V3-Loop genotypes do not predict maraviroc susceptibility of CCR5-tropic virus or clinical response through week 48 in HIV-1-infected, treatment-experienced persons receiving optimized background regimens.

Viruses from 15 of 35 maraviroc-treated participants with virologic failure and CCR5-tropic (R5) virus in the MOTIVATE studies at Week 24 had reduced maraviroc susceptibility. On-treatment amino acid changes were observed in the viral envelope glycoprotein 120 third variable (V3)-loop stems and tips and differed between viruses. No amino acid change reliably predicted reduced susceptibility, indicating that resistance was genetic context-dependent. Through Week 24, poor adherence was associated with maraviroc-susceptible virologic failure, whereas reduced maraviroc susceptibility was associated with suboptimal background regimen activity, highlighting the importance of overall regimen activity and good adherence. Predictive values of pretreatment V3-loop sequences containing these Week 24 mutations or other variants present at >3% in pretreatment viruses of participants with virologic failure at Week 48 were retrospectively assessed. Week 48 clinical outcomes were evaluated for correlates with pretreatment V3-loop CCR5-tropic sequences from 704 participants (366 responders; 338 virologic failures [83 with R5 virus with maraviroc susceptibility assessment]). Seventy-five amino acid variants with >3% prevalence were identified among 23 V3-loop residues. Previously identified variants associated with resistance in individual isolates were represented, but none were associated reliably with virologic failure alone or in combination. Univariate analysis showed virologic-failure associations with variants 4L, 11R, and 19S (P < 0.05). However, 11R is a marker for CXCR4 tropism, whereas neither 4L nor 19S was reliably associated with reduced maraviroc susceptibility in R5 failure. These findings from a large study of V3-loop sequences confirm lack of correlation between V3-loop genotype and clinical outcome in participants treated with maraviroc.Clinical trial registration numbers (ClinicalTrials.gov): NCT00098306 and NCT00098722.

Highly prevalent Russian HIV-1 V3-loop sequence variants are susceptible to maraviroc.

INTRODUCTION: Maraviroc inhibits CCR5-tropic HIV-1 across different subtypes in vitro and has demonstrated efficacy in clinical trials. V3-loop amino acid variants observed in individual maraviroc-resistant viruses have not been found to be predictive of reduced susceptibility. Sequence-database searches have demonstrated that approximately 7.3% of viruses naturally encode these variants, raising concerns regarding potential pre-existing resistance. A study from Russia reported that combinations of these same amino acids are present in the V3 loops of the Russian variant subtype A (IDU-A, now A6) with a much greater prevalence (range: 74.4%-92.3%) depending on the combination. However, these studies and database searches did not include phenotypic evaluation. METHODS: Sixteen Russian HIV-1 isolates (including sub-subtype A6 viruses) were assessed for V3 loop sequence and phenotypic susceptibility to maraviroc. RESULTS: All 12 of the A6 viruses and 2/4 subtype B isolates encoded V3-loop variants that have previously been identified in individual virus isolates with reduced susceptibility to maraviroc. However, despite the prevalence of these V3-loop amino acid variants among the tested viruses, phenotypic sensitivity to maraviroc was observed in all instances. Similarly, reduced susceptibility to maraviroc was not found in virus from participants who experienced virologic failure in a clinical study of maraviroc in Russia (A4001101, [NCT01275625]). DISCUSSION: Altogether, these data confirm that the presence of individual or combinations of V3-loop amino acid residues in sub-subtype A6 viruses alone does not predict natural resistance to maraviroc and that V3-loop genotype analysis of R5 virus prior to treatment is not helpful in predicting clinical outcome.

The innate immune response, clinical outcomes, and ex vivo HCV antiviral efficacy of a TLR7 agonist (PF-4878691).

Hepatitis C virus (HCV) infection is an issue of global concern, and studies are ongoing to identify new therapies that are both effective and safe. PF-4878691 is a Toll-like receptor 7 (TLR7) agonist modeled so as to dissociate its antiviral activities from its inflammatory activities. In a proof-of-mechanism study in healthy volunteers who received doses of 3, 6, and 9 mg of PF-4878691 twice a week for 2 weeks, PF-4878691 induced biomarkers of the immune and interferon (IFN) responses in a dose-dependent and dose-frequency-related manner. A novel finding was induction of TLR7 expression in vivo in response to PF-4878691, leading to an amplified biomarker response. A nonresponder at the 9-mg dose had a polymorphism in the IFN-α receptor 1 subunit (Val168Leu). Two subjects who had received 9-mg doses experienced serious adverse events (SAEs), characterized by flu-like symptoms, hypotension, and lymphopenia, leading to early termination of the study. TLR7 stimulation results in a pharmacologic response at levels commensurate with predicted antiviral efficacy, but these doses are associated with SAEs, raising concerns about the therapeutic window of this class of compounds for the treatment of HCV infection.

Progress in HIV vaccine research.

The relentless expansion of the HIV pandemic has demonstrated that the need for a vaccine is desperate. However, the development of an effective vaccine against HIV is a formidable challenge. It is likely that a successful vaccine will have to induce an immune response consisting of not only neutralizing antibodies targeted to conserved epitopes of the viral envelope and cytotoxic T-lymphocytes targeted to a variety of viral antigens, but also local mucosal immunity. Furthermore, a vaccine should induce broad-spectrum immunity covering all HIV subtypes. It is unlikely that a single vaccine will achieve all this, and a combination of vaccines will probably be necessary. Although no efficacious HIV vaccine is available yet, definite progress has been made. It was demonstrated that chimpanzees could be protected from both cell-free and cell-associated HIV challenge. Protection from mucosal challenge was also demonstrated in several studies and limited cross-protection between HIV subtypes was observed in several animal models. In spite of these successes, much remains to be done. Prototype vaccines studied to date have only induced short-lived immune responses and elicited no antibodies able to neutralize clinical isolates of HIV-1. Novel ways of producing HIV-1 envelope antigens may lead to improved antibody responses and raise the chances of a vaccine inducing long-term protective immunity.

HIV/AIDS-related knowledge, attitudes and practices among South African military recruits.

OBJECTIVES: To assess the level of HIV-related knowledge, as well as high-risk behaviour and attitudes towards HIV, in a group of South African National Defence Force (SANDF) recruits. DESIGN: Cross-sectional study. SETTING: Tempe military base in Bloemfontein. SUBJECTS: Three hundred and thirty-nine recruits from one company. OUTCOME MEASURES: HIV-related knowledge, attitudes and practices based on a self-administered questionnaire. RESULTS: All of the recruits were male, and most of them (81.4%) were black. The majority of recruits (98.5%) were between 18 and 24 years old. They had a good level of knowledge regarding HIV and AIDS, with more than 80% giving a correct response in most cases. However, several important misconceptions regarding HIV/AIDS and its transmission still exist. Furthermore, several recruits still practised high-risk behaviour, such as not using condoms with casual or new partners. Most obtained their knowledge regarding HIV/AIDS from schools (34.8%), health and social services (27.1%) and the printed media (17.7%), while only 5.2% stated that they learnt about HIV/AIDS from the SANDF education programmes. CONCLUSION: Efforts towards initiating behaviour changes in military recruits should be intensified, and if necessary education programmes should be adapted to facilitate achievement of this goal.

[Macaque immunization with virions purified from a primary isolate of the human immunodeficiency virus type 1 induced enhancement antibodies]. / L'immunisation du macaque avec les virions purifiés d'un isolat primaire du virus de l'immunodéficience humaine de type 1 induit des anticorps facilitants.

Six Rhesus macaques were hyperimmunized with either live infectious human immunodeficiency virus type 1 (HIV-1) or with beta-propiolactone--or formalin--inactivated HIV-1. The virus used was HIV-1 BX08, a primary virus isolate grown in human PBMC. Instead of eliciting virus-neutralizing antibodies, this regimen induced antibodies that enhanced HIV-1 infectivity for PBMC by 10 to 90 fold. Enhancement was also seen in a cell-to-cell fusion assay using a Semliki Forest virus replicon to express BX08 gp160 in CD4+, CCR5+ HeLa cell cultures. These observations raise the concern that whole virus particles-based HIV-1 vaccines might elicit enhancing antibodies that could play a facilitating role in the transmission and/or evolution of the disease.

Allelic frequencies of host genetic variants influencing susceptibility to HIV-1 infection and disease in South African populations.

OBJECTIVES: Limited information is available on the prevalence in African populations of host genetic polymorphisms conferring resistance to HIV-1 infection and disease. The objective of this study was to determine the allelic frequencies in South African populations of the chemokine receptor gene variants CCR5delta32, CCR5m303 and CCR2b-641 and the CXCR4 ligand gene variant SDF1-3'A. METHOD: Cross-sectional study to determine the prevalence of these gene variants in South African subjects of African and European descent. RESULTS: The CCR5delta32 genetic variant is rare in individuals of African origin, having an allelic frequency of 0.1% (n = 1247) compared with 9.8% (n = 144) in Caucasians. The CCR5m303 mutation was not detected in Africans (n = 687), whereas an allelic frequency of 0.9% (n = 145) was identified in Caucasians. The frequency of CCR2b-641 allele was 13.1% (n = 180) in Africans, which was significantly higher that the 7.2% (n = 146) detected in Caucasians. Finally the allelic frequency of the SDF1-3'A gene variant was only 1.0% (n = 198) in Africans compared with 19.8% (n = 145) in Caucasians. CONCLUSIONS: These results indicate that genetic polymorphisms conferring resistance to HIV-1 infection are rare in the South African Black population. Except for the CCR2b-641 gene variant, individuals of African origin also had a much lower prevalence of genetic variants associated with prolonged disease progression.

Could bats act as reservoir hosts for Rift Valley fever virus?

The inter-epizootic reservoir host of Rift Valley fever virus (RVFV) remains unknown, although the namaqua rock rat, Aethomys namaquensis, as well as bats have been implicated. Bats can be asymptomatically infected with rabies, as well as several arboviruses; the possibility that they can act as host for RVFV therefore exists. To examine this possibility, 350 different samples (brain, liver, salivary glands and brown fat) obtained from 150 bats (comprising seven species) were tested for RVFV antigen using an enzyme linked immunosorbent assay (ELISA). None of the samples tested positive, but the ELISA proved to have limited sensitivity (> or = 10(3) TCID50/ml). In order to determine whether bats could be infected with RVFV, one Miniopterus schreibersii and two Eptesicus capensis bats were inoculated by the oral or intramuscular route with 100 ml and 30 ml, respectively, of a RVFV suspension with a titre of 10(6) TCID50/ml. None of the bats developed any clinical signs. A low concentration of RVFV antigen was found in the liver and urine of M. schreibersii, but not in brain tissue. A third E. capensis bat was inoculated by the intramuscular route and sacrificed on day 18. A low level of antigen was detected in the brown fat. These results demonstrate that bats can be infected with RVFV, and that further studies should be done to determine the potential of different bat species to act as reservoir hosts for RVFV during inter-epizootic periods.

Nutritional status of HIV-1 seropositive patients in the Free State Province of South Africa: anthropometric and dietary profile.

OBJECTIVE: To evaluate the nutritional intake and status of HIV-1 seropositive patients, as well as the relationship between malnutrition and disease stage. DESIGN: A cross-sectional study. SETTINGS: The Immunology Clinic at the Pelonomi Hospital in Bloemfontein, South Africa. SUBJECTS: Eighty-one HIV/AIDS patients in different stages of disease were recruited consecutively from January to May 1995. Eleven of these patients were followed in 1997. MAIN OUTCOME MEASURES: Anthropometric data including current weight, height, triceps skinfold thickness, mid-upper-arm circumference, body mass index and bone-free arm muscle area were collected. Nutrient intake was estimated using a diet history in combination with a standardised food frequency questionnaire. The patients were divided into 3 groups according to their CD4+ T cell counts. RESULTS: The men were leaner (BMI = 18.9) than the women (BMI = 22.7) and patients with a CD4+ T cell count < 200 (stage III) tended to have the lowest median values for all anthropometric measurements. More than half the patients had a low intake (< 67% of the recommended dietary allowances) of vitamin C, vitamin B6, vitamin D, vitamin A, calcium, iron and zinc. CONCLUSIONS: The results confirms that HIV/AIDS patients from this population are malnourished. There was, however, no association between disease stage and nutritional status. Nutritional supplementation of HIV/AIDS patients should be considered, as this might lead to improved immune function in these patients.

Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus type 1 in South Africa.

A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A-D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n = 33); or heteroduplex mobility assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.

Correlation among total lymphocyte count, absolute CD4+ count, and CD4+ percentage in a group of HIV-1-infected South African patients.

Depletion of CD4+ T cells is one of the hallmarks of progression of HIV-1 infection. However, measurement of the CD4+ T-cell count is expensive and often unavailable in less developed areas. Previous studies have suggested that the total lymphocyte count (TLC) can be used to predict a low absolute CD4+ T-cell count. To determine the relationship between TLC and CD4+ T-cell count in HIV-1-infected South African patients, 2777 HIV-1-seropositive patients visiting the Immunology clinic at the Pelonomi Hospital in Bloemfontein, South Africa from April 1991 to April 1997 were included in the study. In total, 3237 observations were used to determine sensitivity, specificity, and likelihood ratios, with 95% confidence intervals, of various cutpoints of the TLC to predict an absolute CD4+ T-cell count of <200 cells/mm3, CD4+ percentage <20%, and CD4+ percentage <15%. Spearman rank correlations were calculated between TLC and CD4+ T cells, CD4+ percentage and CD8+ T cells, as well as between CD4+ and CD8+ T cells. Results demonstrated that a TLC of 2 x 10(9)/L or less had a sensitivity of 90.3% to detect patients with a CD4+ T-cell count of <200 cells/mm3, but a specificity of only 53.7%. When the TLC cutoff value was lowered, specificity increased but sensitivity decreased. For the observations as a group, a correlation (r = 0.704) between CD4+ T-cell count and TLC was demonstrated, but if the patients were divided into three groups according to their CD4+ T-cell count, this correlation weakened considerably. Therefore, although TLC shows a correlation with CD4+ T-cell count, it is not a good predictor of the CD4+ T-cell count in this population and should preferably not be used in the clinical care of HIV/AIDS patients.

PIP: Measurement of CD4 T-cell counts to monitor progression of HIV-1 infection is expensive and often unavailable in developing countries. Previous studies have suggested that the total lymphocyte count (TLC) can be used to predict a low absolute CD4 T-cell count. This possibility was explored in a study of 2777 HIV-1-positive patients attending the immunology clinic at the Pelonomi Hospital in Bloemfontein, South Africa, in 1991-97. A total of 3237 paired observations were used to determine the sensitivity, specificity, and likelihood ratios of various TLC cutoff points to predict absolute CD4 T-cell counts. A TLC of 2x109/l or less had a sensitivity of 90.3% to detect patients with a CD4 T-cell count of less than 200 cells/cu. mm, but a specificity of only 53.7%. When the TLC cutoff value was lowered, specificity increased but sensitivity decreased. Overall, there was a correlation between CD4 T-cell count and TLC (r = 0.704); however, this correlation was weakened considerably when patients were stratified into three groups according to their CD4 T-cell count. These findings suggest that use of TLC to predict the CD4 T-cell count should not be used in the clinical care of HIV/AIDS patients. Rather, informed decision making based on the clinical condition and risk factors for developing opportunistic infections is recommended.

Detection of Mycobacterium tuberculosis in serum samples using the polymerase chain reaction.

Conventional methods for the diagnosis of Mycobacterium tuberculosis infections have serious limitations. To determine whether amplification of M. tuberculosis DNA in serum by the polymerase chain reaction (PCR) might be a useful additional diagnostic tool, we tested 329 clinical specimens using primers specific for the IS6110 insertion sequence of the M. tuberculosis complex. The samples consisted of 30 serum samples from healthy controls, 114 serum samples from patients with diagnoses other than tuberculosis (including immunosuppressive disorders), 59 samples from patients with a clinical picture suggestive of tuberculosis, and 78 serum samples from patients with proven M. tuberculosis infection. Both serum, and representative samples from anatomical regions suspected of being infected, were collected from a further 48 patients for comparison with serum PCR. Serum PCR identified 72/78 (92%; 95% confidence interval CI: 84%-97%) patients with proven tuberculosis, and 49/59 (83%; 95% CI: 71%-92%) patients with suspected tuberculosis. In the group of patients with other diagnoses, 30/114 (26%; 95% CI: 18%-34%) tested positive, while none of the specimens from the healthy control group were positive (95% CI: 0%-12%). Serum PCR results also compared favourably with other clinical specimens obtained from the same patient. Serum PCR can, therefore, be a useful additional technique for the early diagnosis of M. tuberculosis infection, but it does not necessarily indicate active infection.

Genital infection of female chimpanzees with human immunodeficiency virus type 1.

To develop an animal model for mucosal HIV-1 infection, adult chimpanzees were inoculated without trauma by depositing the virus inoculum at the entrance to the cervical canal with a rigid catheter to which flexible tubing was attached. By this procedure, persistent infections were established in some chimpanzees with various infectious doses of either cell-associated HIV-1LAI(IIIB) (peripheral blood mononuclear cells from an infected chimpanzee) or with cell-free HIV-1 strains representing subtypes B and E, but not with a subtype A strain. Although some animals did not become infected until after the second or third cervicovaginal exposure, one chimpanzee was clearly infected after one exposure by several criteria, including virus isolation, but this animal did not seroconvert. A second chimpanzee appeared to be resistant to infection despite repeated mucosal exposures at irregular intervals. However, lymphocytes from both of these animals exhibited low-level proliferative responses to HIV-1 but not SIV antigens. Despite these apparently abortive or latent infections, after exposure to HIV-1 by the intravenous route, both animals developed systemic infections and seroconverted. Overall, 8 of 10 chimpanzees were infected systemically after one to three cervicovaginal exposures to HIV-1LAI(IIIB). The results indicate that (1) HIV-1 productive infection of female chimpanzees by the cervicovaginal route generally requires more than one exposure, just as with humans; (2) low level infections without seroconversion can be established after mucosal exposure to HIV; and (3) vaccine efficacy studies involving a single virus challenge of immunized chimpanzees by the cervicovaginal route probably will not be possible.