Julius Oyugi

Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi.

Many people who remain persistently seronegative despite frequent HIV exposure have HIV-specific immune responses. The study of these may provide information about mechanisms of natural protective immunity to HIV-1. We describe the specificity of cytotoxic T lymphocyte responses to HIV in seronegative prostitutes in Nairobi who are apparently resistant to HIV infection. These women have had frequent exposure to a range of African HIV-1 variants, primarily clades A, C, and D, for up to 12 yr without becoming infected. Nearly half of them have CTL directed towards epitopes previously defined for B clade virus, which are largely conserved in the A and D clade sequences. Stronger responses are frequently elicited using the A or D clade version of an epitope to stimulate CTL, suggesting that they were originally primed by exposure to these virus strains. CTL responses have been defined to novel epitopes presented by HLA class I molecules associated with resistance to infection in the cohort, HLA-A*6802 and HLA-B18. Estimates using a modified interferon-gamma Elispot assay indicate a circulating frequency of CTL to individual epitopes of between 1:3,200 and 1:50,000. Thus, HIV-specific immune responses-particularly cross-clade CTL activity- may be responsible for protection against persistent HIV infection in these African women.

Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection

Certain human leukocyte antigens, by presenting conserved immunogenic epitopes for T cell recognition, may, in part, account for the observed differences in human immunodeficiency virus type 1 (HIV-1) susceptibility. To determine whether HLA polymorphism influences HIV-1 susceptibility, a longitudinal cohort of highly HIV-1-exposed female sex workers based in Nairobi, Kenya, was prospectively analyzed. Decreased HIV-1 infection risk was strongly associated with possession of a cluster of closely related HLA alleles (A2/6802 supertype; incidence rate ratio [IRR], 0.45; 95% confidence interval [CI], 0.27-0.72; P=.0003). The alleles in this supertype are known in some cases to present the same peptide epitopes for T cell recognition. In addition, resistance to HIV-1 infection was independently associated with HLA DRB1*01 (IRR, 0.22; 95% CI, 0.06-0.60; P=.0003), which suggests that anti-HIV-1 class II restricted CD4 effector mechanisms may play an important role in protecting against viral challenge. These data provide further evidence that resistance to HIV-1 infection in this cohort of sex workers is immunologically mediated.

CD8(+) lymphocytes respond to different HIV epitopes in seronegative and infected subjects.

HIV-1-specific cytotoxic T-lymphocyte (CTL) responses have been detected at a low frequency in many HIV-1-exposed, persistently seronegative (HEPS) subjects. However, it is unclear how CTLs could protect against HIV acquisition in HEPS subjects, when high levels of circulating CTL fail to prevent disease progression in most seropositive subjects. To address this issue we studied CD8(+) lymphocyte responses to a panel of HIV-1 CTL epitopes in 91 HEPS and 87 HIV-1-infected Nairobi sex workers. HIV-specific responses in seropositive women focused strongly on epitopes rarely or never recognized in HEPS subjects, who targeted epitopes that were subdominant or unrecognized in infected women. These differences in epitope specificity were restricted by only those HLA class I alleles that are associated with a reduced risk of HIV-1 infection in this cohort. Late seroconversion in HEPS donors was associated with a switch in epitope specificity and/or immunodominance to those epitopes preferentially recognized by HIV-1-infected women. The likelihood of detecting HIV-1-specific responses in HEPS women increased with the duration of viral exposure, suggesting that HIV-1-specific CD8(+) responses are acquired over time. The association between differential recognition of distinct CTL epitopes and protection from HIV-1 infection may have significant implications for vaccine design.

HIV-1-specific cellular immune responses among HIV-1-resistant sex workers.

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, Students 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.

Broadly cross-reactive HIV-specific cytotoxic T-lymphocytes in highly-exposed persistently seronegative donors.

HIV-specific cytotoxic T-lymphocytes (CTL) are believed to play a key part in the control of virus levels throughout HIV infection. An important goal of a potential prophylactic vaccine against HIV is therefore to elicit a strong CTL response which is broadly cross-reactive against a diverse range of HIV strains. We have detected HIV-specific CTL in two groups of highly-exposed but persistently seronegative female sex workers in Africa which show extensive cross-reactivity between different viral sequences. In a small group of women exposed to both HIV-1 and HIV-2 in Gambia, studied over 4 years, we have repeatedly detected HLA-B35-restricted CTL which exhibit cross-reactivity between the HIV-1 and HIV-2 sequences of the CTL epitopes. In women with particularly intense exposure to what are likely to be multiple clades of HIV-1 in Nairobi Kenya, we have detected CTL directed towards epitopes conserved between HIV-1 clades. In neither group is there any evidence that variation in CCR5 sequence or expression is responsible for their apparent resistance to HIV infection. However, in seropositive donors from Oxford infected with African strains of HIV-1, we have defined CTL responses which are specific for particular clades and have mapped some unique A clade CTL epitopes, together with others to highly-conserved regions of the virus. Further information about the extent of cross-reactive CTL immunity will be important for future vaccine design and evaluation.

Human Leukocyte Antigen (HLA) B*18 and Protection against Mother-to-Child HIV Type 1 Transmission

Human leukocyte antigen (HLA) molecules regulate the cellular immune system and may be determinants of infant susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Molecular HLA typing for class I alleles was performed on infants followed in a Kenyan perinatal cohort. Early HIV-1 infection status was defined as infection occurring at birth or month 1, while late infection via breast milk was defined as first detection of HIV-1 after 1 month of age. Likelihood ratio tests based on a proportional hazards model adjusting for maternal CD4 T cell count and HIV-1 viral load at 32 weeks of gestation were used to test associations between infant allelic variation and incident HIV-1 infection. Among 433 infants, 76 (18%) were HIV-1 infected during 12 months of follow-up. HLA B*18 was associated with a significantly lower risk of early HIV-1 transmission [relative risk (RR) = 0.26; 95% confidence interval (CI) 0.04-0.82], and none of the 24 breastfeeding infants expressing HLA B*18 who were uninfected at month 1 acquired HIV-1 late via breast milk. We observed a trend toward increased early HIV-1 acquisition for infants presenting HLA A*29 (RR = 2.0; 95% CI 1.0-3.8) and increased late HIV-1 acquisition via breast milk for both Cw*07 and Cw*08 (RR = 4.0; 95% CI 1.0-17.8 and RR = 7.2; 95% CI 1.2-37.3, respectively). HLA B*18 may protect breast-feeding infants against both early and late HIV-1 acquisition, a finding that could have implications for the design and monitoring of HIV-1 vaccines targeting cellular immune responses against HIV-1.

Identification of preferential CD4 + T-cell targets for HIV infection in the cervix

A better understanding of the cellular targets of HIV infection in the female genital tract may inform HIV prevention efforts. Proposed correlates of cellular susceptibility include the HIV co-receptor CCR5, peripheral homing integrins, and immune activation. We used a CCR5-tropic pseudovirus to quantify HIV entry into unstimulated endocervical CD4(+) T cells collected by cytobrush. Virus entry was threefold higher into cervix-derived CD4(+) T cells than blood, but was strongly correlated between these two compartments. Cervix-derived CD4(+) T cells expressing CD69, α(4)β(7), or α(4)β(1) were preferential HIV targets; this enhanced susceptibility was strongly correlated with increased CCR5 expression in α(4)β(7)(+) and CD69(+) CD4(+) T cells, and to a lesser extent in α(4)β(1)(+) CD4(+) T cells. Direct binding of gp140 to integrins was not observed, integrin inhibitors had no effect on virus entry, and pseudotypes with an env that preferentially binds α(4)β(7) still demonstrated enhanced entry into α(4)β(1)(+) cells. In summary, a rapid and sensitive HIV entry assay demonstrated enhanced susceptibility of activated endocervical CD4(+) T cells, and those expressing α(4)β(7) or α(4)β(1). This may relate to increased CCR5 expression by these cell subsets, but did not appear to be due to direct interaction of α(4)β(7) or α(4)β(1) with HIV envelope.

Long-term survivors in Nairobi: complete HIV-1 RNA sequences and immunogenetic associations

To investigate African long-term survivors (LTSs) infected with non-subtype B human immunodeficiency virus type 1 (HIV-1), we obtained full-length HIV-1 RNA sequences and immunogenetic profiles from 6 untreated women enrolled in the Pumwani Sex Worker Cohort in Nairobi, Kenya. There were no discernible sequence changes likely to cause attenuation. CCR2-V64I, an immunogenetic polymorphism linked to LTSs, was detected in 4 women, all of whom carried the HLA B58 allele. Further investigation of 99 HIV-1-infected Nairobi women found an association between CCR2-V64I and HLA B58 (P=.0048). Studying the interaction among immunogenetics, immune responses, and viral sequences from all HIV-1 subtypes may increase our understanding of slow HIV-1 disease progression.

A Common CD4 Gene Variant Is Associated with an Increased Risk of HIV-1 Infection in Kenyan Female Commercial Sex Workers

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-1(IIIB) 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.

Antagonistic effect of alkaloids and saponins on bioactivity in the quinine tree (Rauvolfia caffra sond.): further evidence to support biotechnology in traditional medicinal plants

BackgroundThe Quinine tree (Rauvolfia caffra) is used as a medicinal plant among traditional communities in many countries to manage tumors and other diseases associated with oxidative stress. To validate indigenous knowledge and possibly position this herb for technology uptake and utilization, we established the level of antioxidant activity in R. caffra, and probed for the presence of associated phytochemicals.MethodsAntioxidant activity was determined on 1,1-diphenyl-2-picrylhydrazyl (DPPH) while major phytochemicals were identified by multiple tests on methanol fractions.ResultsR. caffra showed promise as a cure, with antioxidant activity comparable to the commercially used drug quercetin (R. caffra = 79.7% ±1.9; quercetin = 82.6% ± 2.0). However, we found two phytochemicals with possible antagonistic effect: co-occurrence of alkaloids and saponins significantly reduced antioxidant activity (alkaloids only = 63%; alkaloids plus saponins = 15%; steroids, terpenoids and cardiac glycosides = 82%), thus alkaloids and saponins should be exclusive to each other in drug formulations.ConclusionsAntagonistic relationship among phytochemicals would affect the efficacy of crude extracts as used in traditional medicine. Unlike in herbal medicine, use of modern biotechnology in extraction, purification and design of optimal combinations will ensure efficient drug formulations with optimum bioactivity and minimum toxicity. Metabolic pathway engineering under a controlled environment may optimize availability of desired compounds.