After the recent approval of various anti-influenza drugs and rapid diagnosis kits for influenza infection by the Ministry of Health, Labor, and Welfare of Japan, it has become easier to diagnose this infection. Along with the developments in diagnostic methods and treatment of the infection, influenza vaccination programs have been actively applied in HIV-1-infected individuals. Influenza virus infection may be more prolonged in individuals with immunodeficiency1 and can cause a transient increase in plasma HIV-1 viral load (VL)2 that might become relevant to the clinical course of HIV-1 infection.2,3 Therefore, influenza vaccine has been generally recommended for HIV-1-infected patients,4-6 as is already stated in the guidelines of the Advisory Committee on Immunization Practices.7 Few studies have reported the protective effect of such vaccination in patients with HIV-1 infection, however. Previous studies demonstrated that the number of CD4 T cells (CD4 count) could predict the efficacy of and/or antibody response to the vaccine but did not clearly demonstrate the correlation between the vaccine efficacy and HIV VL.1,8-15
Activated memory CD4+ T cells are the predominant target of HIV-1,16 and the antibody response to hemagglutinin (HA) is T-cell dependent.17-19 Therefore, highly active antiretroviral therapy (HAART) may reconstitute the immune function of not only the antibody responses but T helper (Th)-cell responses. In this large prospective clinical study, we investigated the clinical efficacy of influenza vaccine in HIV-1-infected patients and correlated it with the immune response to the vaccine as determined by increased antibody titer and/or HA-specific CD4 T cells.
MATERIALS AND METHODS
Study Design and Participants
A 0.5-mL dose of single-shot trivalent influenza subunit vaccine, which contains 15 μg of influenza virus strains A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shanton/7/87, was prepared for adults in the 2002 through 2003 winter season in Japan. All HIV-1-infected patients who consulted the outpatient clinic of the AIDS Clinical Center at the International Medical Center of Japan from November 1 to December 27, 2002 were advised to receive the vaccine, although the final decision was left to the individual. In previous seasons, nearly half of HIV-1-infected patients received influenza vaccine in our clinic. This study was designed to be prospective in nature but nonrandomized. Only individuals, vaccinated and nonvaccinated, who understood the purpose of the study were enrolled, without any incentives. To keep selective bias to a minimum, all vaccinated and consecutive first-come 100 nonvaccinated patients were asked to participate in this study. All study participants gave informed consent, and the institutional ethical committee approved this study (protocol IMCJ-141). Twenty-six hospital staff members who were vaccinated with the same vaccine batch were enrolled as healthy immunized controls after consenting to participate in this study. Among them, 4 had no anti-influenza antibodies before vaccination. All participants were asked to visit to our clinic at least at week 0, 8, and/or 16 after enrollment to allow the withdrawal of 17 mL of blood at each visit for analysis of immunologic responses and routine examinations, including CD4 count and HIV VL.
Definition and Diagnosis of Influenza Virus Infection
In this study, influenza infection (illness) was defined if the patient had flulike symptoms associated with at least 1 adjunct diagnosis such as a serologic or virologic diagnosis. Flulike symptoms were defined as a fever of ≥38.0°C combined with 2 of the following 5 clinical symptoms: cough, rhinitis, myalgia, sore throat, and headache. All participants were asked to visit the clinic if they developed flulike symptoms. To avoid a bias in the clinical diagnosis, a history of influenza vaccination was written out on a separate colored sheet, which was removed from medical records before the outpatient clinic physician attended and examined the patient. The serologic diagnosis was defined as a >4-fold rise in anti-influenza antibody titer compared with before and 4 weeks after the symptoms. In addition, a change of the antibody titer from <10 to 40 U was defined as a 4-fold rise. Patients who had only the antibody rise but no flulike symptoms were not considered to have influenza-related illness. The virologic diagnosis was made by means of viral culture and/or a Rapidvue influenza test kit (Quidel, San Diego, CA) using a nasal or throat swab.
At each visit, CD4 T cells were enumerated by standard flow cytometry and HIV VL was measured using the Roche Amplicor assay kit, version 1.5 (Roche Diagnostic Systems, Branchburg, NJ). Antibody responses to each of the 3 individual vaccine components were examined by the standard hemagglutinin inhibition (HAI) assay.20 Titers ≥40 U were defined as protective, and a >4-fold rise in the antibody titer was considered an adequate response in previously antibody-negative patients.
For assessment of HA-specific CD4 T-cell responses, intracellular γ-interferon (IFN) production was examined by flow cytometry using the method described previously.21,22 Because of the limited availability of peripheral blood mononuclear cells (PBMCs), we analyzed the H1-specific CD4 T cells only. Because fresh PBMCs must be used for this assay, as a result of a labor limitation, only the first 10 participants per day were examined on any particular day. Briefly, HA was purified from influenza virus strain, A/New Caledonia/20/99 (H1N1), as described previously.23 PBMCs were isolated from the fresh heparinized blood and cultured (2 × 106 cells/mL) with diluted H1 plus anti-CD28 antibody (1 μg/mL) or medium alone for 16 hours at 37°C. Brefeldin A (10 μg/mL) was added to each sample in the final 5 hours of incubation. After 16 hours of stimulation, the cells were collected and stained with anti-CD4 allophycocyanin antibody (Beckman Coulter, Fullerton, CA) and anti-CD69-fluorescent isothiocyanate antibody (Becton Dickinson). Subsequently, the cells were fixed and permeabilized to examine for the intracellular production of γ-IFN as described previously.21,22 The flow cytometry analysis was performed by means of the FACSCalibur fluorescence-activated cell sorter with CellQuest software (BD Biosciences, San Jose, CA), and 10,000 CD4 T cells were collected for each analysis.
The data on HA-specific CD4 T cells are presented as the arithmetic mean ± SEM. The data on anti-HA antibody titer are presented as the geometric mean. Statistical analyses were performed using StatView 5.0 software (Abacus Concepts, Berkeley, CA). Differences in the proportion of influenza virus infection between vaccinated and nonvaccinated groups were analyzed by the χ2 test. Multiple logistic regression analysis was used to identify factors that contributed to protection against influenza illness. For the analyses of immune responses, participants were stratified by their CD4 count or HIV VL. Changes in antibody titer and HA-specific CD4 T cells were analyzed using the Kruskal-Wallis test or the Mann-Whitney U test. In all tests, a P value <0.05 was considered significant.
During the period of vaccination, 626 HIV-1-infected patients visited our clinic, and 332 of these received the vaccine, whereas 294 did not. Among them, 317 of those vaccinated and 87 of 100 approached to participate as nonvaccinated patients agreed to participate in the present study. Consequently, 76 patients dropped out of the study (55 of 317 vaccinated patients and 21 of 87 nonvaccinated patients). There were no characteristic differences at baseline between the analyzed and drop-out patients (data not shown). None of the patients dropped out from the study because of HIV-1 disease progression, and none received anticancer or immunosuppressive agents during this study. The final composition of the study group based on compliance with the study protocol, including visits on the fixed dates, was 262 vaccinated (82.6%) and 66 nonvaccinated (75.9%) patients (Fig. 1). Table 1 summarizes the baseline characteristics of the participants.
Efficacy of Influenza Vaccine
The peak of the influenza epidemic of the 2002 through 2003 winter season in Japan was documented during the fourth week of January 2003 and was predominantly caused by influenza A/H3N2. The prevalence of influenza infection in this season was the third highest in the last decade.24 In this study, 30 participants were diagnosed as having definitive influenza illness (5 patients with A/H1N1 strain, 16 with A/H3N2 strain, and 9 with B strain). Six patients were confirmed to have an influenza illness by flulike symptoms, positive viral cultures, positive influenza test kit results, and a >4-fold rise in antibody titer (1 with H1N1 strain, 1 with H3N2 strain, and 4 with B strain); 3 by the symptoms, positive viral cultures, and antibody rise (2 with H1N1 strain and 1 with H3N2 strain); 5 by the symptoms, influenza test kit results, and antibody rise (1 with H1N1 strain, 2 with H3N2 strain, and 2 with B strain); and 16 by the antibody rise between the symptoms (1 with H1N1 strain, 12 with H3N2 strain, and 3 with B strain). In total, 16 of 262 vaccinated patents had influenza illness (6.1%, confidence interval [CI]: 0.04-0.1) and 14 of 66 nonvaccinated patients had the illness (21.2%, CI: 0.13-0.35). The difference in the incidence between the 2 groups was significant (P < 0.001). The relative risk (RR) of influenza illness in vaccinated patients was 0.29 (CI: 0.14-0.55; P < 0.001) compared with nonvaccinated patients (Table 2). Eight patients who had a >4-fold rise in anti-H3 antibody titers between week 8 and week 16 without any clinical symptoms were not regarded as having influenza illness.
In patients with a CD4 count >200 cells/μL, the incidence of influenza illness in vaccinated patients (6.2%) was significantly lower than in nonvaccinated patients (21.0%) (P < 0.001). Conversely, in patients with a CD4 count <200 cells/μL, the same comparison showed no significant difference. Nevertheless, the incidences of influenza illness in vaccinated (5.9%) and nonvaccinated (22.2%) patients were the same as the incidence in patients with a CD4 count >200 cells/μL. Therefore, this analysis had lack of power because of the small number of nonvaccinated patients in this stratum. In vaccinated and nonvaccinated patients, the differences in the incidence were significant in patients with HAART (P < 0.002) and without HAART (P < 0.05) (see Table 2). When CD4 count was entered as a continuous variable, multivariate analysis using the logistic regression model identified vaccination (P < 0.001) and CD4 count (P < 0.05) but not HIV VL as independent predictors of influenza illness in HIV-1-infected patients.
In patients with influenza illness, 4 of 16 vaccinated patients and 4 of 14 nonvaccinated patients received an anti-influenza drug. None of the patients with influenza illness developed pneumonia that required treatment or hospitalization during the study period. Vaccination did not significantly change the HIV VL or CD4 count at weeks 8 and 16.
Anti-Hemagglutinin Antibody Responses Before and After Vaccination
HAI antibody titers against HA antigens (H1 and H3) were tested before and 8 and 16 weeks after vaccination (Table 3). To evaluate the effect of the single-shot influenza vaccine, subjects were divided into 2 groups based on the HAI titer before vaccination: the baseline HAI antibody-negative and antibody-positive groups. Furthermore, we excluded from this analysis the 13 patients who received the vaccination but had influenza illness (5 with H1N1 strain and 8 with H3N2 strain) during the study period so as to evaluate the antibody responses by the vaccination. The 8 patients who showed a >4-fold rise in anti-H3 antibody titers between week 8 and week 16 without any clinical symptoms were also excluded from this analysis, because the antibody rise in these cases was thought to be caused by influenza virus but not by vaccination. In the baseline HAI-negative group, the antibody responses to both antigens were significantly different compared with those in stratified HIV-1-infected patients by CD4 count (<200 cells/μL and ≥200 cells/μL; P < 0.05) at week 8 and week 16. These titers were low compared with those of the healthy immunized controls in both strata, however. In those with a CD4 count <200 cells/μL, 12 (27.9%) of 43 patients and 12 (32.4%) of 37 patients showed more than a 4-fold rise in the antibody responses against anti-H1 and anti-H3, respectively. In contrast, in those patients with a CD4 count >200 cells/μL, 62 (44.6%) of 139 patients and 61 (46.9%) of 130 patients showed a >4-fold rise in the antibody responses against anti-H1 and anti-H3, respectively. Although differences in the percentages of patients who showed both anti-H1 (P = 0.05) and anti-H3 (P = 0.12) antibody responses of the different CD4 strata were only marginal, there was a tendency for the single-shot vaccination to be more effective in terms of antibody responses in patients with a CD4 count >200 cells/μL. The antibody responses in both groups were not influenced by HIV VL (<100 copies/mL and ≥100 copies/mL; data not shown).
In the baseline HAI antibody-positive group, HAI titers to both antigens remained high and the sustainability of the antibody titers in HIV-1-infected patients was similar to those of the healthy controls, irrespective of CD4 counts (see Table 3). In terms of the antibody rise, in those with a CD4 count <200 cells/μL, 5 of 8 patients and 1 of 6 patients showed more than a 4-fold rise in the antibody response against anti-H1 and anti-H3. Conversely, in those with a CD4 count >200 cells/μL, 16 of 67 patients and 19 of 73 patients showed more than a 4-fold rise.
Anti-H1 and Anti-H3 Antibody Responses in Patients With Influenza Illness Despite Vaccination
A total of 16 patients (5 with H1N1 strain, 8 with H3N2 strain, and 3 with B strain) had influenza illness among the vaccinated group during this study period. In the 5 patients with H1N1 illness, 3 were baseline anti-H1 antibody-negative and 2 had the antibody. Among the 3 baseline anti-H1 antibody-negative patients, 2 were infected before week 8 and 1 was infected after week 8. In the patient infected after week 8, no anti-H1 antibody was detected at week 8. In each of the 2 baseline anti-H1 antibody-positive patients, the titer was 20 U. Both patients were infected before week 8. In the 8 patients with H3N2 illness, 6 were baseline anti-H3 antibody-negative and 2 were positive for the antibody. In the 6 baseline anti-H3 antibody-negative patients, all were infected after week 8. Among these 6 patients, 4 were negative for anti-H3 antibody at week 8, whereas 2 had a 4-fold rise in the antibody before infection. In each of the 2 baseline anti-H3 antibody-positive patients, the titer was 20 U. Both patients were infected after week 8. Anti-H3 antibody at week 8 was increased to 40 U (a 2-fold rise) only in 1 patient. Overall, among the 9 infected patients (1 with H1N2 strain and 8 with H3N2 strain) in whom the antibody responses at week 8 could be evaluated, only 2 had a >4-fold rise of the antibody response before infection.
H1-Specific CD4 T-Cell Response Before and After Vaccination in Baseline Anti-H1 Antibody-Negative Subjects
H1-specific CD4 T-cell responses at week 8 were HIV VL dependent (P < 0.005) but not CD4 count dependent (Fig. 2A). Therefore, H1-specific CD4 T-cell responses were significantly increased by vaccination in HAART-treated patients (P = 0.001), because HIV VL was decreased by HAART (see Fig. 2B). In contrast, responses of HAI antibody titer were not different between HAART-treated and antiretroviral-naive patients (see Fig. 2C).
Comparison of Immune Responses to H1 Antigen at Week 8 Between Influenza A/H1N1-Infected and -Uninfected Patients
Five individuals were infected with influenza A/H1N1 during this season. HAI antibody titers at 8 weeks after the vaccination were not different between the infected and uninfected individuals. In contrast, H1-specific CD4 T-cell responses at week 8 were significantly low in the infected persons compared with those in the uninfected persons (P < 0.05; Fig. 3).
Our prospective study confirmed many conclusions of previously reported small studies. First, we confirmed the protective effect of influenza vaccine in HIV-1-infected patients.8-15 Second, anti-H1-specific and anti-H3-specific antibody responses were examined in HIV-1-infected patients after vaccination, and the responses were confirmed to be dependent on CD4 counts.8-11
To clarify the efficacy of a single-shot vaccination, we divided the participants by the positivity of anti-H1- and anti-H3-specific antibodies before vaccination and found that in baseline antibody-negative HIV-1-infected patients, the antibody responses to the single-shot vaccination were less effective than those in healthy patients. In contrast, however, in baseline antibody-positive HIV-1-infected patients, the antibody responses were similar or more effective than those in the healthy controls and the titers exceeded >40 U in most cases, irrespective of CD4 count. Previous studies demonstrated that an antibody titer >40 U could be used as an index of vaccine protection.12,25 In our study, the antibody titer was <40 U in most patients who became infected with influenza. Considered together, these results suggest that the antibody response may support the clinical efficacy of influenza vaccination. Kroon et al8 reported that postvaccination antibody titers were higher in previously vaccinated HIV-1-infected patients than in nonvaccinated patients, although the difference was not significant. In the present study, the antibody titers showed a better response in individuals positive at baseline for anti-HA antibody than in those negative for the antibody. Furthermore, the response was well sustained, irrespective of CD4 count. Thus, it is conceivable that annual vaccination is specifically important for all HIV-1-infected patients. Sustainability of the antibody titer raised by the vaccination is to be followed in a future study.
In the immunologic part of our study, we examined antibody responses and specific CD4 T cells. The antibody response was almost the same as that reported previously8,9; the response correlated with the CD4 count. In contrast, specific CD4 T cells were much more influenced by HIV VL than by CD4 count.1,8-15 Therefore, the specific CD4 T cells were higher in patients treated with HAART than in those untreated. This result indicates that HAART improves HA-specific CD4 T cells like in other infections,21 or, in other words, the heightened cellular response to the influenza vaccine suggests functional reconstitution of the immune system after HAART.
Our data indicate that the specific CD4 T-cell responses may be related to HIV VL. The specific CD4 T-cell response needs antigen presentation by dendritic cells.26 HIV-1 infection impairs the function of antigen presentation of dendritic cells.27 Therefore, specific CD4 T-cell responses may be profoundly decreased in patients with a high HIV VL.
It is interesting to note that the percentage of H1-specific CD4 T cells at week 8 was significantly lower in influenza A/H1N1-infected patients. It is conceivable that the response of HA-specific CD4 T cells at week 8 can predict the efficacy of influenza vaccine. Influenza-specific CD4 T cells provide help (as Th cells) to B cells for the production of antibody to influenza HA and neuraminidase28,29 and also promote the generation of virus-specific CD8+ cytotoxic T lymphocytes (CTLs).26,30-33 Therefore, the specific CD4 T cell must have a protective role. This concept would be more reliable if we had analyzed H3-specific CD4 T cells rather than H1-specific CD4 T cells, because influenza A/H3N2 was the predominant subtype in this season. Further studies are necessary to elucidate this point.
Our study was designed as a prospective but nonrandomized study, because influenza vaccine has been already recommended for HIV-1-infected patients.7 Practically, the number of nonvaccinated patients who did not participate in our study was higher than that of vaccinated patients (13% of nonvaccinated patients vs. 4.5% of vaccinated patients), and the violation rate of the study protocol was higher in nonvaccinated patients than in vaccinated patients (24.1% vs. 17.4%). Thus, 262 (78.9%) of 332 vaccinated patients and 66 (66%) of 100 nonvaccinated patients were analyzed in this study. Although a relatively high proportion of patients failed to complete the protocol, the main reason for the drop out may have been the lack of incentives and the need to visit our clinic on a fixed date for blood sampling. The vaccinated and nonvaccinated groups were well balanced in terms of baseline characteristics, however. Finally, we believe that the selection bias of participants, if any, is negligible.
In conclusion, our prospective study in a large population demonstrated that influenza vaccine provides protection of HIV-1-infected patients. In baseline antibody-negative patients, the antibody responses to the vaccination were significant in those patients with a CD4 count >200 cells/μL compared with those with a CD4 count <200 cells/μL. In contrast, in baseline antibody-positive patients, good antibody responses were observed, irrespective of CD4 counts. Annual vaccination of HIV-1-infected patients is thus recommended. Therapy with HAART improves the cellular immune response to influenza HA.
The authors are indebted to Dr. N. Sugaya from the Keiyu Hospital for helpful suggestions regarding this study and to Drs. T. Gotanda and Y. Suzuki from the Kitasato Institute Research Center for Biologicals for kindly providing purified HA obtained from influenza virus A/New Caledonia/20/99. The authors thank Dr. N. Ishizuka, International Medical Center of Japan, for special advice on interpretation of the statistical analyses. We also thank F. Negishi and Y. Takahashi for sample stock, preparation, and technical support.
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Members of the HIV/Influenza Vaccine Study Team include the following individuals from the International Medical Center of Japan: Yoshihiro Hirabayashi, MD, PhD, Natsuo Tachikawa, MD, Ikumi Genka, MD, PhD, Miwako Honda, MD, Hiroyuki Gatanaga, MD, PhD, Hirohisa Yazaki, MD, Junko Tanuma, MD, Akihiro Ueda, MD, Kuniko Yoshida, MD, and Yasuhiro Suzuki, MD, PhD.