Rupert Kaul

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.

HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers.

OBJECTIVESnMost HIV-1 transmission is sexual; therefore, immune responses in the genital mucosa may be important in mediating protection against HIV infection. This study examined HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan female sex workers.nnnMETHODSnHIV-1-specific immune responses were compared in HIV-1-resistant and HIV-1-infected sex workers, and in lower risk uninfected women. Cervical and vaginal samples from each group were tested for HIV-1-specific IgA and IgG by enzyme immunoassay. Systemic T-helper lymphocyte cell responses to HIV-1 envelope peptide epitopes were assayed using an interleukin 2 bioassay. HIV-1 risk-taking behaviours were assessed using standardized questionnaires.nnnRESULTSnHIV-1-specific IgA was present in the genital tract of 16 out of 21 (76%) HIV-1-resistant sex workers, five out of 19 (26%) infected women, and three out of 28 (11%) lower risk women (P < 0.0001). Among lower risk women, the presence of HIV-1-specific IgA was associated with HIV-1 risk-taking behaviour. Systemic T-helper lymphocyte responses to HIV-1 envelope peptides were present in 11 out of 20 (55%) HIV-1-resistant women, four out of 18 (22%) infected women, and one out of 25 (4%) lower risk women (P < 0.001). T-helper lymphocyte responses did not correlate with the presence or titre of virus-specific mucosal IgA in any study group.nnnCONCLUSIONSnHIV-1-specific IgA is present in the genital tract of most HIV-1-resistant Kenyan sex workers, and of a minority of lower risk uninfected women, where it is associated with risk-taking behaviour. These data suggest a role for mucosal HIV-1-specific IgA responses in HIV-1 resistance, independent of host cellular responses.

Mucosal and Plasma IgA from HIV-1-Exposed Uninfected Individuals Inhibit HIV-1 Transcytosis Across Human Epithelial Cells

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

Recombination following superinfection by HIV-1.

Background: There is increasing recognition of recombinant HIV-1 strains globally, but it has been unclear whether recombination results from superinfection during untreated, chronic infection. Objective: To search for evidence of recombination and superinfection in Africa, where multiple HIV-1 subtypes facilitate identification of strains. Methods: Serial blood samples from highly exposed, chronically infected women in Nairobis Pumwani sex workers cohort were examined. Serial, complete HIV-1 RNA sequence analyses were performed for seven untreated long-term survivors. Sequences were subjected to computational analysis. Results: One woman had evidence of both superinfection and recombination. Complete HIV-1 RNA sequences were first derived from plasma obtained in 1986, when the woman had been HIV seropositive for at least 21 months; this sequence was entirely subtype A. The sequences obtained from plasma in 1995 and 1997, however, were subtype A/C recombinants with a SimPlot demonstrating that the subtype A fragment in 1995 and 1997 was derived from the original 1986 A sequence. Heteroduplex tracking assays demonstrated that the subtype C sequences were not detectable as minor species in 1986. Conclusion: Intersubtype recombination took place between the original non-recombinant subtype A strain and the superinfecting subtype C strain in an untreated, chronically infected woman. This finding helps to explain the rising prevalence of recombinant HIV-1 worldwide. Recombination resulting from superinfection with diverse strains may pose problems for eliciting broad immune responses necessary for an effective vaccine.

New insights into HIV-1 specific cytotoxic T-lymphocyte responses in exposed, persistently seronegative Kenyan sex workers

A clearer understanding of HIV-1 specific immune responses in highly-exposed, persistently seronegative (HEPS) subjects is important in developing models of HIV-1 protective immunity. HIV-1 specific cytotoxic T-lymphocytes (CTL) have been described in a cohort of HEPS Kenyan sex workers, and recent work has further elucidated these responses. CTL specific for HIV-1 Env were found in the blood of over half the sex workers meeting criteria for HIV resistance, and in some women recognized unmapped epitopes. The proportion of women with Env-specific CTL increased with the duration of uninfected HIV exposure, suggesting that these responses were acquired over time. CD8+ lymphocyte responses directed against predefined HIV-1 CTL epitopes from various HIV-1 genes were found in the blood and genital tract of >50% resistant sex workers, at a ten-fold lower frequency than in infected subjects. The epitope specificity of CD8+ responses differs between HEPS and HIV infected women, and in HEPS the maintenance of responses appears to be dependent on persistent HIV exposure. Several HIV-1 resistant sex workers have become HIV infected over the past 6 years, possibly related to waning of pre-existing HIV-specific CTL, and infection has often been associated with a switch in the epitope specificity of CD8+ responses. These findings suggest that vaccine-induced protective HIV immunity is a realistic goal, but that vaccine strategies of boosting or persistent antigen may be necessary for long-lived protection.

Functional HIV-1 specific IgA antibodies in HIV-1 exposed, persistently IgG seronegative female sex workers

Although HIV-specific cellular immune responses are found in a number of HIV highly-exposed, persistently seronegative (HEPS) cohorts, late seroconversion can occur despite pre-existing cytotoxic T lymphocytes (CTL), suggesting that a protective HIV vaccine may need to induce a broader range of HIV-specific immune responses. Low levels of HIV-specific IgA have been found in the genital tract and plasma of the majority of Nairobi HEPS sex workers and appeared to be independent of HIV-specific cellular responses. IgA purified from genital tract, saliva and plasma of most HEPS sex workers were able to neutralize infection of PBMC by a primary (NSI) clade B HIV isolate, as well as viral isolates from clades A and D, which predominate in Kenya. In addition, these IgA were able to inhibit transcytosis of infective HIV virions across a transwell model of the human mucosal epithelium in an HIV-specific manner. Preliminary work in other HEPS cohorts has suggested the recognition of different gp41 epitopes in HEPS and HIV-infected subjects. Although present at low levels, these IgA demonstrated cross-clade neutralizing activity and were able to inhibit HIV mucosal transcytosis, suggesting an important functional role in protection against HIV infection.

How important is the 'quality' of the cytotoxic T lymphocyte (CTL) response in protection against HIV infection?

Cytotoxic T lymphocyte (CTL) responses have been associated with protection from HIV-1 infection in people with a high degree of exposure to HIV and who show no serological evidence of HIV infection (HEPS, highly exposed persistently seronegative). However, it remains unclear how protective CTL responses could apparently develop in a minority of people, whilst the great majority of HIV-infected people make strong CTL responses yet progress to AIDS and death. In this paper we review the data which supports the hypothesis that the quality of the T-cell response, rather than its magnitude, may be an important factor that merits further investigation.

HIV-1 RNA Dysregulates the Natural TLR Response to Subclinical Endotoxemia in Kenyan Female Sex-Workers

Background Subclinical endotoxemia has been reported in HIV-1 infected persons and may drive systemic immune activation and pathogenesis. Proinflammatory responsiveness to endotoxin (LPS) is mediated by Toll-like receptor 4 (TLR4). We therefore examined the association between plasma LPS levels, HIV RNA, and TLR4 expression and cytokine responses in the blood of HIV infected and uninfected participants in a cohort of female sex-workers in Kenya. Methodology/Principal Findings Ex vivo plasma and peripheral blood mononuclear cells (PBMC) were assessed for LPS and TLR mRNA, respectively. The effects of HIV single stranded RNA, a TLR8 ligand, on TLR4 and LPS signaling were further assessed in short term PBMC culture. Both HIV uninfected and infected subjects frequently had low detectable LPS levels in their plasmas. Significantly increased LPS levels were associated with chronic HIV-1 infection, both treated and untreated, but not with other acute or semi-chronic conditions reported. In HIV-uninfected subjects, TLR4 mRNA expression levels correlated inversely with plasma LPS levels, suggesting chronic endotoxin ‘tolerance’ in vivo. A similar effect of reduced TLR4 mRNA was seen in short term PBMC culture after stimulation with LPS. Interestingly, the apparent in vivo tolerance effect was diminished in subjects with HIV infection. Additionally, pre-stimulation of PBMC with LPS lead to proinflammatory (TNF-α) tolerance to subsequent LPS stimulation; however, pre-treatment of PBMC with HIV single-stranded RNA40, could enhance TLR4-mediated LPS responsiveness in vitro. Conclusions/Significance Thus, dysregulation of endotoxin tolerance by HIV-1 RNA may exacerbate HIV chronic immune activation and pathogenesis.

New Insights into Host Factors in HIV-1 Pathogenesis

Although AIDS-related morbidity and mortality were dramatically reduced by combination antiretroviral therapy (Lederman and Valdez, 2000xLederman, M.M. and Valdez, H. JAMA. 2000; 284: 223–228Crossref | PubMedSee all References(Lederman and Valdez, 2000), current therapeutic strategies have limitations. Cumbersome regimens and a plethora of drug side effects lead to poor compliance, and viruses bearing drug resistance mutations now account for a significant number of new infections. Early optimism that antiretroviral therapy could eradicate HIV-1 was tempered on observing the rapid viral relapse that occurs when therapy is discontinued. In consideration of these factors, an argument has been made for delaying therapy in asymptomatic, HIV-infected individuals with adequate CD4+ T cell counts. In this approach, treatment for these patients would be reserved until later in the disease, when the benefits are likely to outweigh the problems of side-effects and viral resistance.There is debate as to whether antiretroviral therapy helps or harms the immune system in its battle with the virus. Suppression of plasma viremia in chronic infection is usually associated with loss or reduction of HIV-1-specific CTL responses (Altfeld et al., 2001xAltfeld, M., Rosenberg, E.S., Shankarappa, R., Mukherjee, J.S., Hecht, F.M., Eldridge, R.L., Addo, M.M., Poon, S.H., Phillips, M.N., Robbins, G.K. et al. J. Exp. Med. 2001; 193: 169–180Crossref | PubMed | Scopus (314)See all References(Altfeld et al., 2001) (see Table 1Table 1) , which could reduce the ability of the immune system to control viral replication when drug therapy fails. Th responses are rarely restored following antiretroviral therapy in chronic infection, even with maximal viral suppression. Structured treatment interruptions (STI), whereby therapy is withheld for carefully controlled intervals in the hope of allowing limited viral replication to “reprime” the cellular immune system, could combine the benefits of viral suppression without the disadvantages of losing the CTL response (Montaner, 2000xMontaner, L.J. Mod. Trends Immunol. 2000; 22: 92–96Abstract | Full Text | Full Text PDF | Scopus (37)See all References(Montaner, 2000). Is there any other way of restoring immune competence to people with chronic HIV-1 infection? Interleukin-2 (IL-2) is the main cytokine produced by Th cells, and has been widely used in HIV therapy. IL-2 can lead to dramatic improvement in CD4+ T cell counts but this is not necessarily translated into clinical benefit or improved immune competence (Lederman and Valdez, 2000xLederman, M.M. and Valdez, H. JAMA. 2000; 284: 223–228Crossref | PubMedSee all References(Lederman and Valdez, 2000). IL-2 could also activate latently infected CD4+ T cells, “flushing out” HIV-1 from its hiding places, thereby allowing antiretroviral drugs to reduce the latent viral burden to levels that could be controlled by host cellular immune responses alone. However, in practice, rapid viral rebound is still seen upon discontinuation of therapy. Similarly, attempts to boost immune responses against the virus with strategies such as therapeutic vaccination with HIV immunogens or infusion of HIV-specific T cells have not so far resulted in long-lasting immunological or clinical benefit, although these efforts are continuing.Table 1Impact of the Timing of Antiretroviral Therapy on Viral Parameters and Components of the Host HIV-Specific Cellular Immune ResponseEarly therapy** ± STI††Late therapy‡‡Plasma viremia↓↓↓↓↓↓CD4 count/mm3↑↑↑Anti-HIV CTL activityearly ↑, gradual ↓gradual ↓Anti-HIV T-helper activity↑↑generally not detectedAnti-HIV CD8+ noncytotoxic activity↓/↑↑Cumulative in vivo HIV replicationlowvery highHIV viral diversitylowhigh*Antiretroviral therapy initiated within 6 months of primary infection.†Supervised treatment interruption.‡Antiretroviral therapy more than 6 months after primary infection.If patients can be recognized very early in HIV infection, there are clear immunological benefits from prompt institution of combination drug therapy. Early therapy during acute HIV-1 infection leads to preserved Th responses (Oxenius et al. 2000xOxenius, A., Price, D.A., Easterbrook, P.J., OCallaghan, C.A., Kelleher, A.D., Whelan, J.A., Sontag, G., Sewell, A.K., and Phillips, R.E. Proc. Natl. Acad. Sci. USA. 2000; 97: 3382–3387Crossref | PubMed | Scopus (323)See all References, Altfeld et al. 2001xAltfeld, M., Rosenberg, E.S., Shankarappa, R., Mukherjee, J.S., Hecht, F.M., Eldridge, R.L., Addo, M.M., Poon, S.H., Phillips, M.N., Robbins, G.K. et al. J. Exp. Med. 2001; 193: 169–180Crossref | PubMed | Scopus (314)See all References), which are not seen if therapy is delayed by as little as six months (Oxenius et al., 2000xOxenius, A., Price, D.A., Easterbrook, P.J., OCallaghan, C.A., Kelleher, A.D., Whelan, J.A., Sontag, G., Sewell, A.K., and Phillips, R.E. Proc. Natl. Acad. Sci. USA. 2000; 97: 3382–3387Crossref | PubMed | Scopus (323)See all References(Oxenius et al., 2000). The breadth and magnitude of CTL responses detected after early initiation of therapy may be lower than usually seen in chronic infection (Altfeld et al., 2001xAltfeld, M., Rosenberg, E.S., Shankarappa, R., Mukherjee, J.S., Hecht, F.M., Eldridge, R.L., Addo, M.M., Poon, S.H., Phillips, M.N., Robbins, G.K. et al. J. Exp. Med. 2001; 193: 169–180Crossref | PubMed | Scopus (314)See all References(Altfeld et al., 2001). This could be because early therapy, by rapidly reducing initial viremia, also leads to reduced viral diversity: this would reduce the likelihood of CTL escape (Allen et al., 2000xAllen, T.M., OConnor, D.H., Jing, P., Dzuris, J.L., Mothe, B.R., Vogel, T.U., Dunphy, E., Liebl, M.E., Emerson, C., Wilson, N. et al. Nature. 2000; 407: 386–390Crossref | PubMed | Scopus (226)See all References(Allen et al., 2000), and thereby the requirement for a broad and/or intense host CTL response. The combination of early therapy and STI may be particularly valuable in primary HIV-1 infection (Rosenberg et al., 2000xRosenberg, E.S., Altfeld, M., Poon, S.H., Phillips, M.N., Wilkes, B.M., Eldridge, R.L., Robbins, G.K., DAquila, R.T., Goulder, P.J., and Walker, B.D. Nature. 2000; 407: 523–526Crossref | PubMed | Scopus (811)See all References(Rosenberg et al., 2000). Although some degree of plasma viral rebound was seen in all subjects during the initial STIs in this study, there were substantial improvements in the breadth and magnitude of both HIV-1-specific CTL and Th responses, and a few patients were eventually able to stop therapy altogether. As even carefully controlled HIV-1 replication may cause long-term damage, the hope is that therapeutic vaccination with constructs designed to elicit a cellular immune response may prove a safer alternative.The future generation of HIV therapies consists largely of agents aiming to inhibit the critical early interactions between the virus and its target cell, either acting at the level of CD4 or the chemokine receptor or targeting envelope glycoproteins, with a view to blocking attachment or fusion. Several promising compounds have been identified (reviewed in DSouza et al., 2000xDSouza, M.P., Cairns, J.S., and Plaeger, S.F. JAMA. 2000; 284: 215–222Crossref | PubMedSee all ReferencesDSouza et al., 2000) and clinical trials are now underway, although it is likely to be some time before this will translate into a new class of antiretroviral drugs.Malim Emmerman 2001xMalim, M.H. and Emmerman, M. Cell. 2001; 104: 469–472Abstract | Full Text | Full Text PDF | PubMed | Scopus (140)See all ReferencesMalim Emmerman 2001

Disproportionately High Semen Shedding of HIV Is Associated with Compartmentalized Cytomegalovirus Reactivation

Semen transmission of human immunodeficiency virus (HIV) drives the global pandemic. HIV loads are generally lower in semen than in blood, but semen loads may be disproportionately high in a subgroup of men. HIV loads in semen exceeded those in blood in 9 (35%) of 26 of antiretroviral therapy-naive men, and disproportionately high shedding was strongly associated with compartmentalized semen cytomegalovirus (CMV) reactivation (odds ratio [OR], 10.5; P<.01). Overall, 17 of 26 participants were shedding CMV in semen. Semen levels of HIV and CMV were closely correlated (r=0.5; P<.01), independently of blood HIV load and CD4(+) T cell count. Prevention of CMV reactivation warrants further study as a possible strategy to reduce semen shedding of HIV.