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Interleukin-2 Increases CD4+ Lymphocyte Numbers but Does Not Enhance Responses to Immunization: Results of A5046s
  To ascertain whether CD4+ lymphocyte increases induced by interleukin (IL)2 enhanced in vivo immune responses, 38 human immunodeficiency virus (HIV)infected patients who had received highly active antiretroviral therapy (HAART) or HAART and IL-2 for at least 60 weeks were immunized with tetanus toxoid, inactivated glycoprotein 120depleted HIV-1, and hepatitis A and B vaccines. Despite dramatic increases in CD4+ lymphocyte counts, IL-2 did not enhance immunization responses. Whether cyclic high-dose IL-2 administration to HIV-1 infected subjects will confer clinical benefit is currently under study in 2 phase 3 clinical trials
The Journal of Infectious Diseases 2003;187:320-325 Hernan Valdez, Ronald Mitsuyasu2 Alan Landay, Anne D. Sevin,,a Ellen S. Chan, John Spritzler, Spyros A. Kalams, Richard B. Pollard, John Fahey, Lawrence Fox, Ann Namkung, Scharla Estep, Ronald Moss, David Sahner, and Michael M. Lederman
A regimen of intermittent high-dose interleukin (IL) 2 increases naive and memory CD4+ lymphocyte numbers. Only limited information is available about the effects of IL-2 on functional immune enhancement. To ascertain whether IL-2 induced increases in CD4+ lymphocytes enhance immune competence in vivo, we immunized patients who had been receiving HAART or HAART and IL-2 and compared their immune responses. Although IL-2 recipients had substantially higher CD4+ lymphocyte counts, immune responses after immunization were not enhanced.
In the AIDS Clinical Trials Group (ACTG) 328 study, HIV-1infected subjects with CD4+ lymphocyte counts of 50350 cells/L who were naive to protease inhibitors and to at least 1 nucleoside reverse-transcriptase inhibitor (NRTI) were treated with 2 NRTIs and indinavir. Patients who had plasma HIV-1 RNA levels <5000 copies/mL after 11 weeks were randomly assigned on a 1 : 1 : 1 ratio to also receive either IL-2, 9 MIU/day by intravenous infusion for 5 days every 8 weeks, or IL-2, 7.5 MIU/day subcutaneously twice daily for 5 days every 8 weeks, or to continue to receive only HAART. After at least 60 weeks, patients with HIV-1 RNA levels 2000 copies/mL could enroll in the A5046s substudy; these patients continued to receive the assigned treatment. The A5046s substudy included 38 patients. Twenty-five patients were receiving HAART and IL-2, and 13 were receiving only HAART; the median duration of treatment on substudy entry was 98 weeks (interquartile range [IQR], 89107 weeks); patients in the IL-2 arm received a median of 10 IL-2 cycles before enrollment into A5046s.
ACTG 328 subjects who were receiving IL-2 entered the A5046s substudy 4 weeks after their last IL-2 cycle; they continued to receive IL-2 every 8 weeks. At baseline (entry into A5046s), serum antibody levels were determined, and skin tests for delayed-type hypersensitivity (DTH) reactions and T lymphocyte proliferation assays (LPA) were performed. After 4872 h, patients returned to the study center to have DTH responses read and were then immunized with TT, inactivated gp120-depleted HIV-1, hepatitis B (if the patient was seronegative and uninfected) and hepatitis A (if the patient was seronegative). Additional evaluations and immunizations were performed at weeks 8, 16, and 24, using ACTG consensus methods. Patients received a second dose of TT, inactivated gp120-depleted HIV-1, and hepatitis A and B vaccines at week 8, and a third dose of inactivated gp120-depleted HIV-1 and hepatitis B vaccine at week 16. All immunizations and immunologic evaluations for patients receiving IL-2 were performed 4 weeks after the patient's last IL-2 cycle.
The median CD4+ lymphocyte count before initiation of HAART for subjects participating in the substudy was 238 cells/L; at immunization (A5046s baseline), the median CD4+ lymphocyte count was higher among IL-2 recipients than among subjects who received only HAART (865 vs. 445 CD4+ lymphocytes/L; P < .03). Before HAART, IL-2 recipients had a higher proportion of activated CD4+ lymphocytes (17% vs. 10%; P < .03) and higher plasma virus loads (4.6 vs. 3.7 log10 copies/mL; P = .057) than did subjects who received only HAART, but these differences were not present at immunization. Seventy-one percent of patients had plasma virus loads <50 copies/mL at immunization, and the proportions of subjects with plasma virus loads below this level in each group were comparable at each evaluation.
None of the patients in the HAART-only arm and 1 patient in the HAART/IL-2 arm developed an LPA response to native HIV p24. When we analyzed native p24 responses using less stringent criteria (SI >3 and >3-fold increase from baseline), 31% of HAART-only recipients responded, compared with 12% of HAART/IL-2 recipients (P = .378). The magnitude of the responses tended to be greater in HAART-only recipients. When the less strict definition of response was used, 70% of HAART-only recipients had a response to whole inactivated virus (HZ321) at any time after immunization, compared with 24% of HAART/IL-2 recipients (P = .049). The magnitude of proliferative responses to HZ321 tended to be larger in HAART-only recipients than in HAART/IL-2 recipients.
Eleven of 13 HAART-only recipients and 21 of 25 HAART/IL-2 recipients received 2 tetanus immunizations. Although the percentages of HAART-only recipients (46%) and HAART/IL-2 recipients (20%) who developed LPA responses to TT were not significantly different, patients in the HAART-only arm tended to have larger LPA responses after immunization.
There were no differences between groups in the percentage of subjects who had antibody responses to inactivated gp120-depleted HIV-1 (increase in p24-binding antibodies) or to hepatitis B virus or tetanus immunizations at any time. There was no significant difference between groups when we compared the percentages of patients who developed detectable antibody levels after hepatitis A virus immunization at any single time point. However, when we compared the proportions of patients who developed antibody responses to hepatitis A virus immunization any time point after immunization, 88% of HAART-only recipients and 36% of HAART/IL-2 recipients had responses (P < .04).
Because T helper dysfunction may contribute to CD8+ T cell dysfunction in HIV disease, we investigated whether induction of HIV-specific proliferative responses (largely a CD4+ T cell response) would enhance HIV-specific CD8+ lymphocyte responses. We identified 3 patients who had consistent responses to HIV-1 antigen and 4 patients who did not respond who had viably stored cells. We tested a median of 26 peptides in patients who had responses and a median of 22 peptides in patients who did not have responses at each time point after immunization when viable cells were available. CD8+ lymphocytes of patients who had responses to HIV-1 antigen recognized a median of 1.3 peptides, whereas CD8+ lymphocytes of patients who did not have responses recognized 0.5 peptides (P > .1). In contrast, lymphocytes of chronically HIV-1infected subjects who start HAART with early-stage HIV disease typically recognize a median of 6.5 of 21 cytotoxic T lymphocyte epitopes (S.A.K., unpublished data).
Immunization responses are an in vivo reflection of the ability of the host's immune system to mount defensive responses to a microbial challenge; this is supported by the observation that HIV-1infected subjects who respond to immunization have a more favorable disease course. We tested immunization responses to investigate whether IL-2induced increases in CD4+ lymphocytes enhanced immune function in vivo. Although patients who were receiving HAART and IL-2 had nearly twice as many CD4+ lymphocytes at the time of immunization as did patients who received only HAART, the response to immunization among HAART/IL-2 recipients was no better than that among HAART-only recipients. The results for other predictors of responses to immunization in patients with HIV-1 infection either were comparable between groups or favored IL-2 recipients. Several explanations can be postulated to account for these unexpected findings.
Patients who received HAART and IL-2 tended to have higher plasma virus loads and a larger proportion of activated CD4+ lymphocytes before initiation of HAART. Higher plasma virus loads and heightened CD4+ lymphocyte activation at the time of immunization (but not before initiation of HAART) have predicted poorer responses to immunization. We do not think that minor disparities in either of these indices before initiation of HAART accounted for the failure to find an effect of IL-2 on immunization responses, because neither predicted responses in a multivariate model. Earlier studies that compared the immune responses of patients receiving combination NRTI regimens with or without IL-2 showed that IL-2 increases the magnitude of responses to mitogens and antigens but not the proportion of patients who have responses. Thus, cyclic IL-2 treatment may expand responses already present but may not facilitate generation of new responses.
Lower doses of IL-2 close to the time of immunization have been used successfully as a vaccine adjuvant. Our data suggest that high doses of IL-2 used to expand circulating CD4+ lymphocyte populations do not enhance and may actually diminish immunization responses. High doses of IL-2 result in mitogen-like activation and expansion of CD4+ lymphocytes. It is conceivable that these activated and proliferating cells are not capable of maintaining the rounds of high-level antigen-driven proliferation that are necessary to mount a response to immunization.
To explore whether insufficient CD4+ lymphocyte help underlies impaired HIV-specific cytotoxic T lymphocyte activity in patients with HIV-1 infection, we compared HIV-specific CD8+ lymphocyte responses in subjects who did and subjects who did not develop lymphoproliferative responses to HIV after immunization with inactivated gp120-depleted HIV-1. We failed to find a difference in the breadth of CD8+ lymphocyte responses to HIV peptides (as assessed by ELISPOT). This suggests that induction of CD4+ lymphocyte help by immunization of HIV-infected persons may not be sufficient to enhance HIV-specific CD8+ lymphocyte responses in persons with advanced HIV infection.
In conclusion, we found that, in subjects who start HAART during moderately advanced HIV-1 infection, coadministration of cyclic high-dose IL-2 did not enhance responses to immunization and may have actually attenuated these responses. Thus, the dramatic CD4+ lymphocyte increases that were seen in patients who received IL-2 did not result in better in vivo immune responses. Whether cyclic high-dose IL-2 administration to HIV-1infected subjects will confer clinical benefit is currently under study in 2 phase 3 clinical trials.
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