Abstract
Background
Delayed-type hypersensitivity (DTH) test responsiveness is associated with HIV disease progression; however it is unknown whether other immune markers, such as hepatitis B virus (HBV) vaccine seroresponse, also predict HIV outcomes.
Methods
Eligible participants received HBV vaccine after HIV diagnosis, had non-anergic DTH testing at the time of last HBV vaccination, and available post-vaccine HBV antibody responses. The risk of progression to AIDS or death from the time of last HBV vaccination was evaluated.
Results
Of 369 eligible participants with non-anergic DTH responses, 148 (40%) were HBV vaccine responders. In a multivariate model adjusted for age, CD4 count, viral load, and number of vaccinations, HBV vaccine non-responders had an increased risk of progression to AIDS or death (HR 1.81; 95% CI, 1.03–3.19).
Conclusions
HBV vaccine seroresponses were independent of DTH responses which suggest that non-response to HBV vaccine is not solely due to cell-mediated immune dysfunction in HIV-infected persons.
Keywords: Delayed-Type Hypersensitivity, Hepatitis B vaccine, HIV, AIDS, anergy
INTRODUCTION
The natural history of HIV infection results in a progressive decline in immune function and, if untreated, ultimately leads to the development of Acquired Immunodeficiency Syndrome (AIDS) and death. The advent of potent, well-tolerated antiretroviral therapy (ART) has significantly improved life expectancy in HIV-infected patients[1]. Despite the successes of modern ART regimens, both AIDS and serious non-AIDS events can occur, even at relatively preserved CD4 cell counts[2–4]. In addition to measurement of CD4 counts, other clinically relevant immune correlates are necessary to better understand HIV pathogenesis and predict disease progression.
Delayed-type hypersensitivity (DTH) testing is a tool that can be used to evaluate cell-mediated immunity (CMI) in clinical settings[5]. Cutaneous anergy to recall antigens is a predictor of HIV disease progression independent of viral load (VL) and CD4 count[6, 7]. Whether markers for other aspects of immune function also predict HIV outcomes over and above DTH has not been completely studied. We previously demonstrated that hepatitis B virus (HBV) vaccine responsiveness can also be used to predict future AIDS events or death[8]. Although HBV vaccine is a T-cell dependent antigen, a positive seroresponse to HBV vaccine requires other aspects of immune function including antigen presentation of the peptide-based vaccine as well as B-cell activity[9–11]. We evaluated the relationships between the humoral response to HBV vaccine, CMI by DTH responses, and progression to clinical AIDS and death in the U.S. Military HIV Natural History Study (NHS).
METHODS
The NHS is a prospective observational cohort comprised of more than 5500 military members, dependents, and beneficiaries with HIV-1 infection that are followed at 7 military medical treatment facilities in the U.S. since 1986. Participants enrolled were age 18 or older and provided signed, written consent in this IRB-approved study and are evaluated approximately every 6 months.
In this retrospective study, participants were required to be HBV vaccine naïve prior to their HIV diagnosis with no history of HBV infection. Participants were also required to have received at least one dose of HBV vaccine and have DTH testing results at the time of last vaccination. Participants were classified as either vaccine responders or non-responders based on antibody to HBV surface antigen (anti-HBs) ≥10 or <10 IU/L, respectively. Since anti-HBs can wane ≥2 years after vaccination in HIV-infected adults[12], only participants with measured anti-HBs between 1–12 months after last vaccination were included to prevent misclassification. DTH testing was performed according to standardized NHS protocols as previously described[6, 13, 14]. A total of 0.1 mL of each antigen was applied intradermally to the forearm by Mantoux method and a positive test was defined as ≥5 mm of induration after 48 hours. The most recent antigens and concentrations used per protocol consisted of tetanus toxoid (Lederle 1.6Lf/mL; 1:100 dilution), mumps (Connaught, 40 CFU/mL), trichophyton (Holister-Stier, 1:500 dilution), and candida (Walter Reed Army Institute of Research, 200 PNU/mL). Participants received a panel of 3–4 antigens, with the majority receiving 3 antigens as trichophyton was removed from the market. DTH responses were categorized by the number of positive skin tests: anergic (0), partial anergic (1), or non-anergic (≥2) as previously described[13]. The subgroup with non-anergic DTH responses was further studied for clinical outcomes based on HBV vaccine seroresponse.
HBV vaccine non-responder and responder characteristics were compared by chi-squared or Fisher’s Exact tests. AIDS outcomes were defined by 1993 Centers for Disease Control and Prevention criteria, with the exception of CD4 count[15]. Vaccine responders were followed from the time of their last HBV vaccination to the development of AIDS, death, or last study visit. Risk of progression for the combined endpoint of AIDS or death from the time of last HBV vaccination was evaluated with multivariate Cox regression and time to AIDS or death was assessed by Kaplan-Meier survival methods.
RESULTS
A total of 507 NHS participants vaccinated from 1986–2005 met inclusion criteria, of which 65 (12.8%) had anergic DTH responses, 73 (14.4%) had partially anergic DTH responses, and 369 (72.8%) had non-anergic DTH responses. The proportion with a positive HBV vaccine response did not differ by DTH category: 31%, 30%, and 40% for anergic, partially anergic, and non-anergic, respectively (P=0.13). Participants with non-anergic DTH responses were further investigated for HIV disease progression based on HBV vaccine seroresponse after last vaccination (Table 1). A greater proportion of participants were classified as non-responders (59.9%) compared to responders (40.1%), but demographic characteristics including age, gender, and race/ethnicity were similar between subgroups. Most participants in both groups were treated with ART at time of last vaccination and received ≥3 vaccine doses. At the time of last HBV vaccination, the median CD4 count and VL for non-responders compared to responders was 451cells/uL (IQR 318–613) versus 573 cells/uL (IQR 418–737; P<0.001) and 3.33 versus 2.30 log10 copies/mL, respectively (P<0.001 for both). VL at last vaccination was unavailable for 138 (37.4%) of study participants since clinical VL assessments were not routinely performed prior to 1996.
Table 1.
Characteristics of Participants with Non-anergic DTH Test Results at the Time of Last HBV Vaccination by Vaccine Response Category
| Characteristic | N | Anti-HBs < 10 IU/L N = 221 |
Anti-HBs ≥ 10 IU/L N = 148 |
P |
|---|---|---|---|---|
|
| ||||
| Age (years) | 369 | 31 (27–36) | 31 (26–37) | 0.76 |
|
| ||||
| Gender, Male | 369 | 202 (91) | 124 (84) | 0.03 |
|
| ||||
| Ethnicity | 369 | 0.85 | ||
| Caucasian | 96 (43) | 59 (40) | ||
| African American | 98 (44) | 66 (45) | ||
| Other | 27 (12) | 23 (16) | ||
|
| ||||
| CD4 Count (cells/uL) | 360 | 451 (318–613) | 573 (418–737) | <0.001 |
|
| ||||
| CD4 Count Category (cells/uL) | 360 | <0.001 | ||
| <200 | 23 (10) | 6 (4) | ||
| 200–499 | 103 (47) | 49 (33) | ||
| ≥500 | 90 (41) | 89 (60) | ||
|
| ||||
| HIV VL (log10 copies/mL) | 231 | 3.33 (2.30–4.38) | 2.30 (2.21–3.85) | <0.001 |
|
| ||||
| HIV VL category (copies/mL) | 369 | <0.001 | ||
| ≤400 | 37 (17) | 46 (31) | ||
| >400 | 85 (38) | 63 (43) | ||
| Unknown | 99 (45) | 39 (26) | ||
|
| ||||
| Number of Vaccinations | 369 | 0.09 | ||
| 1–2 | 102 (46) | 55 (37) | ||
| ≥3 | 119 (54) | 93 (63) | ||
|
| ||||
| On ART at Vaccination | 369 | 182 (82) | 102 (69) | <0.01 |
|
| ||||
| Year of Vaccination | 369 | <0.001 | ||
| <1996 | 134 (61) | 58 (39) | ||
| ≥1996 | 87 (39) | 90 (61) | ||
|
| ||||
| Follow-up (years) | 369 | 5.4 (2.5–10.1) | 6.7 (3.0–11.2) | 0.16 |
| Total (patient-years) | 1,463 | 1,071 | ||
|
| ||||
| Total Number of AIDS or Death Events | 369 | 68 | 17 | <0.001 |
DTH, delayed-type hypersensitivity; HBV, hepatitis B virus; anti-HBs, antibody to HBV surface antigen; VL, viral load; ART, antiretroviral therapy
Data are number (%) or median (interquartile range) unless otherwise specified
Participants were followed for a median of 5.4 years (IQR 2.5–10.1) in the non-responder group and 6.7 years (IQR 3.0–11.2) in the responder group for a total of 1,463 and 1,071 person-years, respectively (P=0.16). In participants with non-anergic DTH responses, 85 (23%) progressed to AIDS or death, including occurrence in 68 (31%) HBV vaccine non-responders and 17 (11%) responders (P<0.001)). In preliminary models, gender, year of vaccination, and receiving ART at vaccination were not significantly associated with AIDS or death risk. The risk of progression to AIDS or death for HBV vaccine non-responders and responders was assessed by multivariate analysis adjusted for age, CD4 cell count, HIV VL, and number of vaccine doses (Table 2). HBV vaccine non-responders were found to have an increased risk of progression to AIDS or death (HR, 1.81; 95% CI, 1.03–3.19). The time to AIDS or death was significantly greater for HBV vaccine responders compared to non-responders (Figure 1; P<.001).
Table 2.
Final Age-adjusted Multivariate Model for Risk of Progression to AIDS or Death Among Those with Non-anergic DTH Test Results
| Characteristic | HR (95% CI) | P |
|---|---|---|
|
| ||
| CD4 count category (cells/uL) | ||
| <200 | 9.10 (4.66 – 17.75) | <0.001 |
| 200–499 | 2.11 (1.20 – 3.71) | 0.01 |
| ≥500 | Referent | |
|
| ||
| HIV VL category (copies/mL) | ||
| ≤400 | 0.13 (0.03 – 0.55) | <0.01 |
| >400 | 0.66 (0.33 – 1.35) | 0.26 |
| Unknown | Referent | |
|
| ||
| Number of Vaccinations | ||
| 1–2 | 1.70 (1.07 – 2.69) | 0.02 |
| ≥ 3 | Referent | |
|
| ||
| HBV Vaccine Response | ||
| Anti-HBs <10 IU/L | 1.81 (1.03 – 3.19) | 0.04 |
| Anti-HBs ≥10 IU/L | Referent | |
DTH, delayed-type hypersensitivity; HR, hazard ratio; CI, confidence interval; VL, viral load; HBV, hepatitis B virus; anti-HBs, antibody to HBV surface antigen
Gender, year of vaccination, and receiving ART at vaccination, were all not significantly associated with AIDS or death risk in preliminary models
Figure 1.
Time to AIDS or Death for Those with Non-anergic DTH Test Results by HBV Vaccine Response (P<0.001 by log rank)
DISCUSSION
In a large cohort of HIV-infected adults, we found that most participants with non-anergic DTH responses to recall antigens did not demonstrate protective seroresponses to HBV vaccine. We also observed that HBV vaccine response status was a predictor of subsequent AIDS or death among those with non-anergic DTH responses. These findings suggest that non-response to HBV vaccine is not entirely due to CMI dysfunction in individuals infected with HIV, but other immune deficits likely contribute to the lack of anti-HBs response after vaccination.
As an in vivo marker of CMI, DTH responsiveness is enhanced at higher CD4 cell counts. For example, one study showed that responses to 2 or more recall antigens was 86% for those with CD4 >400 cells/uL compared to only 45% for those <400 cells/uL[10]. However, DTH responsiveness has also been shown to be a predictor of HIV disease progression in untreated individuals independent of CD4 cell count[6, 7, 16]. A previous study in our cohort showed that the total number of reactive skin tests (0, 1, or 2–4) stratified patients according to survival time and progression to AIDS[13]. In the current study, we observed that in HIV-infected patients with non-anergic DTH responses, a marker for more favorable CMI, seroresponses to HBV vaccine also had prognostic value. Although DTH testing has long been used in clinical practice for various purposes, such as screening for latent tuberculosis infection, DTH methods are not routinely used in HIV medicine. HBV vaccine seroresponses may be of greater practical use since HBV vaccine is recommended for all HIV-infected persons and anti-HBs, a validated marker for HBV vaccine response, is routinely measured in clinical practice[17, 18].
HBV vaccine response, a surrogate for humoral immune function, may be used in a similar manner as DTH response to help identify other host or viral factors that influence HIV disease course and potential response to specific therapies. The effect of HIV on CMI has been utilized in several prediction models to assess the severity of disease and response to therapy by evaluating CD4 cell count, total lymphocyte count, T-cell subtypes, and DTH response in addition to viral load as independent risk factors[8, 13, 14, 19–21]. In a previous study in our cohort, a greater likelihood of HBV vaccine response was demonstrated in those with higher CD4 cell counts at last vaccination, use of ART, and receipt of ≥3 doses of HBV vaccine[21]. HBV vaccine seroresponse was also associated with progression to AIDS or death, even in those with CD4 counts >500 cells/uL. Since DTH responsiveness is improved at higher CD4 counts, the observation in the current study that HBV vaccine seroresponse remains predictive of HIV disease progression in those with non-anergic DTH testing is consistent with prior findings. Although HBV vaccine is a T-cell dependent antigen, only 40% of HBV vaccine responders were non-anergic by DTH testing which suggests that additional immune factors contribute to the development of anti-HBs response.
As future efforts continue to shed new light on the immunologic relationships in the pathogenesis and progression of HIV infection, HBV vaccine seroresponses may serve as a useful tool for predicting HIV disease progression in addition to other clinical laboratory makers such as CD4 count and VL. Identification of additional factors that affect the innate and adaptive immune components could help tailor treatments, guide future therapies, and provide additional data to predict the course of disease for individuals infected with HIV.
Highlights.
Responsiveness to HBV vaccine and DTH testing can predict HIV disease progression
We evaluated HBV vaccine seroresponses in those with non-anergic DTH results
HBV vaccine response predicted AIDS or death even in non-anergic persons with HIV
Acknowledgments
Support for this work (IDCRP-000-25) was provided by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense (DoD) program executed through the Uniformed Services University of the Health Sciences. This project has been funded in whole, or in part, with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), under Inter-Agency Agreement Y1-AI-5072.
Footnotes
The authors have no conflicts of interest.
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References
- 1.Harrison KM, Song R, Zhang X. Life expectancy after HIV diagnosis based on national HIV surveillance data from 25 states, United States. Journal of acquired immune deficiency syndromes. 2010;53:124–30. doi: 10.1097/QAI.0b013e3181b563e7. [DOI] [PubMed] [Google Scholar]
- 2.Mocroft A, Phillips AN, Gatell J, Horban A, Ledergerber B, Zilmer K, et al. CD4 cell count and viral load-specific rates of AIDS, non-AIDS and deaths according to current antiretroviral use. Aids. 2013;27:907–18. doi: 10.1097/QAD.0b013e32835cb766. [DOI] [PubMed] [Google Scholar]
- 3.Masia M, Padilla S, Alvarez D, Lopez JC, Santos I, Soriano V, et al. Risk, predictors, and mortality associated with non-AIDS events in newly diagnosed HIV-infected patients: role of antiretroviral therapy. Aids. 2013;27:181–9. doi: 10.1097/QAD.0b013e32835a1156. [DOI] [PubMed] [Google Scholar]
- 4.Lucero C, Torres B, Leon A, Calvo M, Leal L, Perez I, et al. Rate and predictors of non-AIDS events in a cohort of HIV-infected patients with a CD4 T cell count above 500 cells/mm(3) AIDS research and human retroviruses. 2013;29:1161–7. doi: 10.1089/aid.2012.0367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Dolan MJ, Kulkarni H, Camargo JF, He W, Smith A, Anaya JM, et al. CCL3L1 and CCR5 influence cell-mediated immunity and affect HIV-AIDS pathogenesis via viral entry-independent mechanisms. Nature immunology. 2007;8:1324–36. doi: 10.1038/ni1521. [DOI] [PubMed] [Google Scholar]
- 6.Birx DL, Brundage J, Larson K, Engler R, Smith L, Squire E, et al. The prognostic utility of delayed-type hypersensitivity skin testing in the evaluation of HIV-infected patients. Military Medical Consortium for Applied Retroviral Research. Journal of acquired immune deficiency syndromes. 1993;6:1248–57. [PubMed] [Google Scholar]
- 7.Gordin FM, Hartigan PM, Klimas NG, Zolla-Pazner SB, Simberkoff MS, Hamilton JD. Delayed-type hypersensitivity skin tests are an independent predictor of human immunodeficiency virus disease progression. Department of Veterans Affairs Cooperative Study Group. The Journal of infectious diseases. 1994;169:893–7. doi: 10.1093/infdis/169.4.893. [DOI] [PubMed] [Google Scholar]
- 8.Landrum ML, Hullsiek KH, O’Connell RJ, Chun HM, Ganesan A, Okulicz JF, et al. Hepatitis B vaccine antibody response and the risk of clinical AIDS or death. PloS one. 2012;7:e33488. doi: 10.1371/journal.pone.0033488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.van den Berg R, van Hoogstraten I, van Agtmael M. Non-responsiveness to hepatitis B vaccination in HIV seropositive patients; possible causes and solutions. AIDS reviews. 2009;11:157–64. [PubMed] [Google Scholar]
- 10.Yao ZQ, Moorman JP. Immune exhaustion and immune senescence: two distinct pathways for HBV vaccine failure during HCV and/or HIV infection. Archivum immunologiae et therapiae experimentalis. 2013;61:193–201. doi: 10.1007/s00005-013-0219-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Goncalves L, Albarran B, Salmen S, Borges L, Fields H, Montes H, et al. The nonresponse to hepatitis B vaccination is associated with impaired lymphocyte activation. Virology. 2004;326:20–8. doi: 10.1016/j.virol.2004.04.042. [DOI] [PubMed] [Google Scholar]
- 12.Lopes VB, Hassing RJ, de Vries-Sluijs TE, El Barzouhi A, Hansen BE, Schutten M, et al. Long-term response rates of successful hepatitis B vaccination in HIV-infected patients. Vaccine. 2013;31:1040–4. doi: 10.1016/j.vaccine.2012.12.047. [DOI] [PubMed] [Google Scholar]
- 13.Dolan MJ, Clerici M, Blatt SP, Hendrix CW, Melcher GP, Boswell RN, et al. In vitro T cell function, delayed-type hypersensitivity skin testing, and CD4+ T cell subset phenotyping independently predict survival time in patients infected with human immunodeficiency virus. The Journal of infectious diseases. 1995;172:79–87. doi: 10.1093/infdis/172.1.79. [DOI] [PubMed] [Google Scholar]
- 14.Blatt SP, Hendrix CW, Butzin CA, Freeman TM, Ward WW, Hensley RE, et al. Delayed-type hypersensitivity skin testing predicts progression to AIDS in HIV-infected patients. Annals of internal medicine. 1993;119:177–84. doi: 10.7326/0003-4819-119-3-199308010-00001. [DOI] [PubMed] [Google Scholar]
- 15.1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR Recommendations and reports : Morbidity and mortality weekly report Recommendations and reports / Centers for Disease Control. 1992;41:1–19. [PubMed] [Google Scholar]
- 16.Blatt SP, McCarthy WF, Bucko-Krasnicka B, Melcher GP, Boswell RN, Dolan J, et al. Multivariate models for predicting progression to AIDS and survival in human immunodeficiency virus-infected persons. The Journal of infectious diseases. 1995;171:837–44. doi: 10.1093/infdis/171.4.837. [DOI] [PubMed] [Google Scholar]
- 17.Aberg JA, Gallant JE, Ghanem KG, Emmanuel P, Zingman BS, Horberg MA. Primary Care Guidelines for the Management of Persons Infected With HIV: 2013 Update by the HIV Medicine Association of the Infectious Diseases Society of America. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2014;58:e1–e34. doi: 10.1093/cid/cit665. [DOI] [PubMed] [Google Scholar]
- 18.McMahon BJ, Dentinger CM, Bruden D, Zanis C, Peters H, Hurlburt D, et al. Antibody levels and protection after hepatitis B vaccine: results of a 22-year follow-up study and response to a booster dose. The Journal of infectious diseases. 2009;200:1390–6. doi: 10.1086/606119. [DOI] [PubMed] [Google Scholar]
- 19.Anastos K, Shi Q, French AL, Levine A, Greenblatt RM, Williams C, et al. Total lymphocyte count, hemoglobin, and delayed-type hypersensitivity as predictors of death and AIDS illness in HIV-1-infected women receiving highly active antiretroviral therapy. Journal of acquired immune deficiency syndromes. 2004;35:383–92. doi: 10.1097/00126334-200404010-00008. [DOI] [PubMed] [Google Scholar]
- 20.Carr A, Chuah J, Hudson J, French M, Hoy J, Law M, et al. A randomised, open-label comparison of three highly active antiretroviral therapy regimens including two nucleoside analogues and indinavir for previously untreated HIV-1 infection: the OzCombo1 study. Aids. 2000;14:1171–80. doi: 10.1097/00002030-200006160-00014. [DOI] [PubMed] [Google Scholar]
- 21.Landrum ML, Huppler Hullsiek K, Ganesan A, Weintrob AC, Crum-Cianflone NF, Barthel RV, et al. Hepatitis B vaccine responses in a large U.S. military cohort of HIV-infected individuals: another benefit of HAART in those with preserved CD4 count. Vaccine. 2009;27:4731–8. doi: 10.1016/j.vaccine.2009.04.016. [DOI] [PMC free article] [PubMed] [Google Scholar]