Analysis of susceptibility to human immunodeficiency virus 1 (HIV-1) by comparative genetics

Summary [résumé français voir ci-dessous] From the beginning of the 20th century the world population has been confronted with the human immune deficiency virus 1 (HIV-1). This virus has the particularity to mutate fast, and could thus evade and adapt to the human host. Our closest evolutionary related organisms, the non-human primates, are less susceptible to HIV-1. In a broader sense, primates are differentially susceptible to various retrovirus. Species specificity may be due to genetic differences among primates. In the present study we applied evolutionary and comparative genetic techniques to characterize the evolutionary pattern of host cellular determinants of HIV-1 pathogenesis. The study of the evolution of genes coding for proteins participating to the restriction or pathogenesis of HIV-1 may help understanding the genetic basis of modern human susceptibility to infection. To perform comparative genetics analysis, we constituted a collection of primate DNA and RNA to allow generation of de novo sequence of gene orthologs. More recently, release to the public domain of two new primate complete genomes (bornean orang-utan and common marmoset) in addition of the three previously available genomes (human, chimpanzee and Rhesus monkey) help scaling up the evolutionary and comparative genome analysis. Sequence analysis used phylogenetic and statistical methods for detecting molecular adaptation. We identified different selective pressures acting on host proteins involved in HIV-1 pathogenesis. Proteins with HIV-1 restriction properties in non-human primates were under strong positive selection, in particular in regions of interaction with viral proteins. These regions carried key residues for the antiviral activity. Proteins of the innate immunity presented an evolutionary pattern of conservation (purifying selection) but with signals of relaxed constrain if we compared them to the average profile of purifying selection of the primate genomes. Large scale analysis resulted in patterns of evolutionary pressures according to molecular function, biological process and cellular distribution. The data generated by various analyses served to guide the ancestral reconstruction of TRIM5a a potent antiviral host factor. The resurrected TRIM5a from the common ancestor of Old world monkeys was effective against HIV-1 and the recent resurrected hominoid variants were more effective against other retrovirus. Thus, as the result of trade-offs in the ability to restrict different retrovirus, human might have been exposed to HIV-1 at a time when TRIM5a lacked the appropriate specific restriction activity. The application of evolutionary and comparative genetic tools should be considered for the systematical assessment of host proteins relevant in viral pathogenesis, and to guide biological and functional studies. Résumé La population mondiale est confrontée depuis le début du vingtième siècle au virus de l'immunodéficience humaine 1 (VIH-1). Ce virus a un taux de mutation particulièrement élevé, il peut donc s'évader et s'adapter très efficacement à son hôte. Les organismes évolutivement le plus proches de l'homme les primates nonhumains sont moins susceptibles au VIH-1. De façon générale, les primates répondent différemment aux rétrovirus. Cette spécificité entre espèces doit résider dans les différences génétiques entre primates. Dans cette étude nous avons appliqué des techniques d'évolution et de génétique comparative pour caractériser le modèle évolutif des déterminants cellulaires impliqués dans la pathogenèse du VIH- 1. L'étude de l'évolution des gènes, codant pour des protéines impliquées dans la restriction ou la pathogenèse du VIH-1, aidera à la compréhension des bases génétiques ayant récemment rendu l'homme susceptible. Pour les analyses de génétique comparative, nous avons constitué une collection d'ADN et d'ARN de primates dans le but d'obtenir des nouvelles séquences de gènes orthologues. Récemment deux nouveaux génomes complets ont été publiés (l'orang-outan du Bornéo et Marmoset commun) en plus des trois génomes déjà disponibles (humain, chimpanzé, macaque rhésus). Ceci a permis d'améliorer considérablement l'étendue de l'analyse. Pour détecter l'adaptation moléculaire nous avons analysé les séquences à l'aide de méthodes phylogénétiques et statistiques. Nous avons identifié différentes pressions de sélection agissant sur les protéines impliquées dans la pathogenèse du VIH-1. Des protéines avec des propriétés de restriction du VIH-1 dans les primates non-humains présentent un taux particulièrement haut de remplacement d'acides aminés (sélection positive). En particulier dans les régions d'interaction avec les protéines virales. Ces régions incluent des acides aminés clé pour l'activité de restriction. Les protéines appartenant à l'immunité inné présentent un modèle d'évolution de conservation (sélection purifiante) mais avec des traces de "relaxation" comparé au profil général de sélection purifiante du génome des primates. Une analyse à grande échelle a permis de classifier les modèles de pression évolutive selon leur fonction moléculaire, processus biologique et distribution cellulaire. Les données générées par les différentes analyses ont permis la reconstruction ancestrale de TRIM5a, un puissant facteur antiretroviral. Le TRIM5a ressuscité, correspondant à l'ancêtre commun entre les grands singes et les groupe des catarrhiniens, est efficace contre le VIH-1 moderne. Les TRIM5a ressuscités plus récents, correspondant aux ancêtres des grands singes, sont plus efficaces contre d'autres rétrovirus. Ainsi, trouver un compromis dans la capacité de restreindre différents rétrovirus, l'homme aurait été exposé au VIH-1 à une période où TRIM5a manquait d'activité de restriction spécifique contre celui-ci. L'application de techniques d'évolution et de génétique comparative devraient être considérées pour l'évaluation systématique de protéines impliquées dans la pathogenèse virale, ainsi que pour guider des études biologiques et fonctionnelles

Use of a combined ex vivo/in vivo population approach for screening of human genes involved in the human immunodeficiency virus type 1 life cycle for variants influencing disease progression.

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (n = 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (n = 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of PML, TSG101, and PPIA as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/mul to <200 CD4 T cells/mul.

Orosomucoid (alpha1-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation.

BACKGROUND AND OBJECTIVE: Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. METHODS: Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. RESULTS: Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. CONCLUSION: ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

To say or not to say: a qualitative study on the disclosure of their condition by human immunodeficiency virus-positive adolescents.

PURPOSE: Human immunodeficiency virus (HIV)-positive adolescents face a number of challenges in dealing with their disease, treatment, and developmental tasks. This qualitative study describes some of the reasons why, and the extent to which, adolescents may or may not disclose their condition to others. METHODS: A semistructured interview lasting 40-110 minutes was conducted with each of 29 adolescents 12-20 years old, 22 female and seven male) living in Switzerland. Interviews were tape recorded and transcribed verbatim. The analysis of the content of interviews allowed us to identify salient topics (e.g., disclosure), which were then explored in detail. RESULTS: Of 29 participants, eight had not disclosed their condition to anyone outside the family, 19 had disclosed it to good friends, and 16 had disclosed it to some teachers. Four participants had engaged in public disclosure, and six of 10 sexually active teenagers disclosed their status to their partners. The attitudes toward disclosure among younger adolescents were mostly related to those of the parents, particularly the mother. Older adolescents, engaged in their search for autonomy, tended to decide independently what to say and to whom. Although foster/adoptive parents would often encourage disclosure, biological parents, especially HIV-positive mothers, insisted on not disclosing the adolescent's status for fear of stigma. CONCLUSION: The health care team should systematically address the issue of disclosure with the adolescent and his family (or foster parents), the aim being to balance the right of the adolescent and that adolescent's family to maintain privacy against the concerns of sexual partners, as well as the adolescent's interest in divulging HIV status to relatives, school staff, and friends.

Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection.

Isolated primary human cells from different donors vary in their permissiveness-the ability of cells to be infected and sustain the replication of human immunodeficiency virus type 1 (HIV-1). We used replicating HIV-1 and single-cycle lentivirus vectors in a population approach to identify polymorphic steps during viral replication. We found that phytohemagglutinin-stimulated CD4(+) CD45RO(+) CD57(-) T cells from healthy blood donors (n = 128) exhibited a 5.2-log-unit range in virus production. For 20 selected donors representing the spectrum of CD4 T-cell permissiveness, we could attribute up to 42% of the total variance in virus production to entry factors and 48% to postentry steps. Efficacy at key intracellular steps of the replicative cycle (reverse transcription, integration, transcription and splicing, translation, and budding and release) varied from 0.71 to 1.45 log units among donors. However, interindividual differences in transcription efficiency alone accounted for 64 to 83% of the total variance in virus production that was attributable to postentry factors. While vesicular stomatitis virus G protein-mediated fusion was more efficacious than CCR5/CD4 entry, the latter resulted in greater transcriptional activity per proviral copy. The phenotype of provirus transcription was stable over time, indicating that it represents a genetic trait.

Analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stem-loop structure.

Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein. Frameshift activities are thought to be tightly regulated. Analysis of gag p1 sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop. Expression in Saccharomyces cerevisiae of p1-beta-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability [Delta G degrees] ranging from -23.0 to -4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (Delta G degrees, -23.5 kcal/mol). Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication. In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit.

Comparison of human and rhesus macaque T-cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens.

Rhesus macaques (Macaca mulatta) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-gamma) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4(+) and CD8(+) T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-gamma, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-gamma T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved.

Silencing of both beta-TrCP1 and HOS (beta-TrCP2) is required to suppress human immunodeficiency virus type 1 Vpu-mediated CD4 down-modulation.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein interacts with CD4 within the endoplasmic reticula of infected cells and targets CD4 for degradation through interaction with beta-TrCP1. Mammals possess a homologue of beta-TrCP1, HOS, which is also named beta-TrCP2. We show by coimmunoprecipitation experiments that beta-TrCP2 binds Vpu and is able to induce CD4 down-modulation as efficiently as beta-TrCP1. In two different cell lines, HeLa CD4+ and Jurkat, Vpu-mediated CD4 down-modulation could not be reversed through the individual silencing of endogenous beta-TrCP1 or beta-TrCP2 but instead required the two genes to be silenced simultaneously.

Interleukin-1 receptor antagonist gene polymorphism and circulating levels of human immunodeficiency virus type 1 RNA in Brazilian women.

Interleukin-1 receptor antagonist (IL-1ra) gene polymorphisms in 83 human immunodeficiency virus (HIV)-seropositive women were evaluated. Fourteen of the subjects (16.9%) were homozygous for IL-1ra allele 2 (IL-1RN*2). These women had a lower median level of HIV RNA than did women homozygous for allele 1 (IL-1RN*1) (P = 0.01) or heterozygous for both alleles (P = 0.04). Among 46 subjects not receiving antiretroviral treatment, HIV levels were also reduced in IL-1RN*2 homozygous individuals (P < 0.05). There was no relation between IL-1ra alleles and CD4 levels.

Determinants of vaccine immunity in the cohort of human immunodeficiency virus-infected children living in Switzerland.

BACKGROUND: Human immunodeficiency virus (HIV)-infected children are at increased risk of infections caused by vaccine preventable pathogens, and specific immunization recommendations have been issued. METHODS: A prospective national multicenter study assessed how these recommendations are followed in Switzerland and how immunization history correlates with vaccine immunity. RESULTS: Among 87 HIV-infected children (mean age: 11.1 years) followed in the 5 Swiss university hospitals and 1 regional hospital, most (76%) had CD4 T cells >25%, were receiving highly active antiretroviral treatment (79%) and had undetectable viral load (60%). Immunization coverage was lower than in the general population and many lacked serum antibodies to vaccine-preventable pathogens, including measles (54%), varicella (39%), and hepatitis B (65%). The presence of vaccine antibodies correlated most significantly with having an up-to-date immunization history (P<0.05). An up-to-date immunization history was not related to age, immunologic stage, or viremia but to the referral medical center. CONCLUSIONS: All pediatricians in charge of HIV-infected children are urged to identify missing immunizations in this high-risk population.

Impact of phenotype definition on genome-wide association signals: empirical evaluation in human immunodeficiency virus type 1 infection.

Discussion on improving the power of genome-wide association studies to identify candidate variants and genes is generally centered on issues of maximizing sample size; less attention is given to the role of phenotype definition and ascertainment. The authors used genome-wide data from patients infected with human immunodeficiency virus type 1 (HIV-1) to assess whether differences in type of population (622 seroconverters vs. 636 seroprevalent subjects) or the number of measurements available for defining the phenotype resulted in differences in the effect sizes of associations between single nucleotide polymorphisms and the phenotype, HIV-1 viral load at set point. The effect estimate for the top 100 single nucleotide polymorphisms was 0.092 (95% confidence interval: 0.074, 0.110) log(10) viral load (log(10) copies of HIV-1 per mL of blood) greater in seroconverters than in seroprevalent subjects. The difference was even larger when the authors focused on chromosome 6 variants (0.153 log(10) viral load) or on variants that achieved genome-wide significance (0.232 log(10) viral load). The estimates of the genetic effects tended to be slightly larger when more viral load measurements were available, particularly among seroconverters and for variants that achieved genome-wide significance. Differences in phenotype definition and ascertainment may affect the estimated magnitude of genetic effects and should be considered in optimizing power for discovering new associations.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

Awareness of human immunodeficiency virus screening guidelines is low in emergency departments of western Switzerland.

Background: Human immunodeficiency virus (HIV) prevalence in Switzerland is 0.4% and 30% of HIV patients are diagnosed with CD4 counts <200 cells/microliter. In 2010, the Swiss Federal Office of Public Health (SFOP) published updated guidelines regarding Physician- Initiated Counseling and Testing (PICT) for HIV. In the new guidelines, when acute HIV infection is suspected or HIV is among the differential diagnoses, an HIV test is performed without risk assessment nor prior counseling, unless the patient specifically refuses it. Counseling and verbal consent are still required when the patient asks for an HIV test or belongs to a high risk group. Whist HIV testing in the emergency departments (ED) is recommended, only 1% of patients are currently screened. Lack of awareness among physicians has been cited in the literature as the first barrier to guideline implementation. Objectives: To test if physicians working in EDs of 5 large teaching hospitals in western Switzerland, admitting 175,000 patients / year, were aware of the updated SFOP guidelines. Methods: A survey was delivered to 167 ED physicians in the summer of 2011. The survey consisted of 26 vignettes designed to test whether physicians would request an HIV test according to the new guidelines and if they knew when the PICT strategy was allowed or counseling required. Finally, physicians were asked the number of HIV tests they had requested in the previous 4 weeks, and if they were aware of the new HIV guidelines. Results are presented as mean and standard deviation, median and interquartile range (IQR), or as proportions; Student's t test was used to compare continuous variables; Results: 143 physicians returned the survey (86%); mean age was 32 ± 8 years, and median postgraduate experience of 6 years (IQR 3-12); 52% were male and 17% were attendings. The percentage of correct responses was 60 ± 13% with no difference between attendings and residents (p = 0.31); 2 of the 3 questions with the lowest scores were failure to recognize situations in which HIV testing was indicated, and the third one a failure to recognize acute HIV infection. 82% of physicians were not aware of the new guidelines. The median number of test requests was 1 (IQR 0-2, range 1-10). Conclusion: ED physicians are not aware of current HIV screening guidelines published by the SFOP, and rarely perform HIV tests. An information campaign is required if ED physicians are expected to play a significant role in the reduction of undiagnosed HIV patients.

A new multidisciplinary home care telemedicine system to monitor stable chronic human immunodeficiency virus-infected patients: a randomized study.

Background: Antiretroviral therapy has changed the natural history of human immunodeficiency virus (HIV) infection in developed countries, where it has become a chronic disease. This clinical scenario requires a new approach to simplify follow-up appointments and facilitate access to healthcare professionals. Methodology: We developed a new internet-based home care model covering the entire management of chronic HIV-infected patients. This was called Virtual Hospital. We report the results of a prospective randomised study performed over two years, comparing standard care received by HIV-infected patients with Virtual Hospital care. HIV-infected patients with access to a computer and broadband were randomised to be monitored either through Virtual Hospital (Arm I) or through standard care at the day hospital (Arm II). After one year of follow up, patients switched their care to the other arm. Virtual Hospital offered four main services: Virtual Consultations, Telepharmacy, Virtual Library and Virtual Community. A technical and clinical evaluation of Virtual Hospital was carried out. Findings: Of the 83 randomised patients, 42 were monitored during the first year through Virtual Hospital (Arm I) and 41 through standard care (Arm II). Baseline characteristics of patients were similar in the two arms. The level of technical satisfaction with the virtual system was high: 85% of patients considered that Virtual Hospital improved their access to clinical data and they felt comfortable with the videoconference system. Neither clinical parameters [level of CD4 + T lymphocytes, proportion of patients with an undetectable level of viral load (p = 0.21) and compliance levels 90% (p = 0.58)] nor the evaluation of quality of life or psychological questionnaires changed significantly between the two types of care. Conclusions: Virtual Hospital is a feasible and safe tool for the multidisciplinary home care of chronic HIV patients. Telemedicine should be considered as an appropriate support service for the management of chronic HIV infection.