subset of sequences came formed six clusters of ten or more individuals, as well as many smaller clusters, based on the genetic relatedness of their HIV viruses. The researchers then used information on the date when each sample was collected and a “relaxed clock” approach (which accounts for the possibility that different sequences evolve at different rates) to determine dated phylogenies (patterns of genetic relatedness that indicate when gene sequences change) for the clusters. These phylogenies indicated that at least in one in four transmissions between the individuals in the large clusters occurred within 6 months of infection, and that most of the transmissions within each cluster occurred over periods of 3–4 years during the late 1990s.
What Do These Findings Mean?
This phylodynamic reconstruction of the HIV transmission network among MSM in a London clinic indicates that the HIV epidemic in this population has been episodic with multiple clusters of transmission occurring during the late 1990s, a time when the number of HIV infections in this population doubled. It also suggests that transmission of the virus during the early stages of HIV infection is likely to be an important driver of the epidemic. Whether these results apply more generally to the MSM population at risk for transmitting or acquiring HIV depends on whether the patients in this study are representative of that group. Additional studies are needed to determine this, but if the patterns revealed here are generalizable, then this quantitative description of HIV transmission dynamics should help in the design of strategies to strengthen HIV prevention among MSM.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050050.
Read a related PLoS Medicine Perspective article
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including a list of organizations that provide information for gay men and MSM
The US Centers for Disease Control and Prevention provides information on HIV/AIDS and on HIV/AIDS among MSM (in English and Spanish)
Information is available from Avert, an international AIDS charity, on HIV, AIDS, and men who have sex with men
The Center for AIDS Prevention Studies (University of California, San Francisco) provides information on sexual networks and HIV prevention
The US National Center for Biotechnology Information provides a science primer on molecular phylogenetics
UK Collaborative Group on HIV Drug Resistance maintains a database of resistance tests
HIV i-Base offers HIV treatment information for health-care professionals and HIV-positive people
The NIH-funded HIV Sequence Database contains data on genetic sequences, resistance, immunology, and vaccine trials
doi:10.1371/journal.pmed.0050050
PMCID: PMC2267814  PMID: 18351795
12.  Deep Molecular Characterization of HIV-1 Dynamics under Suppressive HAART 
PLoS Pathogens  2011;7(10):e1002314.
In order to design strategies for eradication of HIV-1 from infected individuals, detailed insight into the HIV-1 reservoirs that persist in patients on suppressive antiretroviral therapy (ART) is required. In this regard, most studies have focused on integrated (proviral) HIV-1 DNA forms in cells circulating in blood. However, the majority of proviral DNA is replication-defective and archival, and as such, has limited ability to reveal the dynamics of the viral population that persists in patients on suppressive ART. In contrast, extrachromosomal (episomal) viral DNA is labile and as a consequence is a better surrogate for recent infection events and is able to inform on the extent to which residual replication contributes to viral reservoir maintenance. To gain insight into the diversity and compartmentalization of HIV-1 under suppressive ART, we extensively analyzed longitudinal peripheral blood mononuclear cells (PBMC) samples by deep sequencing of episomal and integrated HIV-1 DNA from patients undergoing raltegravir intensification. Reverse-transcriptase genes selectively amplified from episomal and proviral HIV-1 DNA were analyzed by deep sequencing 0, 2, 4, 12, 24 and 48 weeks after raltegravir intensification. We used maximum likelihood phylogenies and statistical tests (AMOVA and Slatkin-Maddison (SM)) in order to determine molecular compartmentalization. We observed low molecular variance (mean variability ≤0.042). Although phylogenies showed that both DNA forms were intermingled within the phylogenetic tree, we found a statistically significant compartmentalization between episomal and proviral DNA samples (P<10−6 AMOVA test; P = 0.001 SM test), suggesting that they belong to different viral populations. In addition, longitudinal analysis of episomal and proviral DNA by phylogeny and AMOVA showed signs of non-chronological temporal compartmentalization (all comparisons P<10−6) suggesting that episomal and proviral DNA forms originated from different anatomical compartments. Collectively, this suggests the presence of a chronic viral reservoir in which there is stochastic release of infectious virus and in which there are limited rounds of de novo infection. This could be explained by the existence of different reservoirs with unique pharmacological accessibility properties, which will require strategies that improve drug penetration/retention within these reservoirs in order to minimise maintenance of the viral reservoir by de novo infection.
Author Summary
In the majority of HIV-1 positive patients, antiretroviral therapy (ART) effects a sustained reduction in plasma viremia to below detectable levels. Despite this, replication competent viruses persist and fuel viremia if antiretroviral treatment is interrupted. This viral persistence stands in the way of viral eradication through ART. While this ability to persist in the face of therapy is generally considered to be attributable to a reservoir of latently infected cells, there is debate as to how this reservoir is maintained and in particular, whether there is replenishment of the reservoir by low level, residual replication. Novel antiviral agents targeting the viral integrase offer tools to explore the viral reservoirs that persist in the face of ART and we have shown that raltegravir perturbs these reservoirs as evidenced by an accumulation of episomal DNA upon rategravir intensification (Buzon et al., 2010). Through “deep sequencing” technology, we have longitudinally analyzed the genotypes of HIV episomes and integrated HIV DNA to evaluate whether they represent interrelated sequences or whether they have distinct origins. Statistical methods showed molecular compartmentalization, among and within episomal and integrated HIV-1 DNA samples, and suggest that episomal DNA in PBMC originates from a cellular/anatomic reservoir that is not revealed by sequencing of proviral DNA in PBMC in this study. These, and other data, suggest that ongoing replication, which can be blocked by adding raltegravir, occurs from proviruses that are genetically distinguishable from those detected at >1% frequency in these circulating blood cells.
doi:10.1371/journal.ppat.1002314
PMCID: PMC3203183  PMID: 22046128
13.  Nevirapine- Versus Lopinavir/Ritonavir-Based Initial Therapy for HIV-1 Infection among Women in Africa: A Randomized Trial 
PLoS Medicine  2012;9(6):e1001236.
In a randomized control trial, Shahin Lockman and colleagues compare nevirapine-based therapy with lopinavir/ritonavir-based therapy for HIV-infected women without previous exposure to antiretroviral treatment.
Background
Nevirapine (NVP) is widely used in antiretroviral treatment (ART) of HIV-1 globally. The primary objective of the AA5208/OCTANE trial was to compare the efficacy of NVP-based versus lopinavir/ritonavir (LPV/r)-based initial ART.
Methods and Findings
In seven African countries (Botswana, Kenya, Malawi, South Africa, Uganda, Zambia, and Zimbabwe), 500 antiretroviral-naïve HIV-infected women with CD4<200 cells/mm3 were enrolled into a two-arm randomized trial to initiate open-label ART with tenofovir (TDF)/emtricitabine (FTC) once/day plus either NVP (n = 249) or LPV/r (n = 251) twice/day, and followed for ≥48 weeks. The primary endpoint was time from randomization to death or confirmed virologic failure ([VF]) (plasma HIV RNA<1 log10 below baseline 12 weeks after treatment initiation, or ≥400 copies/ml at or after 24 weeks), with comparison between treatments based on hazard ratios (HRs) in intention-to-treat analysis. Equivalence of randomized treatments was defined as finding the 95% CI for HR for virological failure or death in the range 0.5 to 2.0. Baseline characteristics were (median): age = 34 years, CD4 = 121 cells/mm3, HIV RNA = 5.2 log10copies/ml. Median follow-up = 118 weeks; 29 (6%) women were lost to follow-up. 42 women (37 VFs, five deaths; 17%) in the NVP and 50 (43 VFs, seven deaths; 20%) in the LPV/r arm reached the primary endpoint (HR 0.85, 95% CI 0.56–1.29). During initial assigned treatment, 14% and 16% of women receiving NVP and LPV/r experienced grade 3/4 signs/symptoms and 26% and 22% experienced grade 3/4 laboratory abnormalities. However, 35 (14%) women discontinued NVP because of adverse events, most in the first 8 weeks, versus none for LPV/r (p<0.001). VF, death, or permanent treatment discontinuation occurred in 80 (32%) of NVP and 54 (22%) of LPV/r arms (HR = 1.7, 95% CI 1.2–2.4), with the difference primarily due to more treatment discontinuation in the NVP arm. 13 (45%) of 29 women tested in the NVP versus six (15%) of 40 in the LPV/r arm had any drug resistance mutation at time of VF.
Conclusions
Initial ART with NVP+TDF/FTC demonstrated equivalent virologic efficacy but higher rates of treatment discontinuation and new drug resistance compared with LPV/r+TDF/FTC in antiretroviral-naïve women with CD4<200 cells/mm3.
Trial registration
ClinicalTrials.gov NCT00089505
Please see later in the article for the Editors' Summary
Editors' Summary
Background
About 34 million people (mostly living in low- or middle-income countries) are currently infected with HIV, the virus that causes AIDS. HIV destroys CD4 lymphocytes and other immune cells, leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-infected people died within 10 years of infection. Then, in 1996, antiretroviral therapy (ART)—cocktails of drugs that attack different parts of HIV—became available. For people living in affluent countries, HIV/AIDS became a chronic condition. But, because ART was expensive, for people living in developing countries, HIV/AIDS remained a fatal illness. In 2006, the international community set a target of achieving universal access to ART by 2010 and, although this target has not been reached, by the end of 2010, 6.6 million of the estimated 15 million people in need of ART in developing countries were receiving one of the ART regimens recommended by the World Health Organization (WHO) in its 2010 guidelines.
Why Was This Study Done?
A widely used combination for the initial treatment of HIV-infected people (particularly women) in resource-limited settings is tenofovir and emtricitabine (both nucleotide reverse transcriptase inhibitors; reverse transcriptase is essential for HIV replication) and nevirapine (NVP, a non-nucleoside reverse transcriptase inhibitor). However, little is known about the efficacy of this NVP-based ART combination. Moreover, its efficacy and toxicity has not been compared with regimens containing lopinavir/ritonavir (LPV/r). LPV/r, which inhibits the viral protease that is essential for HIV replication, is available in resource-limited settings but is usually reserved for second-line treatment. LPV/r-based ART is more expensive than NVP-based ART but if it were more effective or better tolerated than NVP-based ART, then first-line treatment with LPV/r-based ART might be cost-effective in resource-limited settings. Conversely, evidence of the clinical equivalence of NVP-based and LPV/r-based ART would provide support for NVP-based ART as an initial therapy. In this randomized equivalence trial, the researchers compare the efficacy and toxicity of NVP-based and LVP/r-based initial therapy for HIV infection among antiretroviral-naïve African women. In a randomized trial, patients are assigned different treatments by the play of chance and followed to compare the effects of these treatments; an equivalence trial asks whether the effects of two treatments are statistically equivalent.
What Did the Researchers Do and Find?
The researchers followed 500 antiretroviral-naïve HIV-infected women with a low CD4 cell count living in seven African countries, half of whom received NVP-based ART and half of whom received LPV/r-based ART, for an average of 118 weeks and recorded the time to virologic failure (the presence of virus in the blood above pre-specified levels) or death among the participants. Forty-two women in the NVP arm reached this primary endpoint (37 virologic failures and five deaths) compared to 50 women in the LPV/r arm (43 virologic failures and seven deaths), a result that indicates equivalent virologic efficacy according to preset statistical criteria. During the initial assigned treatment, similar proportions of women in both treatment arms developed serious drug-related signs and symptoms and laboratory abnormalities. However, whereas 14% of the women in the NVP arm discontinued treatment because of adverse effects, none of the women in the LPV/r arm discontinued treatment. Finally, nearly half of the women tested in the NVP arm but only 15% of the women tested in the LVP/r arm had developed any drug resistance at the time of virologic failure.
What Do These Findings Mean?
These findings indicate that, among HIV-infected, treatment-naïve African women, initial NVP-based ART is as effective as LPV/r-based ART in terms of virologic failure and death although more women in the NVP arm discontinued treatment or developed new drug resistance than in the LPV/r arm. Several limitations of this study may affect the accuracy of these findings. In particular, some of the study participants may have been exposed to single-dose NVP during childbirth to prevent mother-to-child transmission of HIV; in a parallel randomized trial, the researchers found that LPV/r-based ART was superior to NVP-based ART among women with prior exposure to single-dose NVP. Moreover, the duration of the current study means the long-term effects of the two treatments cannot be compared. Nevertheless, these findings support the WHO recommendation of NVP-based ART with careful early toxicity monitoring as an initial affordable and effective HIV treatment regiment in resource-limited settings, until access to better-tolerated and more potent regimens is possible.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001236.
Information is available from the US National Institute of Allergy and Infectious Diseases on all aspects of HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, and summaries of recent research findings on HIV care and treatment (in several languages)
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including detailed information on HIV treatment and care (in English and Spanish)
WHO provides information about universal access to AIDS treatment (in English, French and Spanish); its 2010 ART guidelines can be downloaded
More information about this trial, the OCTANE trial, is available
MedlinePlus provides detailed information about nevirapine and lopinavir/ritinovir (in English and Spanish)
Patient stories about living with HIV/AIDS are available through Avert; the nonprofit website Healthtalkonline also provides personal stories about living with HIV, including stories about taking anti-HIV drugs and the challenges of anti-HIV drugs
doi:10.1371/journal.pmed.1001236
PMCID: PMC3373629  PMID: 22719231
14.  Development of a Standardized Screening Rule for Tuberculosis in People Living with HIV in Resource-Constrained Settings: Individual Participant Data Meta-analysis of Observational Studies 
PLoS Medicine  2011;8(1):e1000391.
Haileyesus Getahun and colleagues report the development of a simple, standardized tuberculosis (TB) screening rule for resource-constrained settings, to identify people living with HIV who need further investigation for TB disease.
Background
The World Health Organization recommends the screening of all people living with HIV for tuberculosis (TB) disease, followed by TB treatment, or isoniazid preventive therapy (IPT) when TB is excluded. However, the difficulty of reliably excluding TB disease has severely limited TB screening and IPT uptake in resource-limited settings. We conducted an individual participant data meta-analysis of primary studies, aiming to identify a sensitive TB screening rule.
Methods and Findings
We identified 12 studies that had systematically collected sputum specimens regardless of signs or symptoms, at least one mycobacterial culture, clinical symptoms, and HIV and TB disease status. Bivariate random-effects meta-analysis and the hierarchical summary relative operating characteristic curves were used to evaluate the screening performance of all combinations of variables of interest. TB disease was diagnosed in 557 (5.8%) of 9,626 people living with HIV. The primary analysis included 8,148 people living with HIV who could be evaluated on five symptoms from nine of the 12 studies. The median age was 34 years. The best performing rule was the presence of any one of: current cough (any duration), fever, night sweats, or weight loss. The overall sensitivity of this rule was 78.9% (95% confidence interval [CI] 58.3%–90.9%) and specificity was 49.6% (95% CI 29.2%–70.1%). Its sensitivity increased to 90.1% (95% CI 76.3%–96.2%) among participants selected from clinical settings and to 88.0% (95% CI 76.1%–94.4%) among those who were not previously screened for TB. Negative predictive value was 97.7% (95% CI 97.4%–98.0%) and 90.0% (95% CI 88.6%–91.3%) at 5% and 20% prevalence of TB among people living with HIV, respectively. Abnormal chest radiographic findings increased the sensitivity of the rule by 11.7% (90.6% versus 78.9%) with a reduction of specificity by 10.7% (49.6% versus 38.9%).
Conclusions
Absence of all of current cough, fever, night sweats, and weight loss can identify a subset of people living with HIV who have a very low probability of having TB disease. A simplified screening rule using any one of these symptoms can be used in resource-constrained settings to identify people living with HIV in need of further diagnostic assessment for TB. Use of this algorithm should result in earlier TB diagnosis and treatment, and should allow for substantial scale-up of IPT.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
In 2009, 1.7 million people died from tuberculosis (TB)—equating to 4,700 deaths a day—including 380,000 people living with HIV. TB remains the most common cause of death in people living with HIV and compared to people without HIV, people living with HIV are more than 20 times more likely to develop TB. Furthermore, TB infection may occur at any stage of HIV disease and is often the initial presentation of underlying HIV infection. Without antiretroviral treatment, up to 50% of people living with HIV who are diagnosed with TB die during the 6–8 months of TB treatment.
Although antiretroviral treatment can reduce the incidence of TB both at the individual and population level, people living with HIV on antiretroviral treatment still have higher TB incidence rates and a higher risk of dying from TB. Therefore, the World Health Organization recommends regular screening for active TB disease in all people living with HIV, so those identified as having active TB disease can be provided with appropriate treatment, and isoniazid preventive therapy (to help mitigate TB morbidity, mortality, and transmission) can be given to vulnerable individuals who do not yet have active TB.
Why Was This Study Done?
There is currently no internationally accepted evidence-based tool to screen for TB in people living with HIV—a serious gap given that the presenting signs and symptoms of TB in people living with HIV are different from those in people without HIV. Therefore, the researchers aimed to develop a simple, standardized TB screening rule for resource-constrained settings, on the basis of the best available evidence that would adequately distinguish between people living with HIV who are very unlikely to have TB from those who require further investigation for TB disease.
What Did the Researchers Do and Find?
The researchers selected 12 studies that met their strict criteria, then asked the authors of these studies for primary data so that they could map individual-level data to identify five symptoms common to most studies. Using a statistical model, the researchers devised 23 screening rules derived from these five symptoms and used meta-analysis methods (bivariate random-effects meta-analysis) and the association of study-level and individual-level correlates (hierarchical summary relative operating characteristic curves) to evaluate the sensitivity and specificity of each tool used in each individual study.
The authors of the selected studies were able to provide data for 29,523 participants, of whom 10,057 were people living with HIV. The dataset included 9,626 people living with HIV who had TB screening and sputum culture performed, of which 8,148 individuals could be evaluated on the five symptoms of interest from nine of 12 studies. TB disease was diagnosed in 5.8% of people living with HIV and the best performing rule was the presence of any one of the following: current cough (any duration), fever, night sweats, or weight loss. The overall sensitivity of the rule was 78.9% and the specificity was 49.6%. However, the sensitivity of the rule increased to 90.1% among participants selected from clinical settings and to 88.0% among those who were not previously screened for TB.
What Do These Findings Mean?
The results of this study suggest that in resource-constrained settings, the absence of current cough, fever, night sweats, and weight loss (all inclusive) can identify those people living with HIV who have a low probability of having TB disease. Furthermore, any one of these symptoms can be used in resource-constrained settings to identify people living with HIV who are in need of further diagnostic assessment for TB.
Despite the limitations of the methodology used in this study, until there are evidence-based and internationally recommended guidelines for the diagnosis and treatment of TB in people living with HIV, use of the algorithm developed and presented in this study could result in earlier TB diagnosis and treatment for people living with HIV and could help to substantially scale-up isoniazid preventive therapy.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000391.
The World Health Organization has information about TB in people living with HIV
The US Centers for Disease Control and Prevention also provide information about TB and HIV coinfection
The World Health Organization also has information about isoniazid preventative therapy
The Stop TB Partnership's TB/HIV Working Group provide information about TB and HIV co-infection
doi:10.1371/journal.pmed.1000391
PMCID: PMC3022524  PMID: 21267059
15.  Polyclonal B Cell Differentiation and Loss of Gastrointestinal Tract Germinal Centers in the Earliest Stages of HIV-1 Infection 
PLoS Medicine  2009;6(7):e1000107.
Studying the effects of early HIV infection on human antibody responses, M. Anthony Moody and colleagues find rapid polyclonal B cell differentiation and structural damage to gut-associated lymphoid tissue.
Background
The antibody response to HIV-1 does not appear in the plasma until approximately 2–5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1–specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4+ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.
Methods and Findings
In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1–specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1–induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.
Conclusions
Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1–induced antibody responses and the delay in plasma antibody responses to HIV-1.
Please see later in the article for Editors' Summary
Editors' Summary
Background
Acquired immunodeficiency syndrome (AIDS) has killed more than 25 million people since 1981 and more than 30 million people are now infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV infects and kills a type of immune system cell called CD4+ T lymphocytes. These cells are needed to maintain a vigorous immune response, so people infected with HIV eventually become susceptible to other infections and develop full-blown AIDS. However, early during HIV infection, other parts of the immune system attempt to fight off the virus. Soon after infection, immune system cells called B lymphocytes begin to produce HIV-specific antibodies (proteins that recognize viral molecules called antigens). The first antibodies to HIV usually appear two to seven weeks after infection; from about 12 weeks after infection, antibodies are made that can kill the specific HIV type responsible for the infection (neutralizing antibodies).
Why Was This Study Done?
Unfortunately, by this time, it is too late for the antibody (“humoral”) immune response to clear HIV from the body. Indeed, the humoral immune response to HIV is very slow; for most viruses, neutralizing antibodies appear within days of infection. To help them design an effective HIV vaccine, scientists need to understand how the virus delays humoral responses to HIV infection (and how it later causes the production of HIV-specific antibodies to decline). Little is known, however, about the early effects of HIV infection on B lymphocytes. These cells are born and mature in the bone marrow. “Naïve” B lymphocytes, each of which carries an antigen-specific receptor (a protein that binds to a specific antigen), then enter the blood and circulate around the body, passing through the “peripheral lymphoid organs”. Exposure to antigens in these organs, which include lymph nodes and gut-associated lymphoid tissues, activates the subset of B lymphocytes that recognize the specific antigens that are present. Finally, with the help of activated T lymphocytes, the activated B lymphocytes proliferate and change (differentiate) into antibody-secreting cells and memory B lymphocytes (which respond more quickly to antigen than naïve B lymphocytes). In this study, the researchers investigate the effects of early HIV-1 infection on B lymphocytes in blood and in gut-associated lymphoid tissues.
What Did the Researchers Do and Find?
The researchers collected blood from patients as early as 17 days after HIV-1 infection and tissue samples from the lower portion of the small intestine (a region rich in gut-associated lymphoid structures called Peyer's patches) from 47 days after infection onward. When they analyzed the B lymphocytes in these samples (which were collected during two trials organized by the US Center for HIV/AIDS Vaccine Immunology [CHAVI]), they found that HIV-1 infection rapidly induced the activation of many different B cells that recognized a variety of antigens (polyclonal activation), as well as the appearance of differentiated B cells in blood and in gut-associated lymphoid tissue. The B lymphocytes that were activated in the gut made HIV-specific antibodies but also antibodies against unrelated antigens (such as flu virus proteins). Finally, the structure of Peyer's patches was altered early in HIV-1 infection. More specifically, most of the lymphoid follicles (organized collections of lymphocytes and antigen-presenting cells) in the Peyer's patches showed signs of damage and T- and B-lymphocyte death and the number of germinal centers (regions in lymphoid follicles in which B lymphocytes proliferate) was reduced.
What Do These Findings Mean?
Although the depletion of gut-associated CD4+ T lymphocytes in early HIV-1 infection is well known, these new results demonstrate the effects of early HIV-1 infection on gut-associated and circulating B lymphocytes. The results of this study are limited by the methods used to analyze the antibodies induced by HIV infection and by only taking tissue samples from one region of the gut. Nevertheless, the findings of polyclonal B-cell activation and damage to gut-associated lymphoid follicles soon after HIV-1 infection may have implications for HIV-1 vaccine design. Specifically, these findings suggest that an effective HIV-1 vaccine will need to ensure that significant levels of neutralizing antibodies are present in people before HIV-1 infection and that other protective immune defenses are fully primed so that, in the event of HIV-1 infection, the virus can be dealt with effectively before it disables any part of the immune system.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000107.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including an article about how HIV-1 infection affects the immune system
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including information on the stages of HIV infection, and on AIDS vaccines (in English and Spanish)
The US Center for HIV/AIDS Vaccine Immunology (CHAVI) Web site provides information on research designed to solve major problems in HIV vaccine development and design
doi:10.1371/journal.pmed.1000107
PMCID: PMC2702159  PMID: 19582166
16.  N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance 
PLoS Medicine  2007;4(12):e335.
Background
The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.
Methods and Findings
The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 × 10−12). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43–4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance.
Conclusions
This study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patient's response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure.
Analyzing HIV sequences from a Canadian cohort, Gilda Tachedjian and colleagues identify a common mutation in a little-studied domain of reverse transcriptase that confers resistance to two drug classes.
Editors' Summary
Background.
In the 1980s, infection with the human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), was a death sentence. Although the first antiretroviral drugs (compounds that block HIV's life cycle) were developed quickly, single antiretrovirals only transiently suppress HIV infection. HIV rapidly accumulates random changes (mutations) in its genetic material, some of which make it drug resistant. Nowadays, there are many different antiretrovirals. Some inhibit the viral protease, an enzyme used to assemble new viruses. Others block reverse transcriptase (RT), which makes replicates of the genes of the virus. Nucleoside/nucleotide RT inhibitors (NRTIs; for example, zidovudine—also called AZT—and lamivudine) and non-nucleoside RT inhibitors (NNRTIs; for example, nevirapine and efavirenz) interfere with the activity of RT by binding to different sites in its so-called “DNA polymerase domain,” the part of the enzyme that constructs copies of the viral genes. Highly active antiretroviral therapy (HAART), which was introduced in the mid 1990s, combines several antiretrovirals (usually a protease inhibitor and two NRTIs or an NNRTI and two NRTIs) so that the replication of any virus that develops resistance to one drug is inhibited by the other drugs in the mix. When treated with HAART, HIV infection is usually a chronic, stable condition rather than a fatal disease.
Why Was This Study Done?
Unfortunately, HIV that is resistant to drugs still develops in some patients. To improve the prevention and management of drug resistance, a better understanding of the mutations that cause resistance is needed. Resistance to RT inhibitors usually involves mutations in the DNA polymerase domain that reduce the efficacy of NRTIs (including thymidine analogue mutations—also known as TAMs—and lamivudine-resistance mutations) and NNRTIs. Blood tests that detect these resistance mutations (genotype tests) have been used for several years to guide individualized selection of HIV drugs. Recently, however, mutations outside the DNA polymerase domain have also been implicated in resistance to RT inhibitors. In this study, the researchers have used data and samples collected since the mid 1990s by Canada's British Columbia Centre for Excellence in HIV/AIDS to investigate the clinical relevance of a mutation called N348I. This mutation changes an asparagine (a type of amino acid) to an isoleucine in a region of RT known as the connection domain. The researchers have also investigated how this mutation causes resistance to RT inhibitors in laboratory tests.
What Did the Researchers Do and Find?
The researchers analyzed the first two-thirds of the RT gene in viruses isolated from a large number of the Centre's patients. Virus carrying the N348I mutation was present in less than one in 100 patients whose HIV infection had never been treated, but in more than one in 10 treatment-experienced patients. The mutation appeared early in therapy, often in viruses that had TAMs, a lamivudine-resistance mutation called M184V/I, and/or NNRTI resistance mutations. Patients treated with zidovudine and nevirapine were 2.6 times more likely to have the N348I mutation than patients not treated with these drugs. Furthermore, the appearance of the N348I mutation often coincided with an increase in viral load, although other mutations that appeared at a similar time could have contributed to this increase. When the researchers introduced the N348I mutation into HIV growing in the laboratory, they found that it decreased the susceptibility of the virus to zidovudine and to NNRTIs.
What Do These Findings Mean?
These findings show that the treatment of patients with RT inhibitors can select a drug-resistant HIV variant that has a mutation outside the enzyme's DNA polymerase domain. Because this N348I mutation, which is commonly selected in vivo and has also been seen in other studies, confers resistance to two classes of RT inhibitors and can emerge early during therapy, it could have a large impact on patient responses to antiviral regimens that contain zidovudine and nevirapine. Although these findings do not show that the N348I mutation alone causes treatment failure, they may have implications for genotypic and phenotypic resistance testing, which is often used to guide treatment decisions. At present, genotype tests for resistance to RT inhibitors look for mutations only in the DNA polymerase domain of RT. This study is the first to demonstrate that it might be worth looking for the N348I mutation (and for other mutations outside the DNA polymerase domain) to improve the ability of genotypic and phenotypic resistance tests to predict treatment outcomes.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040335.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including links to fact sheets (in English, French, and Spanish) about antiretrovirals, and chapters explaining antiretroviral resistance testing
NAM, a UK registered charity, provides information about all aspects of HIV and AIDS, including fact sheets on types of HIV drugs, drug resistance, and resistance tests (in English, Spanish, French, Portuguese, and Russian)
The US Centers for Disease Control and Prevention provides information on HIV/AIDS and on treatment (in English and Spanish)
AIDSinfo, a service of the US Department of Health and Human Services provides information for patients on HIV and its treatment
doi:10.1371/journal.pmed.0040335
PMCID: PMC2100143  PMID: 18052601
17.  Human Immunodeficiency Virus Type 1 Genomic RNA Sequences in the Female Genital Tract and Blood: Compartmentalization and Intrapatient Recombination 
Journal of Virology  2005;79(1):353-363.
Investigation of human immunodeficiency virus type 1 (HIV-1) in the genital tract of women is crucial to the development of vaccines and therapies. Previous analyses of HIV-1 in various anatomic sites have documented compartmentalization, with viral sequences from each location that were distinct yet phylogenetically related. Full-length RNA genomes derived from different compartments in the same individual, however, have not yet been studied. Furthermore, although there is evidence that intrapatient recombination may occur frequently, recombinants comprising viruses from different sites within one individual have rarely been documented. We compared full-length HIV-1 RNA sequences in the plasma and female genital tract, focusing on a woman with high HIV-1 RNA loads in each compartment who had been infected heterosexually and then transmitted HIV-1 by the same route. We cloned and sequenced 10 full-length HIV-1 RNA genomes from her genital tract and 10 from her plasma. We also compared viral genomes from the genital tract and plasma of four additional heterosexually infected women, sequencing 164 env and gag clones obtained from the two sites. Four of five women, including the one whose complete viral sequences were determined, displayed compartmentalized HIV-1 genomes. Analyses of full-length, compartmentalized sequences made it possible to document complex intrapatient HIV-1 recombinants that were composed of alternating viral sequences characteristic of each site. These findings demonstrate that the genital tract and blood harbor genetically distinct populations of replicating HIV-1 and provide evidence that recombination between strains from the two compartments contributes to rapid evolution of viral sequence variation in infected individuals.
doi:10.1128/JVI.79.1.353-363.2005
PMCID: PMC538688  PMID: 15596829
18.  Impaired Hepatitis C Virus-Specific T Cell Responses and Recurrent Hepatitis C Virus in HIV Coinfection 
PLoS Medicine  2006;3(12):e492.
Background
Hepatitis C virus (HCV)-specific T cell responses are critical for spontaneous resolution of HCV viremia. Here we examined the effect of a lymphotropic virus, HIV-1, on the ability of coinfected patients to maintain spontaneous control of HCV infection.
Methods and Findings
We measured T cell responsiveness by lymphoproliferation and interferon-γ ELISPOT in a large cohort of HCV-infected individuals with and without HIV infection. Among 47 HCV/HIV-1-coinfected individuals, spontaneous control of HCV was associated with more frequent HCV-specific lymphoproliferative (LP) responses (35%) compared to coinfected persons who exhibited chronic HCV viremia (7%, p = 0.016), but less frequent compared to HCV controllers who were not HIV infected (86%, p = 0.003). Preservation of HCV-specific LP responses in coinfected individuals was associated with a higher nadir CD4 count (r2 = 0.45, p < 0.001) and the presence and magnitude of the HCV-specific CD8+ T cell interferon-γ response (p = 0.0014). During long-term follow-up, recurrence of HCV viremia occurred in six of 25 coinfected individuals with prior control of HCV, but in 0 of 16 HIV-1-negative HCV controllers (p = 0.03, log rank test). In these six individuals with recurrent HCV viremia, the magnitude of HCV viremia following recurrence inversely correlated with the CD4 count at time of breakthrough (r = −0.94, p = 0.017).
Conclusions
These results indicate that HIV infection impairs the immune response to HCV—including in persons who have cleared HCV infection—and that HIV-1-infected individuals with spontaneous control of HCV remain at significant risk for a second episode of HCV viremia. These findings highlight the need for repeat viral RNA testing of apparent controllers of HCV infection in the setting of HIV-1 coinfection and provide a possible explanation for the higher rate of HCV persistence observed in this population.
HIV infection impairs the immune response to HCV. Even individuals who have cleared HCV infection remain at significant risk for a second episode of HCV viremia.
Editors' Summary
Background.
Because of shared transmission routes (contaminated needles, contaminated blood products, and, to a lesser extent, unprotected sex), a large proportion of HIV-infected individuals (estimates range between 25% and 33%) are also infected with the hepatitis C virus (HCV). In most but not all individuals infected with HCV, the virus infection is chronic and causes liver disease that can eventually lead to liver failure. Disease progress is slow; it often takes decades until infected individuals develop serious liver disease. In people infected with both HCV and HIV, however, liver disease caused by HCV often appears sooner and progresses faster. As highly active antiretroviral therapy (HAART) and prophylaxis of opportunistic infections increase the life span of persons living with HIV, HCV-related liver disease has become a major cause of hospital admissions and deaths among HIV-infected persons.
Why Was This Study Done?
A sizable minority of people who are infected with HCV manage to control the virus and never get liver disease, and scientists have found that these people somehow mounted a strong immune response against the hepatitis C virus. CD4+ T cells, the very immune cells that are infected and destroyed by HIV, play an important role in this immune response. The goal of the present study was to better understand how infection with HIV compromises the specific immune response to HCV and thereby the control of HCV disease progression.
What Did the Researchers Do and Find?
The researchers recruited four groups of patients, 94 in total, all of whom were infected with HCV. Two groups comprised patients who were infected with HIV as well as HCV, with either high or undetectable levels of HCV (30 patients in each group). The two other groups included patients not infected with HIV, either with high or undetectable levels of HCV (17 patients in each group). The researchers focused on the individuals who, despite coinfection with HIV, were able to control their HCV infection. They found that those individuals managed to maintain relatively high levels of CD4+ T cells that specifically recognize HCV. However, a quarter of these patients (six out of 25) failed to keep HCV levels down for the entire observation period of up to 2.5 years; their blood levels of HCV rose substantially, most likely due to recurrence of the previously suppressed virus (the researchers could not be certain that none of the patients had become infected again after a new exposure to HCV-contaminated blood, but there was no evidence that they had engaged in risky behavior). The rise of HCV levels in the blood of the relapsed patients coincided with a drop in overall CD4+ T cell numbers. Following relapse in these individuals, HCV did not return to undetectable levels during the study. During the same period none of the 16 HIV-uninfected people with controlled HCV infection experienced a recurrence of detectable HCV.
What Do These Findings Mean?
Despite the relatively small numbers of patients, these results suggest that recurrence of HCV after initial control of the virus is more likely in people who are coinfected with HIV, and that HCV control is lost when CD4+ T cell counts fall. This is one more reason to test all HIV-positive patients for HCV coinfection. Coinfected patients, even those who seem to be controlling HCV and would not automatically receive HCV treatment, should be regularly tested for a rise of HCV levels. In addition, maintaining CD4+ T cells at a high level might be particularly important for those patients, which means that doctors might consider starting HAART therapy earlier than is generally recommended for HIV-infected individuals. Additional studies are needed to support these recommendations, however, especially as this study did not follow the patients long enough to determine the consequences of the observed loss of control of HCV.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030492.
AIDS Treatment Data Network factsheet on HIV/HCV coinfection
US CDC factsheet on HIV/HCV coinfection
American Liver Foundation, information on HIV and HCV
MedlinePlus pages on HCV
doi:10.1371/journal.pmed.0030492
PMCID: PMC1705826  PMID: 17194190
19.  Compartmentalization and Clonal Amplification of HIV-1 Variants in the Cerebrospinal Fluid during Primary Infection▿  
Journal of Virology  2009;84(5):2395-2407.
Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is a severe neurological disease that affects a subset of HIV-1-infected individuals. Increased compartmentalization has been reported between blood and cerebrospinal fluid (CSF) HIV-1 populations in subjects with HAD, but it is still not known when compartmentalization arises during the course of infection. To assess HIV-1 genetic compartmentalization early during infection, we compared HIV-1 populations in the peripheral blood and CSF in 11 primary infection subjects, with analysis of longitudinal samples over the first 18 months for a subset of subjects. We used heteroduplex tracking assays targeting the variable regions of env and single-genome amplification and sequence analysis of the full-length env gene to identify CSF-compartmentalized variants and to examine viral genotypes within the compartmentalized populations. For most subjects, HIV-1 populations were equilibrated between the blood and CSF compartments. However, compartmentalized HIV-1 populations were detected in the CSF of three primary infection subjects, and longitudinal analysis of one subject revealed that compartmentalization during primary HIV-1 infection was resolved. Clonal amplification of specific HIV-1 variants was identified in the CSF population of one primary infection subject. Our data show that compartmentalization can occur in the central nervous system (CNS) of subjects in primary HIV-1 infection in part through persistence of the putative transmitted parental variant or via viral genetic adaptation to the CNS environment. The presence of distinct HIV-1 populations in the CSF indicates that independent HIV-1 replication can occur in the CNS, even early after HIV-1 transmission.
doi:10.1128/JVI.01863-09
PMCID: PMC2820937  PMID: 20015984
20.  The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions Within the V2 Loop of gp120 
AIDS Research and Human Retroviruses  2012;28(11):1444-1457.
Abstract
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
doi:10.1089/aid.2012.0103
PMCID: PMC3484815  PMID: 23035746
21.  Efficacy of Short-Course AZT Plus 3TC to Reduce Nevirapine Resistance in the Prevention of Mother-to-Child HIV Transmission: A Randomized Clinical Trial 
PLoS Medicine  2009;6(10):e1000172.
Neil Martinson and colleagues report a randomized trial of adding short-course zidovudine+lamivudine to reduce drug resistance from single-dose nevirapine used to prevent mother-to-child transmission of HIV.
Background
Single-dose nevirapine (sdNVP)—which prevents mother-to-child transmission of HIV—selects non-nucleoside reverse-transcriptase inhibitor (NNRTI) resistance mutations in the majority of women and HIV-infected infants receiving it. This open-label, randomised trial examined the efficacy of short-course zidovudine (AZT) and lamivudine (3TC) with sdNVP in reducing NNRTI resistance in mothers, and as a secondary objective, in infants, in a setting where sdNVP was standard-of-care.
Methods and Findings
sdNVP alone, administered at the onset of labour and to the infant, was compared to sdNVP with AZT plus 3TC, given as combivir (CBV) for 4 (NVP/CBV4) or 7 (NVP/CBV7) days, initiated simultaneously with sdNVP in labour; their newborns received the same regimens. Women were randomised 1∶1∶1. HIV-1 resistance was assessed by population sequencing at: baseline, 2, and 6 wk after birth. An unplanned interim analysis resulted in early stopping of the sdNVP arm. 406 pregnant women were randomised and took study medication (sdNVP 74, NVP/CBV4 164, and NVP/CBV7 168). HIV-1 resistance mutations emerged in 59.2%, 11.7%, and 7.3% of women in the sdNVP, NVP/CBV4, and NVP/CBV7 arms by 6 wk postpartum; differences between NVP-only and both NVP/CBV arms were significant (p<0.0001), but the difference between NVP/CBV4 and NVP/CBV7 was not (p = 0.27). Estimated efficacy comparing combined CBV arms with sdNVP was 85.6%. Similar resistance reductions were seen in infants who were HIV-infected by their 6-wk visit.
Conclusions
A short course of AZT plus 3TC, supplementing maternal and infant sdNVP, reduces emergent NNRTI resistance mutations in both mothers and their infants. However, this trial was not powered to detect small differences between the CBV arms.
Trial registration
www.ClinicalTrials.gov NCT 00144183
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Currently, about 33 million people are infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV can be treated with combination antiretroviral therapy (ART), commonly three individual antiretroviral drugs that together efficiently suppress the replication of the virus. HIV infection of a child by an HIV-positive mother during pregnancy, labor, delivery, or breastfeeding is called mother-to-child transmission (MTCT). In 2007, an estimated 420,000 children were newly infected with HIV, the majority through MTCT. Most of these mothers and children live in sub-Saharan Africa where child and maternal mortality rates are high and mortality in HIV-infected children is extremely high. MTCT is preventable and there is a global commitment, agreed at the UN General Assembly Session on HIV/AIDS in 2001, to reduce the proportion of infants infected with HIV by 50% by 2010.
Why Was This Study Done?
In many resource-limited settings, MTCT is prevented by giving a single dose of nevirapine (an antiretroviral drug which has a long duration in the body and protects the fetus during labor and delivery only) to HIV-infected women in labor and also to a baby within 72 hours of birth. However, nevirapine, a non-nucleoside reverse-transcriptase inhibitor (NNRTI), which suppresses the replication of the virus, is associated with increased resistance of HIV, in mother and child, to NNRTI. This resistance reduces the effectiveness of future treatments of both mother and child with combination ART that includes an NNRTI; such regimens are the mainstay for long-term treatment of HIV in developing countries. The researchers investigated whether giving other antiretroviral drugs with nevirapine, during labor and delivery, to both mother and her newborn reduced the chances of them developing resistance to NNRTIs.
What Did the Researchers Do and Find?
The researchers selected 406 HIV-positive pregnant women for study across five sites in South Africa between February 2003 and May 2007. The women and their newborn babies were randomly assigned to receive, either (i) a single dose of nevirapine, (ii) a single dose of nevirapine plus combivir (zidovudine combined with lamivudine) for four days, or (iii) a single dose of nevirapine plus combivir for seven days. At two days, two weeks, and six weeks after delivery blood was collected from mothers and babies. HIV virus from blood samples was analyzed for resistance mutations, and mothers and children with resistance mutations were monitored for a further 96 weeks until no resistance was detected or combination ART (also called “HAART”) was started. Enrollment into the single-dose nevirapine arm was stopped early because a very high rate of NNRTI resistance mutations was found and other investigators reported long-term bad consequences of NNRTI-resistance on subsequent ART. The two nevirapine plus combivir arms were continued. The researchers found that selection of resistance mutations by single-dose nevirapine was reduced in mother and child by the addition of zidovudine and lamivudine for a short period; resistance mutations were found in 59.2% of women who got nevirapine only but only 11.7%, and 7.3% of women treated nevirapine plus four days combivir, and nevirapine plus seven days combivir respectively. A reduction was also seen in new NNRTI resistant mutations in the HIV-infected infants that received combivir. The study did not have enough women to show that there was a real difference between the resistance in the four-day and seven-day combivir regimens.
What Do These Findings Mean?
These findings show that a short-course treatment of zidovudine and lamivudine in addition to a single dose of nevirapine during labor and birth reduces the selection of NNRTI resistance mutations in both mother and child. The drug regimens appeared safe, and easy to provide and adhere to. Preliminary results from this study contributed to a change in clinical practice for the care of pregnant women with HIV; in 2004 the World Health Organisation guidelines introduced a short course of combivir with nevirapine for the management of pregnant HIV-infected women. However, the study had some limitations. It used HIV-positive women who were mainly infected with a subtype of HIV called HIV-1 clade C and who had a lot of virus in their blood. NNRTI resistance after treatment with nevirapine is more common in clade C than in others and this study does not address the effect of these combinations for preventing NNRTI resistance in other HIV subtypes. Also, World Health Organization, national, and international guidelines recommend combination ART during pregnancy, as it decreases HIV transmission from mother to child in the uterus to <2% in resource-limited settings. Although long-term combination treatment may not be available in all locations, this study does not tell us how the short-term combinations during and after delivery tested would compare to longer-term combinations given to pregnant women in reducing both HIV transmission and HIV drug resistance.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000172.
This study is further discussed in a PLoS Medicine Perspective by Lehman et al.
The US Centers for Disease Control and Prevention provide information for HIV treatment and prevention
MedlinePlus provides extensive information on symptoms and treatment for HIV/AIDS as well as access to related clinical trials and medical literature
aidsmap, a nonprofit, nongovernmental organization provides information on HIV and supporting those living with HIV
The World Health Organization gives information on the prevention of mother-to-child transmission of HIV
doi:10.1371/journal.pmed.1000172
PMCID: PMC2760761  PMID: 19859531
22.  Evidence for Limited Genetic Compartmentalization of HIV-1 between Lung and Blood 
PLoS ONE  2009;4(9):e6949.
Background
HIV-1 is frequently detected in the lungs of infected individuals and is likely important in the development of pulmonary opportunistic infections. The unique environment of the lung, rich in alveolar macrophages and with specialized local immune responses, may contribute to differential evolution or selection of HIV-1.
Methodology and Findings
We characterized HIV-1 in the lung in relation to contemporaneous viral populations in the blood. The C2-V5 region of HIV-1 env was sequenced from paired lung (induced sputum or bronchoalveolar lavage) and blood (plasma RNA and proviral DNA from sorted or unsorted PBMC) from 18 subjects. Compartmentalization between tissue pairs was assessed using 5 established tree or distance-based methods, including permutation tests to determine statistical significance. We found statistical evidence of compartmentalization between lung and blood in 10/18 subjects, although lung and blood sequences were intermingled on phylogenetic trees in all subjects. The subject showing the greatest compartmentalization contained many nearly identical sequences in BAL sample, suggesting clonal expansion may contribute to reduced viral diversity in the lung in some cases. However, HIV-1 sequences in lung were not more homogeneous overall, nor were we able to find a lung-specific genotype associated with macrophage tropism in V3. In all four subjects in whom predicted X4 genotypes were found in blood, predicted X4 genotypes were also found in lung.
Conclusions
Our results support a picture of continuous migration of HIV-1 between circulating blood and lung tissue, with perhaps a very limited degree of localized evolution or clonal replication.
doi:10.1371/journal.pone.0006949
PMCID: PMC2736399  PMID: 19759830
23.  Compartmentalized Human Immunodeficiency Virus Type 1 Originates from Long-Lived Cells in Some Subjects with HIV-1–Associated Dementia 
PLoS Pathogens  2009;5(4):e1000395.
Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) shortly after systemic infection and can result in the subsequent development of HIV-1–associated dementia (HAD) in a subset of infected individuals. Genetically compartmentalized virus in the CNS is associated with HAD, suggesting autonomous viral replication as a factor in the disease process. We examined the source of compartmentalized HIV-1 in the CNS of subjects with HIV-1–associated neurological disease and in asymptomatic subjects who were initiating antiretroviral therapy. The heteroduplex tracking assay (HTA), targeting the variable regions of env, was used to determine which HIV-1 genetic variants in the cerebrospinal fluid (CSF) were compartmentalized and which variants were shared with the blood plasma. We then measured the viral decay kinetics of individual variants after the initiation of antiretroviral therapy. Compartmentalized HIV-1 variants in the CSF of asymptomatic subjects decayed rapidly after the initiation of antiretroviral therapy, with a mean half-life of 1.57 days. Rapid viral decay was also measured for CSF-compartmentalized variants in four HAD subjects (t1/2 mean = 2.27 days). However, slow viral decay was measured for CSF-compartmentalized variants from an additional four subjects with neurological disease (t1/2 range = 9.85 days to no initial decay). The slow decay detected for CSF-compartmentalized variants was not associated with poor CNS drug penetration, drug resistant virus in the CSF, or the presence of X4 virus genotypes. We found that the slow decay measured for CSF-compartmentalized variants in subjects with neurological disease was correlated with low peripheral CD4 cell count and reduced CSF pleocytosis. We propose a model in which infiltrating macrophages replace CD4+ T cells as the primary source of productive viral replication in the CNS to maintain high viral loads in the CSF in a substantial subset of subjects with HAD.
Author Summary
Infection of the central nervous system (CNS) with human immunodeficiency virus type 1 (HIV-1) can lead to the development of HIV-1–associated dementia, a severe neurological disease that results in cognitive and motor impairment. Individuals that are chronically infected with HIV-1 sometimes display unique viral variants in their cerebrospinal fluid (CSF) that are not detected in the blood virus population, termed CSF-compartmentalized variants. The cell type that produces CSF-compartmentalized virus throughout the course of infection has not been determined. We used a sensitive assay to detect compartmentalized variants in the CSF of subjects with and without neurological disease, and then measured the decay kinetics of compartmentalized virus when subjects were starting antiretroviral therapy. We found that compartmentalized virus decays rapidly in asymptomatic subjects. Additionally, we detected differential decay (i.e. rapid or slow) in subjects with neurological disease, and this was associated with the number of white blood cells in the CSF. Our data supports a model of HIV-1 infection in the CNS where compartmentalized virus is produced by a long-lived cell type (slow decay), and this virus can be amplified by short-lived cells (rapid decay) that traffic into the CNS, but is increasingly produced from long-lived cells in the immunodeficient state.
doi:10.1371/journal.ppat.1000395
PMCID: PMC2668697  PMID: 19390619
24.  HIV-1 Populations in Semen Arise through Multiple Mechanisms 
PLoS Pathogens  2010;6(8):e1001053.
HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus.
Author Summary
The work described in this report is directed at how HIV-1 viral RNA populations differ between the blood plasma and male genital tract in established infection. This site is of special interest since it is the proximal source of most transmissions of HIV-1. Thus, lessons learned about HIV-1 in the seminal tract are directly relevant to the mechanism of HIV-1 transmission. We have used single genome amplification to generate viral sequences from paired blood and semen samples in men with chronic HIV-1 infection. When compared to viral populations in blood plasma, we observe that virus in the seminal plasma can be equilibrated, clonally-amplified, or compartmentalized. We have also performed a characterization of the cytokine and chemokine milieu in these two compartments. We report a dramatic concentration of immune modulators in the seminal plasma relative to the blood, and these likely enhance the potential for viral replication in this compartment by creating an environment where target cells are kept in an activated state. These data define new and distinct features of virus:host interactions and represent a significant advance in our understanding of HIV-1 replication in the male genital tract.
doi:10.1371/journal.ppat.1001053
PMCID: PMC2924360  PMID: 20808902
25.  Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns. 
Journal of Virology  1994;68(4):2230-2238.
Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered in FIV vaccine and pathogenesis studies.
PMCID: PMC236699  PMID: 8139008

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