Cellular immune responses and susceptibility to HIV-1 superinfection: a case–control study
Blish, Catherine A.a,c; Dogan, Ozge C.a; Jaoko, Walterf; McClelland, R. Scottc,d; Mandaliya, Kishorchandrag; Odem-Davis, Katherine S.a; Richardsonb, Barbra A.b,d,e; Overbaugh, Juliea
aDivision of Human Biology
bVaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center
cDepartment of Medicine
dDepartment of Global Health
eDepartment of Biostatistics, University of Washington, Seattle, Washington, USA
fDepartment of Medical Microbiology, University of Nairobi, Nairobi
gCoast General Hospital, Mombasa, Kenya.
Correspondence to Catherine A. Blish, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Grant Building, S-101D, Stanford, CA 94305-5107, USA. E-mail: firstname.lastname@example.org
Received 29 September, 2011
Revised 3 December, 2011
Accepted 20 December, 2012
A case–control study was performed to determine the effects of HIV-1-specific cellular immune responses on the odds of acquiring a second HIV-1 infection (superinfection). Changes in the frequency of cytokine-producing or cytolytic CD8+ or CD4+ T cells were not associated with significant alterations in the odds of superinfection, suggesting that HIV-1 specific cellular immune responses at the level induced by chronic infection do not appear to significantly contribute to protection from HIV-1 superinfection.
Despite 25 years of research, relatively little is known about the ability of HIV-1-specific immune responses to modulate the risk of infection in humans exposed to HIV-1. Although many studies have shown that cytotoxic T-lymphocytes (CTLs) contribute to the control of an established HIV-1 infection , their role in preventing new infections are challenging to ascertain. Aside from vaccine trials, studies of superinfection provide one of the only settings to evaluate the protective role of HIV-1-specific immune responses in humans exposed to diverse, circulating strains. HIV-1 superinfection occurs when an individual infected with one strain of HIV-1 becomes infected with a second strain from a different source partner . The protective role of preexisting HIV-1-specific immunity in the setting of superinfection remains unclear. In six superinfected individuals examined in prior case studies, at least some of the targeted CTL epitopes were altered in the superinfecting strain in comparison to the initial strain, potentially contributing to the ability of the superinfecting strains to establish infection despite the ongoing CTL response [3–7]. However, no prior studies of superinfection have compared the breadth, magnitude, or polyfunctionality of the T-cell responses among superinfected individuals and individuals who were not superinfected. To better define correlates of protection from a second HIV-1 infection, we undertook the first case–control study to assess HIV-1-specific cellular immune responses in HIV-1-infected women with continued exposure to HIV-1, some of whom went on to become superinfected.
The superinfected cases (n = 12) and nonsuperinfected controls (n = 36) were previously identified among 56 women enrolled in a prospective cohort study of high-risk women [8–10]. Informed consent was obtained from all participants, and the Ethical Review Committees of the University of Nairobi, the University of Washington, and the Fred Hutchinson Cancer Research Center approved this study. None of the individuals were treated with antiretroviral medications or were in an advanced state of immunosuppression at the time of superinfection. Controls (three per case) were matched to cases according to the HIV-1 subtype, the timing of samples in relation to initial infection, and the viral load, as described previously . Cases and controls had similar levels of ongoing reported risk for exposure to HIV-1 during the follow-up period, with an average of 1.46 sex partners per week for the cases and 1.42 sex partners per week for the controls. The sample dates ranged from 21 to 1150 days (median 285) prior to documented superinfection.
We determined the frequency of antigen-specific CD8+ and CD4+ T-cell responses by intracellular cytokine staining (ICS) following stimulation with HIV-1 and cytomegalovirus (CMV) peptide pools, as described previously [12,13]. The CMV pp65 peptides, HIV-1 potential T-cell epitope (PTE) gag peptides, and HIV-1 PTE env peptides were obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health (NIH); phorbol 12-myristte 13-acetate/ionomycin (Sigma-Aldrich, St Louis, Missouri, USA) was used as a positive control. The following antibodies were used: CD107a-PE-Cy5, CD4-Alexa700, CD8-PerCP-Cy5.5, GranzymeB-Alexa700, interferon (IFN)-γ-PE-Cy7, interleukin (IL)-2-APC, macrophage inflammatory protein (MIP)-1β-PE, tumor necrosis factor (TNF)-α-FITC (BD Biosciences, San Jose, California, USA), and CD3-Qdot605 (Molecular Probes, Grand Island, New York, USA). The percentage of CD8+ and CD4+ T cells producing the cytokines IL-2, TNFα, IFNγ, and MIP-1β was determined. Cytolytic activity was also assessed based on expression of CD107a, using Granzyme B expression to confirm that degranulating CD107a+ CD8+ T cells had cytolytic potential . Data were analyzed using FlowJo, version 9.1 (Tree Star Inc., Ashland, Oregon, USA), and background from wells stimulated with dimethyl sulfoxide alone was subtracted. Samples were rejected for 10 of the 48 participants for low viability (<50% after resting and stimulation) and/or for low cell numbers (<1000 live CD4+ or CD8+ T cells), leaving 10 superinfection cases and 28 controls. Of the superinfection cases evaluated, three were superinfected with a virus of the same subtype (intrasubtype superinfection), whereas seven were intersubtype superinfections. Cytokine production and cytolytic activity were similar to that observed in other cohorts of chronically HIV-1-infected individuals [15–20].
To determine whether individuals with deficits in T-cell functions were more likely to be superinfected, the odds of superinfection were evaluated in relation to T-cell function by exact conditional logistic regression (Table 1). For cases, analyses were performed using samples collected at the visit prior to documented superinfection in order to assess immunity at the time point most relevant in terms of exposure to, and lack of protection from, the second virus. The samples for the controls were taken from approximately the same time following initial infection. Statistical analyses were performed using the Open Source statistical package R (http://http://www.r-project.org; ISBN 3–900051-07-0) and LogXact (Cytel Incorporated, Cambridge, Massachusetts, USA). Overall, changes in the frequency of cytokine-producing or cytolytic CD8+ or CD4+ T cells were not associated with significant alterations in the odds of superinfection (Table 1). For instance, the odds of being superinfected were a negligible 1.02 times greater (95% confidence interval 0.95–1.09) for every increase of 0.1 in the percentage of CD8+ T cells expressing CD107a following stimulation with gag peptides, with a P-value of 0.47 (Table 1). Similar results were observed for every other function of CD8+ and CD4+ T cells, including the production of IFNγ, IL-2, MIP-1β, or TNFα, and for every possible combination of functions as determined by Boolean gating (Table 1 and data not shown). Thus, CD8 or CD4 T-cell responses to global HIV-1 peptide pools were not significantly associated with the odds of superinfection.
These findings are admittedly disappointing: there is little evidence that HIV-1-specific cellular immune responses, at least at the level observed during chronic infection, play a significant role in protection from HIV-1 acquisition in the setting of superinfection. Similar results were recently attained in simian immunodeficiency virus-infected macaques in which CTL contributed to the control of chronic infection, but not to protection from superinfection . Although these data do not necessarily mean that HIV-specific T cells are incapable of contributing to protection from HIV-1, they do suggest that protection may require T cells at higher frequency or with different specificity than those found in chronically HIV-1-infected individuals. Further studies of humans exposed to diverse HIV-1 strains are therefore necessary to determine which immune responses, if any, contribute to protection.
C.A.B. designed and implemented the study, supervised and performed the experiments, analyzed and interpreted the data, and wrote the article. O.C.D. performed experiments, analyzed, interpreted the data, and helped write the article. W.J., R.S.M., and K.M. helped to implement the study, particularly running all the components performed in Kenya, and helped to write the article. K.O.-D. and B.A.R. performed the statistical analyses, assisted with study design, and helped to write the article. J.O. helped to design the study, interpret the data, and write the article.
The authors would like to thank the clinical and laboratory staff in Mombasa for their tremendous efforts to recruit and retain the women in the sex worker cohort and for collection and storage of the samples. The authors are also grateful to Helen Horton for helpful comments on the manuscript. The authors also express our gratitude to Stephen De Rosa, Evan Thomas, and Helen Horton for assistance with flow cytometry. The authors gratefully acknowledge the women who participated in the study.
Conflicts of interest
This work was supported by NIH R37-AI038518 to J.O. and NIH K08 AI068424 and CTSA UL1 RR025014 University of Washington Institute of Translational Health Sciences Technology Access Grant to C.A.B. The Mombasa study site was supported in part by the University of Washington Center for AIDS Research (P30 AI027757).
There are no conflicts of interest.
The sponsors of the study had no role in the study design, data collection, data analysis, data interpretation, or in the writing of the report.
1. McMichael AJ, Hanke T. HIV vaccines 1983–2003
. Nat Med
2. Chohan BH, Piantadosi A, Overbaugh J. HIV-1 superinfection and its implications for vaccine design.Curr HIV Res
3. Jost S, Bernard MC, Kaiser L, Yerly S, Hirschel B, Samri A, et al. A patient with HIV-1 superinfection
. N Engl J Med
4. Altfeld M, Allen TM, Yu XG, Johnston MN, Agrawal D, Korber BT, et al. HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus
5. Yang OO, Daar ES, Jamieson BD, Balamurugan A, Smith DM, Pitt JA, et al. Human immunodeficiency virus type 1 clade B superinfection: evidence for differential immune containment of distinct clade B strains
. J Virol
6. Ramos A, Hu DJ, Nguyen L, Phan KO, Vanichseni S, Promadej N, et al. Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users
. J Virol
7. Streeck H, Li B, Poon AF, Schneidewind A, Gladden AD, Power KA, et al. Immune-driven recombination and loss of control after HIV superinfection
. J Exp Med
8. Chohan B, Lavreys L, Rainwater SMJ, Overbaugh J. Evidence for frequent reinfection with human immunodeficiency virus type 1 of a different subtype
. J Virol
9. Piantadosi A, Chohan B, Chohan V, McClelland RS, Overbaugh J. Chronic HIV-1 infection frequently fails to protect against superinfection
. PLoS Pathog
10. Piantadosi A, Ngayo MO, Chohan B, Overbaugh J. Examination of a second region of the HIV type 1 genome reveals additional cases of superinfection
. AIDS Res Hum Retroviruses
11. Blish CA, Dogan OC, Derby NR, Nguyen MA, Chohan B, Richardson BA, et al. HIV-1 superinfection occurs despite relatively robust neutralizing antibody responses
. J Virol
12. Frahm N, Kaufmann DE, Yusim K, Muldoon M, Kesmir C, Linde CH, et al. Increased sequence diversity coverage improves detection of HIV-specific T cell responses
. J Immunol
13. Horton H, Thomas EP, Stucky JA, Frank I, Moodie Z, Huang Y, et al. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination
. J Immunol Methods
14. Migueles SA, Osborne CM, Royce C, Compton AA, Joshi RP, Weeks KA, et al. Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control
15. Yang OO, Daar ES, Ng HL, Shih R, Jamieson BD. Increasing CTL targeting of conserved sequences during early HIV-1 infection is correlated to decreasing viremia
. AIDS Res Hum Retroviruses
16. Rolland M, Heckerman D, Deng W, Rousseau CM, Coovadia H, Bishop K, et al. Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads
. PLoS One
17. Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, et al. CD8+ T-cell responses to different HIV proteins have discordant associations with viral load
. Nat Med
18. Masemola A, Mashishi T, Khoury G, Mohube P, Mokgotho P, Vardas E, et al. Hierarchical targeting of subtype C human immunodeficiency virus type 1 proteins by CD8+ T cells: correlation with viral load
. J Virol
19. Riviere Y, McChesney MB, Porrot F, Tanneau-Salvadori F, Sansonetti P, Lopez O, et al. Gag-specific cytotoxic responses to HIV type 1 are associated with a decreased risk of progression to AIDS-related complex or AIDS
. AIDS Res Hum Retroviruses
20. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, et al. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells
21. Weinfurter JT, May GE, Soma T, Hessell AJ, Leon EJ, Macnair CE, et al. Macaque long-term nonprogressors resist superinfection with multiple CD8+ T cell escape variants of simian immunodeficiency virus
. J Virol
© 2012 Lippincott Williams & Wilkins, Inc.
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Data is temporarily unavailable. Please try again soon.