Kelly S. MacDonald

Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression

The IL-1 family cytokines are regulated on transcriptional and posttranscriptional levels. Pattern recognition and cytokine receptors control pro-IL-1β transcription whereas inflammasomes regulate the proteolytic processing of pro-IL-1β. The NLRP3 inflammasome, however, assembles in response to extracellular ATP, pore-forming toxins, or crystals only in the presence of proinflammatory stimuli. How the activation of gene transcription by signaling receptors enables NLRP3 activation remains elusive and controversial. In this study, we show that cell priming through multiple signaling receptors induces NLRP3 expression, which we identified to be a critical checkpoint for NLRP3 activation. Signals provided by NF-κB activators are necessary but not sufficient for NLRP3 activation, and a second stimulus such as ATP or crystal-induced damage is required for NLRP3 activation.

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.

HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi

Understanding how individuals with a high degree of HIV exposure avoid persistent infection is paramount to HIV vaccine design. Evidence suggests that mucosal immunity, particularly virus-specific CTL, could be critically important in protection against sexually acquired HIV infection. Therefore, we have looked for the presence of HIV-specific CD8+ T cells in cervical mononuclear cells from a subgroup of highly HIV-exposed but persistently seronegative female sex workers in Nairobi. An enzyme-linked immunospot assay was used to measure IFN-γ release in response to known class I HLA-restricted CTL epitope peptides using effector cells from the blood and cervix of HIV-1-resistant and -infected sex workers and from lower-risk uninfected controls. Eleven of 16 resistant sex workers had HIV-specific CD8+ T cells in the cervix, and a similar number had detectable responses in blood. Where both blood and cervical responses were detected in the same individual, the specificity of the responses was similar. Neither cervical nor blood responses were detected in lower-risk control donors. HIV-specific CD8+ T cell frequencies in the cervix of HIV-resistant sex workers were slightly higher than in blood, while in HIV-infected donor cervical response frequencies were markedly lower than blood, so that there was relative enrichment of cervical responses in HIV-resistant compared with HIV-infected donors. HIV-specific CD8+ T cell responses in the absence of detectable HIV infection in the genital mucosa of HIV-1-resistant sex workers may be playing an important part in protective immunity against heterosexual HIV-1 transmission.

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.

Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8 + responses

Resistance to HIV infection in a small group of Kenyan sex workers is associated with CD8+-lymphocyte responses to HIV cytotoxic T-lymphocyte (CTL) epitopes. Eleven prostitutes meeting criteria for HIV resistance seroconverted between 1996 and 1999. The occurrence and specificity of preexisting HIV-1 epitope-specific responses were examined using the IFN-gamma enzyme-linked immunospot assay, and any epitopes recognized were cloned and sequenced from the infecting viral isolate. Immunologic and behavioral variables were compared between late seroconverters and persistently uninfected sex worker controls. HIV-1 CTL epitope responses were present in four of six cases, 5-18 months before seroconversion, and their presence was confirmed by bulk CTL culture. A possible viral escape mutation was found in one of six epitopes. The key epidemiologic correlate of late seroconversion was a reduction in sex work over the preceding year. In persistently uninfected controls, a break from sex work was associated with a loss of HIV-specific CD8+ responses. Late seroconversion may occur in HIV-1-resistant sex workers despite preceding HIV-specific CD8+ responses. Seroconversion generally occurs in the absence of detectable CTL escape mutations and may relate to the waning of HIV-specific CD8+ responses due to reduced antigenic exposure.

Mother-Child Class I HLA Concordance Increases Perinatal Human Immunodeficiency Virus Type 1 Transmission

Major histocompatibility complex (MHC) gene products are expressed on human immunodeficiency virus (HIV)-infected cells and incorporated into the lipid envelope of HIV virions. Macaques immunized with human MHC gene products are protected from simian immunodeficiency virus challenge when the virus is grown in cells expressing the same MHC alleles. To relate these findings to mother-to-child transmission of HIV-1, investigations of whether sharing HLA between mother and infant influenced the risk of transmission of HIV-1 to the child were carried out. Class I HLA concordance was independently associated with a stepwise increase in the risk of perinatal HIV-1 transmission for each additional concordant allele (odds ratio, 2.63; 95% confidence interval, 1.36-5.07; P = .003). Thus, discordant HLA may provide infants with a means of protection against HIV-1 as a result of allogeneic infant anti-maternal MHC immune responses.

Levels of innate immune factors in genital fluids: association of alpha defensins and LL-37 with genital infections and increased HIV acquisition.

Background:Several mucosal innate immune proteins exhibit HIV inhibitory activity and their analogues are potential microbicide candidates. However, their clinical associations and in-vivo role in cervicovaginal host defense against HIV acquisition are poorly defined. Methods:Cervicovaginal secretions (CVSs) were collected from HIV uninfected Kenyan sex workers at enrolment into an HIV prevention trial. After trial completion, CVS from participants acquiring HIV (cases) and matched controls were assessed for levels of innate immune factors and HIV neutralizing capacity, by blinded investigators. Cross-sectional and prospective associations of innate immune factors were examined. Results:CVS contained high levels of defensins (human neutrophil peptide-1-3 and human β defensin-2-3), LL-37 and secretory leukocyte protease inhibitor. Regulated upon activation normal T-cell expressed and secreted levels were lower, and IFNα was undetectable. CVS from 20% of participants neutralized a clade A primary HIV isolate, and 12% neutralized both clade A and C isolates. HIV neutralization was correlated with human neutrophil peptide-1-3 (α-defensins) and LL-37 levels. However, α-defensin and LL-37 levels were increased in participants with bacterial sexually transmitted infections and were independently associated with increased HIV acquisition in multivariate analysis. Conclusions:Despite significant HIV inhibitory activity, cervicovaginal levels of α-defensins and LL-37 were associated with increased HIV acquisition, perhaps due to their association with bacterial sexually transmitted infections.

Prevalent Herpes Simplex Virus Type 2 Infection is Associated with Altered Vaginal Flora and an increased Susceptibility to Multiple Sexually Transmitted Infections

BACKGROUND Prevalent herpes simplex virus type 2 (HSV-2) infection increases human immunodeficiency virus acquisition. We hypothesized that HSV-2 infection might also predispose individuals to acquire other common sexually transmitted infections (STIs). METHODS We studied the association between prevalent HSV-2 infection and STI incidence in a prospective, randomized trial of periodic STI therapy among Kenyan female sex workers. Participants were screened monthly for infection with Neisseria gonorrhoeae and Chlamydia trachomatis, and at least every 6 months for bacterial vaginosis (BV) and infection with Treponema pallidum, Trichomonas vaginalis, and/or HSV-2. RESULTS Increased prevalence of HSV-2 infection and increased prevalence of BV were each associated with the other; the direction of causality could not be determined. After stratifying for sexual risk-taking, BV status, and antibiotic use, prevalent HSV-2 infection remained associated with an increased incidence of infection with N. gonorrhoeae (incidence rate ratio [IRR], 4.3 [95% confidence interval {CI}, 1.5-12.2]), T. vaginalis (IRR, 2.3 [95% CI, 1.3-4.2]), and syphilis (IRR, 4.7 [95% CI, 1.1-19.9]). BV was associated with increased rates of infection with C. trachomatis (IRR, 2.1 [95% CI, 1.1-3.8]) and T. vaginalis (IRR, 8.0 [95% CI, 3.2-19.8]). CONCLUSION; Increased prevalences of HSV-2 infection and BV were associated with each other and also associated with enhanced susceptibility to an overlapping spectrum of other STIs. Demonstration of causality will require clinical trials that suppress HSV-2 infection, BV, or both.

HIV-neutralizing immunoglobulin A and HIV-specific proliferation are independently associated with reduced HIV acquisition in Kenyan sex workers.

Objectives:HIV-neutralizing immunoglobulin A (IgA) and HIV-specific cellular immunity have been described in highly exposed, persistently seronegative (HEPS) individuals, but well controlled studies have not been performed. We performed a prospective, nested case–control study to examine the association of genital IgA and systemic cellular immune responses with subsequent HIV acquisition in high-risk Kenyan female sex workers (FSWs). Design and methods:A randomized trial of monthly antibiotic prophylaxis to prevent sexually transmitted disease/HIV infection was performed from 1998 to 2002 in HIV-uninfected Kenyan FSWs. After the completion of trial, FSWs who had acquired HIV (cases) were matched 1: 4 with persistently uninfected controls based on study arm, duration of HIV-seronegative follow-up, and time of cohort enrolment. Blinded investigators assayed the ability at enrolment of genital IgA to neutralize primary HIV isolates as well as systemic HIV-specific cellular IFNγ-modified enzyme-linked immunospot and proliferative responses. Results:The study cohort comprised 113 FSWs: 24 cases who acquired HIV and 89 matched controls. Genital HIV-neutralizing IgA was associated with reduced HIV acquisition (P = 0.003), as was HIV-specific proliferation (P = 0.002), and these associations were additive. HIV-specific IFNγ production did not differ between case and control groups. In multivariable analysis, HIV-neutralizing IgA and HIV-specific proliferation each remained independently associated with lack of HIV acquisition. Genital herpes (HSV2) was associated with increased HIV risk and with reduced detection of HIV-neutralizing IgA. Conclusion:Genital HIV-neutralizing IgA and systemic HIV-specific proliferative responses, assayed by blinded investigators, were prospectively associated with HIV nonacquisition. The induction of these immune responses may be an important goal for HIV vaccines.