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Background: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution. Methodology/Principal Findings: HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB) sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL) and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions. Conclusions/Significance: Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies. [ABSTRACT FROM AUTHOR]

Studies of human immunodeficiency virus (HIV)-exposed seronegative individuals are crucial to inform vaccine design. In the present study we demonstrated that HIV-exposed seronegative commercial sex workers produce lower levels of proinflammatory cytokines at baseline than HIV-negative control subjects. We also showed that CD4+ T cells of HIV-exposed seronegative commercial sex workers have a characteristically lower level of gene expression that can be seen in differentially expressed genes and systems crucial for HIV replication, such as the T cell receptor pathway and previously identified HIV dependency factors. This apparent lowered activation results in a phenomenon we term "immune quiescence," which may contribute to host resistance to HIV. [ABSTRACT FROM AUTHOR]

Copyright of Biochemistry & Cell Biology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Human immunodeficiency virus (HIV)-resistant commercial sex workers provide a unique opportunity to study correlates of protection associated with natural resistance to HIV infection. Emerging data from studies of these individuals and other uninfected individuals who have been exposed to HIV suggest that low levels of immune activation may contribute to protection against infection. In the present study, HIV-resistant individuals were shown to have reduced frequencies of T cells expressing the activation marker CD69. They were also found to have elevated frequencies of regulatory T (T(reg)) cells, compared with HIV-negative control individuals. By controlling levels of T cell activation, T(reg) cells may contribute to HIV resistance by minimizing the pool of cells susceptible to infection. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV)-resistant commercial sex workers provide a unique opportunity to study correlates of protection associated with natural resistance to HIV infection. Emerging data from studies of these individuals and other uninfected individuals who have been exposed to HIV suggest that low levels of immune activation may contribute to protection against infection. In the present study, HIV resistant individuals were shown to have reduced frequencies of T cells expressing the activation marker CD69. They were also found to have elevated frequencies of regulatory T (Treg) cells, compared with HIV-negative control individuals. By controlling levels of T cell activation, Treg cells may contribute to HIV resistance by minimizing the pool of cells susceptible to infection. [ABSTRACT FROM AUTHOR]

Background. It has been predicted that CD4 C868T, a novel CD4 single-nucleotide polymorphism (SNP) that has been found to be highly prevalent among Africans, changes the tertiary structure of CD4, which may alter susceptibility to human immunodeficiency virus (HIV) infection. Methods. Participants were from a Kenyan cohort and included 87 uninfected and 277 HIV-1-infected individuals. DNA sequencing was used to determine CD4 genotype. A2.01 cells expressing similar levels of either wild-type CD4 or CD4-Trp240 as well as peripheral blood mononuclear cells from uninfected donors were infected with HIV-1IIIB or a Kenyan primary HIV-1 isolate. HIV-1 p24 enzyme-linked immunosorbent assay was used to determine the outcome of infection. Results. CD4 C868T was found to be significantly more prevalent among HIV-1-infected participants than among HIV-1-uninfected participants (P = .002), and C868T was associated with an increased incidence of HIV-1 infection as well (P = .005, log-rank test; P = .009, Wilcoxon test), with an odds ratio of 2.49 (P = .009). Both in vitro and ex vivo models demonstrated a significant association between CD4 C868T and susceptibility to HIV-1 infection (P < .001 and P = .003, respectively). Conclusion. Overall, the present study found a strong correlation between CD4 C868T and increased susceptibility to HIV-1 infection. Given the high prevalence of both HIV infection and CD4 C868T in African populations, the effect of this SNP on the epidemic in Africa could be dramatic. [ABSTRACT FROM AUTHOR]

Background: We analyzed the prevalence of pre-antiretroviral therapy (ART) drug resistance mutations (DRMs) in a Kenyan population. We also examined whether host HLA class I genes influence the development of pre-ART DRMs. Methods: The HIV-1 proviral DNAs were amplified from blood samples of 266 ART-naïve women from the Pumwani Sex Worker cohort of Nairobi, Kenya using a nested PCR method. The amplified HIV genomes were sequenced using next-generation sequencing technology. The prevalence of pre-ART DRMs was investigated. Correlation studies were performed between HLA class I alleles and HIV-1 DRMs. Results: Ninety-eight percent of participants had at least one DRM, while 38% had at least one WHO surveillance DRM. M184I was the most prevalent clinically important variant, seen in 37% of participants. The DRMs conferring resistance to one or more integrase strand transfer inhibitors were also found in up to 10% of participants. Eighteen potentially relevant (p < 0.05) positive correlations were found between HLA class 1 alleles and HIV drug-resistant variants. Conclusions: High levels of HIV drug resistance were found in all classes of antiretroviral drugs included in the current first-line ART regimens in Africa. The development of DRMs may be influenced by host HLA class I-restricted immunity. [ABSTRACT FROM AUTHOR]

Copyright of Biochemistry & Cell Biology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The initial site of exposure to human immunodeficiency virus (HIV)-1 during heterosexual transmission occurs in the genital tract. Although the majority of immunological studies have focused on the immune response to HIV-1 at the systemic level, our understanding of tissue-specific immunity is deficient. The goal of the present study was to characterize T cell populations found in the cervix of women shown to be resistant to infection by HIV-1. Levels of both systemic and cervical mucosal lymphocytes were compared between HIV-1-resistant, HIV-1-uninfected, and HIV-1-infected commercial sex workers (CSWs) as well as HIV-1-uninfected non-CSW control subjects at low risk for exposure. The HIV-1-resistant CSWs had increased cervical CD4+ and CD8+ T cell counts, compared with the HIV-1-uninfected CSWs; importantly, these increases were not reflected in the systemic lymphocyte compartment. There was a 2-fold increase in CD4+ T cell counts in the HIV-1-resistant CSWs, compared with both the HIV-1-infected and the HIV-1-uninfected CSWs. Expression of the HIV-1 coreceptors CCR5 and CXCR4 was also determined, and cytokine and beta chemokine levels in the genital mucosa were assessed. The HIV-1-resistant CSWs had a 10-fold increase in RANTES expression, compared with the HIV-1-uninfected CSWs. This is the first study to show elevated levels of beta chemokines and CD4+ T cells in the genital tracts of women who are exposed to HIV-1 and yet are uninfected. [ABSTRACT FROM AUTHOR]

The immune response of human immunodeficiency virus (HIV)-exposed seronegative (ESN) women may be qualitatively different from that in those infected with HIV (HIV+). In a cohort of female commercial sex workers in Nairobi, Kenya, we found significantly lower (P ≤ .01) levels of CD4+-specific immune activation and apoptosis in the ESN women compared with those in the HIV+-women. Compared with the HIV+-women, a lower proportion of the ESN women showed p24 peptide pool responses by the short-term, CD4+-specific, interferon (IFN)-γ intracellular cytokine staining assay, whereas the proportion showing responses by the long-term, CD8+-depleted T cell proliferation assay was similar. Interestingly, the ESN responders had a 4.5-fold stronger proliferation response (P = .002) than the HIV+ group. These data suggest that, compared with those in HIV+ women, CD4+ T cells in ESN women have a much greater ability to proliferate in response to p24 peptides. [ABSTRACT FROM AUTHOR]

Variation in susceptibility to HIV-1 infection depends on numerous factors, and host genetic variation has been well-described as an important component. As a transcriptional regulator, interferon regulatory factor 1 (IRF-1) plays a key role in both innate and adaptive immunity against viral infection. IRF-1 has also been shown to directly interact with HIV-1 5' LTR and efficiently initiate or amplify HIV-1 replication. By complete gene sequencing, we investigated genetic polymorphism of the IRF-1 gene in an HIV-1-endemic Kenyan population. This population displayed extensive genetic diversity at the IRF-1 locus. Fifty-three single nucleotide polymorphisms (SNPs) were identified in this population, including 26 novel SNPs. Two insertion and one deletion polymorphisms in IRF-1 were also identified. Linkage disequilibrium (LD) among these genetic variations was shown to be common in IRF-1. The functional consequences of these mutations in the context of HIV-1/AIDS remain to be determined. We also identified 35 consistent discrepancies between IRF-1 GenBank sequences and our population based sequencing data, suggesting that the previously submitted GenBank data were not representative of the majority of human IRF-1 sequences. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae cervicitis and human immunodeficiency virus (HIV) type 1 frequently coinfect core transmitter populations, such as female sex workers. Gonococcal cervicitis is associated with increased viral shedding and plasma viremia in HIV-1-infected women and increased HIV-1 susceptibility in uninfected women. We studied the influence of gonococcal cervicitis on CD8(+) interferon (IFN)-gamma responses to HIV-1 and cytomegalovirus (CMV) epitopes in HIV-1-infected and in highly-exposed, persistently seronegative (HEPS) female sex workers. In HIV-1-infected women, gonococcal cervicitis was associated with reduced IFN-gamma responses in bulk CD8(+) lymphocyte populations, and intracellular cytokine staining, combined with class I major histocompatibility complex (MHC)-peptide tetramer studies, demonstrated reduced IFN-gamma production by HIV-1 epitope-specific CD8(+) lymphocytes. In HEPS sex workers, cervicitis was associated with the transient loss of systemic HIV-1-specific CD8(+) responses and with reduced function of CMV-specific CD8(+) lymphocytes. Impaired function of virus-specific CD8(+) lymphocytes may partly explain the deleterious effects of gonococcal cervicitis on HIV-1 immune control and susceptibility. [ABSTRACT FROM AUTHOR]

Summary The goal of the present study was to determine whether there were HIV-1 specific cellular immune responses among a subgroup of women within a cohort of Nairobi prostitutes (n = 1800) who, despite their intense sexual exposure to HIV-1, are epidemiologically resistant to HIV-1 infection. Of the 80 women defined to be resistant, 24 were recruited for immunological evaluation. The HIV-1-specific T-helper responses were determined by IL-2 production following stimulation with HIV-1 envelope peptides and soluble gp120. Cytotoxic T lymphocyte responses were determined by lysis of autologous EBV-transformed B cell lines infected with control vaccinia virus or recombinant vaccinia viruses containing the HIV-1 structural genes env, gag and pol. Resistant women had significantly increased HIV-1 specific T-helper responses, as determined by in vitro IL-2 production to HIV-1 envelope peptides and soluble glycoprotein 120, compared with low-risk seronegative and HIV-1-infected controls (P ≤ 0.01, Student’s t-test). Seven of the 17 (41%) resistant women showed IL-2 stimulation indices ≥ 2.0. HIV-1-specific CTL responses were detected among 15/22 (68.2%) resistant women compared with 0/12 low-risk controls (Chi-squared test, P < 0.001). In the two resistant individuals tested, the CTL activity was mediated by CD8+ effectors. Many HIV-1-resistant women show evidence of HIV-1-specific T-helper and cytotoxic responses. These data support the suggestion that HIV-1-specific T-cell responses contribute to protection against HIV-1 infection. [ABSTRACT FROM AUTHOR]

FREM1 (Fras-related extracellular matrix 1) and its splice variant TILRR (Toll-like interleukin-1 receptor regulator) have been identified as integral components of innate immune systems. The potential involvement of FREM1 in HIV-1 (human immunodeficiency virus 1) acquisition was suggested by a genome-wide SNP (single nucleotide polymorphism) analysis of HIV-1 resistant and susceptible sex workers enrolled in the Pumwani sex worker cohort (PSWC) in Nairobi, Kenya. The studies showed that the minor allele of a FREM1 SNP rs1552896 is highly enriched in the HIV-1 resistant female sex workers. Subsequent studies showed that FREM1 mRNA is highly expressed in tissues relevant to mucosal HIV-1 infection, including cervical epithelial tissues, and TILRR is a major modulator of many genes in the NF-κB signal transduction pathway. In this article, we review the role of FREM1 and TILRR in modulating inflammatory responses and inflammation, and how their influence on inflammatory responses of cervicovaginal tissue could enhance the risk of vaginal HIV-1 acquisition. [ABSTRACT FROM AUTHOR]

Comments on an article by Mahrukh Bamji and colleagues about the presence of palpable lymph nodes in healthy newborns. Survey of palpable lymph nodes in healthy young term infants; Size of the nodes found in the infants; Implications for the assessment of congenital infections.

Among 302 female sex workers in Nairobi, Kenya, who were followed for 17.6 ± 11.1 months, 146 had one or more infections with Chlamydia trachomatis; 102 had uncomplicated cervical infection only, 23 had C. trachomatis pelvic inflammatory disease (PID), and 21 had combined C. trachomatis and Neisseria gonorrhoeae PID. As determined by multivariate logistic regression analysis, risk factors for C. trachomatis PID included repeated C. trachomatis infection (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.3–2.4; P = .0004),antibody to C. trachomatis heat-shock protein 60 (OR, 3.9; CI, 1.04–14.5; P = .04), oral contraceptive use (OR, 0.28; 95% CI, 0.08–0.99; P = .048), and number of episodes of nongonococcal nonchlamydial PID (OR, 1.7; 95% CI, 1.1–2.7; P = .02). Among human immunodeficiency virus (HIV)-seropositive women, a CD4 lymphocyte count of <400/mm3 was an additional independent risk factor for C. trachomatis PID (OR, 21.7; 95% CI, 1.2–383; P = .036); among HLA-typed women, HLA-A31 was independently associated with C. trachomatis PID (OR, 5.6; 95% CI, 1.1–29.4; P = .043). The results suggest an immune-mediated pathogenesis for C. trachomatis PID. [ABSTRACT FROM PUBLISHER]

Female sex workers in Nairobi were prospectively evaluated for risk factors of incident Chlamydia trachomatis infection. Independent risk factors included cervical ectopy (P = .007), gonococcal infection (P = .002), human immunodeficiency virus (HIV) seropositivity (P = .003), HIV seroconversion (P = .001), and duration of prostitution (P = .002). Eighteen different C. trachomatis outer membrane protein (omp1) genotypes were identified, with the allelic composition of the C. trachomatis population changing significantly over time (P = .005). Seventeen of 19 reinfections ⩾6 months apart were with different C. trachomatis ompi genotypes. Women with HIV infection had an increased proportion of visits with C. trachomatis infection (P = .001) and an increased risk of reinfection (P = .008). Overall, the data demonstrate significant fluctuations in the genotype composition of the C. trachomatis population and a reduced rate of same-genotype reinfection consistent with the occurrence of strain-specific immunity. [ABSTRACT FROM PUBLISHER]

Phagocytosis of Treponema pallidum by cytokine-activated macrophages aids bacterial clearance and lesion resolution in early syphilis. To investigate the cytokine profiles of cells infiltrating primary and secondary syphilis lesions, reverse transcription and polymerase chain reaction (RT-PCR) were used to detect cytokine mRNA in 13 lesion biopsies. Both primary and secondary lesions contained mRNA encoding interleukin (IL)-2, interferon-γ (IFN-γ), IL-12p40, and IL-IO. In contrast to a lesion from a patient with recurrent herpes simplex virus type 2, no message for IL-4 could be detected in any of the syphilis lesions, and 10 of 13 had no mRNA for IL-S or IL-13.These findings are consistent with a Thl-predominant local cellular response activating macrophages and support the hypothesis that IFN-γ-activated macrophages are primary effectors in treponeme clearance. [ABSTRACT FROM PUBLISHER]

In a study of human immunodeficiency virus type 1 (HIV-1)-uninfected African prostitutes, 83 (67%) of 124 sero converted to HIV-1. Oral contraceptive use (odds ratio [OR], 3.1; 95% confidence interval (CI], 1.1–8.6;P < .03), genital ulcers (mean annual episodes, 1.32 ± 0.55 in seroconverting women vs. 0.48 ± 0.21 in seronegative women; P < .02) and Chlamydia trachomatis infections (OR, 3.6;CI, 1.3–11.0; P < .02)were associated with increased risk of HIV-1 infection. Condom use reduced the risk of HIV-1 infection (OR, 0.11; CI, 0.05–0.27; P < .0001). Stepwise logistic regression analysis confirmed independent associations between HIV-l infection and oral contraceptive use, condom use, genital ulcers, and C. trachomatis. The presence of other sexually transmitted diseases may in part explain the heterosexual HIV-1 epidemic in Africa and may represent important targets for intervention to control HIV-1 infection. [ABSTRACT FROM PUBLISHER]

Objectives. The purpose of this study was to identify health-care seeking and related behaviors relevant to controlling sexually transmitted diseases in Kenya. Methods. A total 380 patients with sexually transmitted diseases (n=189 men and 191 women) at eight public clinics were questioned about their health-care seeking and sexual behaviors. Results. Women waited longer than men to attend study clinics and were more likely to continue to have sex while symptomatic. A large proportion of patients had sough treatment previously in both the public and private sectors without relief of symptoms, resulting in delays in presenting to study clinics. For women, being married and giving a recent history of selling sex were both independently associated with continuing to have sex while symptomatic. Conclusions. Reducing the transmission of sexually transmitted diseases in Kenya will require improved access, particularly for women, to effective health services, preferably at the point of first contact with the health system. It is also critical to encourage people to reduce sexual activity while symptomatic, seek treatment promptly, and increase condom us. [ABSTRACT FROM AUTHOR]

The factors responsible for the explosive spread of human immunodeficiency virus type 1 (HIV-1) in sub-Saharan Africa continue to be identified and debated. One of the most controversial factors has been male circumcision. This cross-sectional study was conducted to measure the association between circumcision status and infection with HIV-1 among men with genital ulcer disease. Eight hundred and ten men participated in the study, of whom 190 (23%) were HIV-1-positive. A logistic regression model adjusted for behavioral and historical factors showed that HIV-1 positivity was independently associated with being uncircumcised (adjusted odds ratio [OR], 4.8; 95% confidence interval [CI], 3.3–7.2) and with a history of urethral discharge (adjusted OR, 2.0; 95% CI, 1.4–2.8). This association could not be explained by measures of sexual exposure to HIV-1 among this population. Male circumcision should be considered as an intervention strategy for AIDS control. [ABSTRACT FROM PUBLISHER]

The sexual transmission of human immunodeficiency virus type 1 (HIV-1) continues at an alarming rate in sub-Saharan Africa despite the fact that awareness of AIDS is high. One explanation for this alarming rate may be that individuals do not believe that they are personally at risk for AIDS and are not sufficiently motivated to make changes in their behavior. We conducted a cross-sectional study of men with genital ulcer disease to assess their sexual behavior and their perceived risk of AIDS. We studied 787 men between the ages of 17 and 54 years who presented to a referral clinic for sexually transmitted diseases (STDs) in Nairobi, Kenya. Of these 787 men, 188 (24%) were infected with HIV-1. Awareness of AIDS was essentially universal in this population; however, only 64 men (8%) thought that they were personally at risk of developing AIDS. A logistic regression analysis found that men who believed they were personally at risk knew someone with AIDS (odds ratio [OR], 8.9; 95% confidence interval [CI], 4.0–19.7), received information about AIDS from television or video (OR, 3.0; 95% CI, 1.7–5.5), or had previously had an STD (OR, 2.2; 95% CI, 1.2–4.1). Except for a modest increase in condom use, there was no significant difference in sexual behavior between the group who considered themselves to be at risk for AIDS and the group who did not consider themselves to be at risk. The results of this study challenge the current strategies on HIV/AIDS education and prevention for urban men in Kenya. [ABSTRACT FROM PUBLISHER]

We conducted a prospective observational study to determine the clinical features, the degree of immunosuppression, and the prevalence of human immunodeficiency virus type 1 (HIV-1) infection associated with herpes zoster in Kenya. The study included 196 HIV-1-positive individuals and 34 HIV-1-negative individuals between the ages of 16 and 50 years who presented to a referral clinic in Nairobi. Comparison of the clinical characteristics in the two groups found that the duration of illness in the HIV-1-positive group was longer (32 vs. 22 days; P < .001) and that the HIV-1-positive group was more likely to have generalized lymphadenopathy (74% vs. 3%; OR: 12.2; 95% CI: 1.6, 91.7), severe pain (69% vs. 39%; OR: 3.6; 95% CI: 1.7, 7.6), bacterial superinfection (15% vs. 6%; OR: 5.7; 95% CI: 1.3, 25.0), and more than one affected dermatome (38% vs. 18%; OR: 2.8; 95% CI: 1.1, 8.0). Dermatomal distribution of the lesions was similar in the two groups, except for cranial lesions, which occurred exclusively in the HIV-1-positive group. The mean CD4 T lymphocyte count at presentation was 333/mm3 in the HIV-1-positive group and 777/mm3 in the HIV-1-negative group (P < .001). Herpes zoster is often recognized as the initial HIV-1-related illness in Kenya despite the fact that patients have moderate to severe depression of CD4 cell counts at presentation. Although the clinical features of herpes zoster may be more severe in HIV-1-positive individuals, recovery is generally complete and uncomplicated. [ABSTRACT FROM PUBLISHER]

In Nairobi (Kenya) 334 women prostitutes of whom 80.5% were HIV-1 positive, were examined for oral mucosal lesions; 15.6% of seropositive, and 4.6%, of seronegative, women had oral mucosal lesions, predominantly oral candidiasis. In the seropositives, 8.6% had erythematous. 1.1% had hyperplastic candidiasis, and 0.4% had hairly leukoplakia as the only oral lesions: 0.4% had a combination of erythematous and hyperplastic candidiasis. and 1.5% had combinations of pseudomembranous and erythematous candidiasis in the presence of hairly leukoplakia. Of the 211 seropositive women for whom we knew the first date on which a positive serologic test was obtained, the likelihood of developing an oral mucosal lesion was found to be dependent on the duration of seropositivity. The low incidence of oral mucosal lesions in this population may be due to the relatively recent acquisition of HIV-I infection. [ABSTRACT FROM AUTHOR]

In the article, the authors discuss the development of an effective Ebola vaccine by the Canadian National Microbiology Laboratory. Topics include the discovery of the Ebola hemorrhagic fever filovirus in the Democratic Republic of the Congo in 1976, the identification by Jack Rose of a reverse genetics system for vesicular stomatitis virus (VSV) in 1996, and the tool created by Yoshihiro Kawaoka for studying Ebola virus in 1997.