Mucosal Neisseria gonorrhoeae coinfection during HIV acquisition is associated with enhanced systemic HIV-specific CD8 T-cell responses
Sheung, Anthonya; Rebbapragada, Anua; Shin, Lucy YYa; Dobson-Belaire, Wendyb; Kimani, Joshuac; Ngugi, Elizabethd; MacDonald, Kelly Sa,e; Bwayo, Job Jc,†; Moses, Stephenf; Gray-Owen, Scottb; Kaul, Ruperta,c,g; The Kibera HIV Study Group
aDepartment of Medicine, Canada
bDepartment of Medical Genetics, University of Toronto, Ontario, Canada
cDepartment of Medical Microbiology, Kenya
dDepartment of Community Health, University of Nairobi, Nairobi, Kenya
eDepartment of Microbiology, Mount Sinai Hospital, Toronto, Canada
fDepartment of Community Health Sciences and Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
gDepartment of Medicine, University Health Network, Ontario, Canada.
† Job J. Bwayo deceased.
Received 22 December, 2007
Revised 16 May, 2008
Accepted 27 May, 2008
Correspondence to Dr Rupert Kaul, Clinical Science Division, University of Toronto, Medical Sciences Building #6356, Toronto, Ontario M5S 1A8, Canada. Tel: +1 416 978 8607; fax: +1 416 978 8765; e-mail: firstname.lastname@example.org
Background: The host immune response against mucosally acquired pathogens may be influenced by the mucosal immune milieu during acquisition. As Neisseria gonorrhoeae can impair dendritic cell and T-cell immune function, we hypothesized that coinfection during HIV acquisition would impair subsequent systemic T-cell responses.
Methods: Monthly screening for sexually transmitted infections was performed in high risk, HIV seronegative Kenyan female sex workers as part of an HIV prevention trial. Early HIV-specific CD8 T-cell responses and subsequent HIV viral load set point were assayed in participants acquiring HIV, and were correlated with the presence of prior genital infections during HIV acquisition.
Results: Thirty-five participants acquired HIV during follow-up, and 16 out of 35 (46%) had a classical sexually transmitted infection at the time of acquisition. N. gonorrhoeae coinfection was present during HIV acquisition in 6 out of 35 (17%), and was associated with an increased breadth and magnitude of systemic HIV-specific CD8 T-cell responses, using both interferon-gammaγ and MIP-1 beta as an output. No other genital infections were associated with differences in HIV-specific CD8 T-cell response, and neither N. gonorrhoeae nor other genital infections were associated with differences in HIV plasma viral load at set point.
Conclusion: Unexpectedly, genital N. gonorrhoeae infection during heterosexual HIV acquisition was associated with substantially enhanced HIV-specific CD8 T-cell responses, although not with differences in HIV viral load set point. This may have implications for the development of mucosal HIV vaccines and adjuvants.
Sexually transmitted infections (STI) are important cofactors in the acquisition, transmission and progression of HIV-1, which is most commonly acquired across the genital or rectal mucosa during unprotected sex . Ulcerative STI such as Herpes simplex virus type 2 (HSV-2) are thought to facilitate HIV acquisition by causing disruption of normal epithelial barrier, providing a route of entry for HIV , and may also recruit susceptible cells to the genital mucosa . Non-ulcerative STI such as Neisseria gonorrhoeae and Chlamydia trachomatis may also recruit and activate HIV-susceptible cells [1,4]. Given that STIs predispose to HIV acquisition, and that unprotected sex is associated with acquisition of both STI and HIV, it is perhaps not surprising that concurrent STI are very common in individuals with acute HIV infection .
HIV-specific host immunity, although unable to clear infection, plays an important role in partial viral containment [6,7]. In particular, a robust early CD8 T-cell cytotoxic T-cell lymphocyte (CTL) response is crucial in the control of HIV after acute infection, and CTL-mediated immune pressure has been demonstrated to be a major evolutionary force on both SIV and HIV [8–10]. The impact of a concurrent STI on host immune control of newly acquired HIV is not known, but STI can have important immunopathogenetic effects in other settings. During chronic HIV infection, incident infection by N. gonorrhoeae has been associated with transient increases in blood HIV viral load and decreased absolute CD4 T-cell counts , an effect that may relate to gonococcal impairment of HIV-specific CD8 T-cell responses  and CD4 T-cell function . In addition, N. gonorrhoeae inhibits the maturation of dendritic cells, and their ability to prime HIV-specific immune responses . Therefore, it has been hypothesized that host immune impairment due to frequent STI, particularly N. gonorrhoeae, may be partly responsible for the rapid HIV disease progression seen in Nairobi female sex workers (FSW) . If this is the case during chronic HIV infection, then the presence of N. gonorrhoeae infection at the time of HIV acquisition and initial immune priming may be particularly deleterious. However, the effect of mucosal coinfections on immunity against mucosally acquired pathogens has not been well studied.
Despite the potential of N. gonorrhoeae to impair host immunity, peptide mimics of neisserial polysaccharide from N. meningitidis are immunogenic, and are being developed as possible mucosal vaccine adjuvants [16–18]. In addition, gonococcal lipooligosaccharide can induce anti-HIV innate immunity in primary human macrophages, through interferon beta (IFNβ) release induced by toll-like receptor-4 (TLR4) signaling . Although these studies suggest that neisserial induction of innate immunity has the potential to enhance anti-HIV immunity, the genital application of TLR7 and TLR9 agonists was associated with mucosal inflammation, no protection against SIV infection, and higher peak viral loads . Elucidating the impact of concurrent STI on subsequent host immune control of HIV in humans could have significant implications in the control of transmission and treatment of HIV, and provide insight in the context of developing HIV mucosal vaccine adjuvants.
We sought to address this question using a unique repository of cryopreserved viable lymphocytes collected from Kenyan FSW with newly acquired HIV infection, in whom detailed STI diagnostics had been performed prior to HIV acquisition. We performed a blinded analysis of the effect of genital coinfections on acute HIV-specific CD8 T-cell immunity and viral load at set point. Specifically, we hypothesized that the immunomodulatory effects of coinfection by N. gonorrhoeae at the time of HIV acquisition would result in the impaired generation and function of HIV-specific CD8 T-cell responses.
Study participants and diagnostic testing for HIV and STI
Study participants were Kenyan commercial sex workers (CSW) enrolled in a clinical trial of HIV and STI prevention that was performed between 1998 and 2002 . All participants were HIV seronegative at enrolment. Full STI diagnostic testing and therapy was performed at baseline. Specifically, cervical swabs were obtained for N. gonorrhoeae and C. trachomatis PCR (Amplicor PCR Diagnostics, Roche Diagnostics Systems, Ontario, Canada) and for N. gonorrhoeae culture. Vaginal Trichomonas vaginalis culture was performed using In-Pouch-TV (Biomed Diagnostics, San Jose, California, USA), a Gram stain was performed, and blood was drawn for HIV and syphilis serology. If a genital ulcer was present, a swab of the ulcer base was taken for M-PCR detection of HSV-2 and Troponema pallidum (Roche Molecular Systems). Bacterial vaginosis was defined as a Nugent score of 7–10 , and lactobacillus colonization and candidiasis were defined as the finding of any lactobacilli or yeast, respectively, on the Gram-stained specimen. Subsequently, urine was collected every month for PCR detection of N. gonorrhoeae and C. trachomatis. The full STI diagnostic screening protocol was performed every 6 months, and whenever participants presented with genital symptoms. Any infections diagnosed were treated according to Kenyan national guidelines. HIV-1 serology was performed every 3 months. Ethics approval for both the larger trial and this immunological substudy were obtained through the research ethics board of collaborating institutions (Universities of Toronto, Manitoba and Nairobi).
Isolation of peripheral blood mononuclear cells
Blood was transported to an on-site laboratory within 4 h of collection. Peripheral blood mononuclear cells (PBMC) were isolated from blood collected into Acid Citrate Dextran solution A (ACD; BD Bioscience, San Jose, California, USA) by density gradient centrifugation over Ficoll-Paque Plus (Amersham Biosciences, Piscataway, New Jersey, USA) and cryopreserved at −150 °C in fetal bovine serum (FBS) with 10% dimethyl sulfoxide (Fisher Scientific, Fairlawn, New Jersey, USA), as previously described . Blood plasma was flash frozen at −80 °C. PBMC and plasma samples were collected and stored every 3 months during the clinical trial, whenever HIV enzyme-linked immunosorbent assay (ELISA) was performed.
HIV-specific CD8 T-cell intracellular cytokine assays
All assays were performed by research personnel blinded to participant clinical status (presence of a genital infection) and HIV viral load. HIV-specific production of interferon-gamma (IFNγ) and MIP1-beta (MIP1b) by CD8 T cells was examined at the first HIV seropositive visit, using short-term peptide stimulation followed by intracellular cytokine staining. Thawed PBMCs were washed once and resuspended in R10 tissue culture medium. One million PBMCs were spun down and resuspended in 150 μl of R10 with either culture medium alone, staphylococcal enterotoxin B (SEB; Sigma–Aldrich, Oakville, Ontario, Canada), or pooled overlapping HIV peptides obtained from the NIH Reagent Program. Overlapping peptides were based on either clade A (Gag) or clade B (Pol, Env, Rev, Nef, Tat, Vif, Vpr, and Vpu) consensus sequence. Four separate HIV peptide pools were made up spanning HIV-1 Gag, Pol, Env, and the accessory genes (Rev, Nef, Tat, Vif, Vpr, and Vpu; ‘accessory pool’) to a final concentration of 0.1 μg/ml per peptide. A single pool of peptides spanning the entire HIV-1 genome (‘all-HIV’ pool) was also tested, with each peptide at a final concentration of 0.01 μg/ml. PBMC were incubated for 1 h at 37 °C (5% CO2), brefeldin A (Sigma–Aldrich) was added to a final concentration of 10 μg/ml, and cells incubated for an additional 5 h at 37 °C (5% CO2). Assays were stopped at 4 °C overnight, and cells washed once the following day with 1% FBS in phosphate buffered saline (PBS; Invitrogen, Burlington, Ontario, Canada), and permeabilized in BD permeabilizing solution (BD Biosciences, Mississuaga, Ontario, Canada) according to manufacturers' instructions. Fluorescein isothiocynate conjugated anti-CD3, peridinin–chlorophyll–protein-conjugated (PerCP) anti-CD8 conjugated, phycoerythrin conjugated anti-MIP-1β, and allophycocyanin conjugated IFNγ (BD Biosciences) were added to the resuspended cells for 30 min at 4 °C, washed once with 1% FBS and PBS and supernatant decanted. Stained cells were fixed, and flow cytometric acquisition performed using FACS Calibur (BD Biosciences), with results analyzed using the FlowJo software (Tree Star Incorporated, Ashland, Oregon, USA). A positive response was defined as background-corrected cytokine production in the HIV peptide well exceeding 0.05% of gated cells, and exceeding background levels of cytokine production by at least twofold.
HIV-1 viral load measurement and establishment of viral load set point
HIV-1 RNA viral load was assayed using the VERSANT HIV-1 RNA3.0 bDNA assay, with a limit of detection of 50 HIV-1 RNA copies/ml (Bayer Diagnostics, Leverkusen, Germany). A single blood plasma sample collected at least 3 months after the first HIV seropositive visit was used to determine the viral load set point, after appropriate medical therapy of any prior STIs. When plasma samples were available, viral load was also assayed contemporaneously with the early phase PBMC samples used in the CD8 T-cell assays.
All analyses were performed with SPSS version 11.0 software (SPSS Inc., Chicago, Illinois, USA). Associations of continuous variables were determined using a Mann–Whitney U non-parametric test. Comparisons between discrete variables were performed using the χ2 test with calculations of likelihood ratios. Bivariate correlations were calculated using the Pearson two-tailed test.
Study population and clinical characteristics
A total of 466 HIV-uninfected FSW from the Kibera slum of Nairobi were enrolled and followed prospectively during 1998–2002 ; 35 participants acquired HIV despite a very successful sexual risk reduction program . HIV serology was performed every 3 months, and as there is generally a ‘window period’ of several weeks after acute HIV infection before the immunoglobulin G (IgG) ELISA becomes positive, any genital infection diagnosed during the 3 months prior to HIV seroconversion was assumed to have been present or acquired at the time of HIV acquisition. Among CSW acquiring HIV, 16 out of 35 (46%) had a bacterial or protozoan genital tract infection during HIV acquisition, and 2 out of 35 (6%) had multiple genital infections. Specifically, 5 out of 35 (14%) participants were infected by T. vaginalis; 5 out of 35 (14%) by C. trachomatis; 3 out of 35 (9%) by Troponema pallidum; and 6 out of 35 (17%) by N. gonorrhoeae. Fifteen participants (43%) had prior bacterial vaginosis. Almost all participants who acquired HIV were chronically infected by HSV-2 (33/35; 94%). Overall, the mean plasma HIV viral load at set point among women who acquired HIV was 26 309 copies/ml (range; <50–131 223 copies/ml; Table 1).
N. gonorrhoeae infection and subsequent HIV-specific CD8 T-cell responses
Unexpectedly, infection by N. gonorrhoeae at the time of HIV acquisition was associated with significantly broader and stronger systemic HIV-specific CD8 T-cell IFNγ responses (Fig. 1a; gating strategy and representative dot plots shown in Fig. 1d). CD8 responses directed against the peptide pool spanning the entire HIV genome were stronger (1.3 versus 0.3%; Mann–Whitney P = 0.02), as were responses to several HIV gene-specific pools (Gag, 0.5 versus 0.1%, P = 0.03; Env, 0.3 versus 0.1%, P = 0.02; accessory gene pool 1.0 versus 0.2%, P = 0.06). In addition, participants with concurrent N. gonorrhoeae infection were more likely to have a positive response to the HIV whole genome pool (6/6 versus 18/29; likelihood ratios = 5.1; P = 0.02), as well as to the accessory gene (5/6 versus 7/26; likelihood ratios = 6.8; P = 0.01) and Gag (4/6 versus 3/26; likelihood ratios = 7.4; P = 0.007) pools. N. gonorrhoeae infection was associated with recognition of a larger number of HIV peptide pools (2.5 versus 1.0; Mann–Whitney P = 0.009). Background production of IFNγ and responses to non-specific stimulation by the superantigen SEB were not affected by N. gonorrhoeae infection status (data not shown).
Because defining immune function based solely on IFNγ production may incompletely identify HIV-specific CD8 T-cell populations , HIV-specific MIP1b production was also assessed (Fig. 1b). Again, CD8 T-cell responses in the N. gonorrhoeae infected subgroup were significantly enhanced against the peptide pool spanning the entire HIV genome (1.8 versus 0.6%; Mann–Whitney P = 0.02), as well as to several HIV gene-specific pools (Gag, 0.7 versus 0.2%, P = 0.07; accessory gene pool 0.9 versus 0.3%, P = 0.05). In addition, the frequency of HIV-specific CD8 T cells coexpressing both IFNγ and MIP1b was also consistently enhanced in the N. gonorrhoeae infected subgroup (Fig. 1c).
Immune associations of other genital infections during HIV acquisition
It was possible that the N. gonorrhoeae-associated differences in HIV-specific CD8 T-cell immunity were nonspecific, and would be induced by any genital inflammatory process present at the time of HIV acquisition. The effect of other genital infections was therefore assessed, after the exclusion of participants with N. gonorrhoeae infection. Neither C. trachomatis infection (n = 5), T. vaginalis infection (n = 5) nor bacterial vaginosis (n = 17) during HIV acquisition were associated with differences in subsequent HIV-specific IFNγ or MIP1b responses to any HIV peptide pools (all P > 0.1; Fig. 2a–c), and nor was prior syphilis (n = 3; all P > 0.5).
Although HSV-2 coinfection has been associated with impaired HIV-specific CD8 T-cell immunity , virtually all sex worker participants acquiring HIV were coinfected by HSV-2 (33/35; 94%), precluding analysis of HSV-2 serostatus and HIV-specific immunity. Routine screening for asymptomatic HSV-2 genital shedding was not performed, and so the associations of transient genital HSV-2 reactivation on subsequent HIV immune control were not examined.
Associations of HIV viral load
The association of HIV RNA plasma viral load set point with concurrent gonorrhea at the time of HIV acquisition was assessed in the 25 out of 35 participants (71%) with plasma available more than 3 months after the first seropositive visit. The median time from first HIV seropositive visit to viral load set point measurement was 278 days, and did not vary based on gonorrhea infection status (P = 0.3). Despite the differences in HIV-specific CD8 T-cell responses, there was no difference in HIV RNA set point between participants with (n = 5) and without (n = 20) N. gonorrhoeae infection during HIV acquisition (3.8 versus 3.8 log10 copies/ml; P = 0.8).
Early phase HIV viral load contemporaneous with the PBMCs sample used in the CD8 T-cell assay was available for 17 of 35 participants (two coinfected by N. gonorrhoeae). Viral load at this early time point was not correlated with the CD8 T-cell response against any HIV gene pool, using either IFNγ or MIP1b as an output (P > 0.05 for all). However, there was a strong trend to an increased early phase viral load in those participants with N. gonorrhoeae coinfection (5.0 versus 3.7 log10 RNA copies/ml; P = 0.1).
STI cause considerable perturbation of the mucosal immune milieu of the genital tract , and are commonly present during acute HIV infection . However, to study the impact of preexisting STI on HIV immune responses and viral load set point requires preacquisition screening for genital infections, and is logistically very difficult. After following a large cohort of high-risk Kenyan CSW for an average of 2 years, we identified 35 women acquiring HIV. Just under half of the participants acquiring HIV were coinfected with a classical STI at the time of acquisition, N. gonorrhoeae in approximately one-third of cases. We had hypothesized that HIV acquisition by women with concurrent gonorrhea would be associated with the impaired generation and maintenance of subsequent HIV-specific CD8 T-cell responses. However, in this operator-blinded study, we clearly demonstrated that host HIV-specific CD8 T-cell responses were enhanced in N. gonorrhoeae-infected women, who generated both stronger and broader responses. This unexpected observation applied when using both ex vivo IFNγ and MIP1b production as an experimental output, and was seen across a range of individual HIV genes and a pool spanning the entire HIV genome.
The mechanism underlying the N. gonorrhoeae-associated enhancement in HIV-specific CD8 T-cell responses is not clear, and might relate to factors in either the transmitting partner (the male client) or the acquiring partner (the FSW). Enhanced CD8 T-cell responses in the acquiring partner might be an indirect reflection of a higher ‘challenge dose’ of HIV during unprotected sex with a male client who has HIV–STI coinfection, as gonococcal urethritis may increase the level of HIV RNA in semen as much as 10-fold . However, the observed enhancement of CD8 T-cell immune responses in the acquiring partner was specific for N. gonorrhoeae coinfection, and was not seen in women with coinfection by C. trachomatis or T. vaginalis, both of which also cause male urethritis and increased HIV levels in both the semen [28–30] and cervix .
Infection by N. gonorrhoeae may also have influenced antiviral T-cell responses through immunologic or virologic effects in the acquiring partner. Genital infections have been demonstrated to increase local levels of multiple cytokines and chemokines . Type I interferons such IFNα may directly impair local HIV replication in CD4 T cells , perhaps delaying systemic dissemination of virus and allowing more time for the host to prime HIV-specific CD8 T-cell responses. In addition, local increases in Th1-type cytokines such as TNFα and IFNγ, or the immunostimulatory effects of IFNα on dendritic and T cells, might have promoted a more vigorous CD8 T-cell response . Indeed, gonococcal porins are the most represented surface proteins on pathogenic Neisseria, and act as immune adjuvants through the upregulation of B7-2 (CD86) on the antigen presenting cell surface , which can in turn drive MHC-restricted antigen-specific CTL responses in vivo . However, demonstrating whether these mechanisms contributed to immune enhancement was beyond the scope of our study.
Alternatively, active coinfection by N. gonorrhoeae in the acquiring partner might have been associated with a higher peak viral load during acute HIV infection, similar to the effects on plasma viral load during chronic HIV infection . This could then have induced a more robust virus-specific CD8 T-cell response as a reaction to the higher antigen load, without effects on the viral load set point, which was measured after N. gonorrhoeae therapy. Although plasma was not collected frequently enough to define the peak viral load, the strong trend towards a higher plasma viral load in N. gonorrhoeae-infected participants at the earliest available study time point supports this hypothesis. Whether therapy would affect N. gonorrhoeae-associated increases in early phase HIV-specific CD8 T-cell responses is not known.
These findings appear to conflict with earlier in-vivo observations that N. gonorrhoeae infection was associated with impaired function of HIV-specific CD8 T cells during chronic HIV infection , and inhibited T-cell activation and proliferation in vitro . However, these prior reports did not examine N. gonorrhoeae effects at the time of HIV mucosal acquisition, and so would miss any potential effects of N. gonorrhoeae on HIV challenge dose or mucosal immune priming. In addition, the previous in-vivo study examined the effects of gonorrhea in participants with chronic HIV infection , and both HIV infection status and plasma viral load might significantly alter the immune effects of N. gonorrhoeae coinfection .
Despite the observed enhancement of HIV-specific immune responses in N. gonorrhoeae coinfected participants, there was no suggestion of a benefit in terms of protection against HIV acquisition or improved host immune control of HIV. Indeed, incident infection by N. gonorrhoeae in this cohort was associated with a five-fold increased risk of HIV acquisition , perhaps owing to mucosal immune activation with the local recruitment of HIV-susceptible CD4 T lymphocytes and dendritic cells . More surprisingly, the increased strength and breadth of the HIV-specific CD8 T-cell response did not correlate with a reduced HIV plasma viral load set point. As discussed above, this might imply that the observed increase in virus-specific CD8 T cells was simply a reactive response to a higher early HIV plasma viral load in N. gonorrhoeae coinfected participants. Alternatively, as recent studies have demonstrated that CD8 T-cell functions such as degranulation or interleukin 2 production may predict host HIV immune control better than IFNγ production alone , it may be that the immune implications of enhanced CD8 T-cell responses were incompletely defined using just IFNγ and MIP1b production as our functional outputs.
Some limitations were imposed by our study population and methodology. In particular, participant numbers were relatively small, and the descriptive nature of our immune studies means that it was not possible to prove the mechanisms underpinning our findings. The immune effects of several coinfections were analyzed, raising the possibility that the N. gonorrhoeae associations were due to chance alone. However, the enhancement of host CD8 T-cell responses was clearly limited to N. gonorrhoeae, and for this infection the effects were seen across a range of HIV genes and functional outputs. This strongly suggests that this represents a true finding, although it will be important to confirm these findings in other cohorts. The use of overlapping peptides based on consensus clade B sequence (for HIV genes other than Gag) was based on reagent availability. HIV clade A predominates in East Africa , and so the use of clade B peptides may have resulted in underestimation of CD8 T-cell frequencies . However, this should not have affected our observations regarding the impact of N. gonorrhoeae infection on these immune responses.
In conclusion, genital N. gonorrhoeae infection at the time of heterosexual acquisition of HIV was associated with stronger and broader subsequent HIV-specific CD8 T-cell responses, without any observed differences in plasma RNA viral load. Although the mechanism of this effect remains unknown, a better understanding might aid in the development of effective adjuvants for mucosally administered HIV vaccines.
The study was supported by grants from the American Foundation for AIDS Research (R.K., #106502-35-RGGN); the Rockefeller Foundation (S.M., ROG 2000 HE 025); the Canadian Research Chair Programme (R.K.); Ontario HIV Treatment Network (S.D.G.; K.M., Career Scientist); and the Canadian Institutes of Health Research (R.K., #HOP-75350; R.K./S.G.O./K.S.M., #HET-85518; S.M., Investigator Award). In addition, we acknowledge the support of the Nairobi City Council, and give special thanks to all the Kibera study participants for their enthusiasm and support.
R.K. and S.G.O. designed the study. R.K., J.K., E.N., K.S.M., J.J.B. and S.M. codirected the clinical trial that enrolled all participants. A.S., A.R., L.Y.Y.S. and W.D.B. performed experiments. R.K., A.S. and A.R. analyzed the data and wrote the initial manuscript draft. L.Y.Y.S., W.D.B., J.K., E.N., K.S.M., S.M. and S.G.O. provided the revisions resulting in the final manuscript draft.
The Kibera HIV Study Group: Ghent University: Dr Marleen Temmerman, Dr Karolien Fonck.
University of Nairobi: Dr Florence Keli, Grace Kamunyo, Ruth Wanguru, Grace Waithira, Jane Njeri, Isaiah Onyango, Dr Isaac Malonza, Dr Francis Mwangi. University of Toronto: Bing Li, Dr Mark Luscher.
These data were presented in part at the 2007 Keystone Conference on HIV Pathogenesis (Whistler, British Columbia, Canada).
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