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Laboratory of Immunoregulation and Office of Clinical Research, National Institute of Allergy and Infectious Diseases
Clinical Center, Nursing and Patient Care Services, National Institutes of Health
Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
Background.
Yearly influenza vaccination, although recommended for human immunodeficiency virus (HIV)infected individuals, has not received thorough evaluation in the era of antiretroviral therapy. We assessed the impact of HIV disease on B cell responses to influenza vaccination.
Methods.
Sixty-four HIV-infected and 17 HIV-negative individuals received the 20032004 trivalent inactivated influenza vaccine. Frequencies of influenza-specific antibody-secreting cells (ASCs) were measured by enzyme-linked immunospot (ELISPOT) assay, and antibody responses were measured by hemagglutination-inhibition (HI) assay. Memory responses to influenza were measured by ELISPOT assay after polyclonal activation of B cells in vitro.
Results.
Prevaccination HI titers were significantly higher in HIV-negative than in HIV-infected individuals. Peak HI titers and influenza-specific ASC frequencies were directly correlated with CD4+ T cell counts in HIV-infected individuals. Influenza-specific memory B cell responses were significantly lower in HIV-infected than in HIV-negative individuals and were directly correlated with CD4+ T cell counts.
Conclusions.
HIV infection is associated with a weak antibody response to influenza vaccination that is compounded by a poor memory B cell response. CD4+ T cell count is a critical determinant of responsiveness to influenza vaccination, and the contribution of plasma HIV RNA level is suggestive and warrants further investigation.
Human influenza is a common viral respiratory disease that is of growing public health concern because of the established morbidity and mortality associated with the disease [1] and the potential for a pandemic dissemination of new and highly pathogenic strains [2]. Annual influenza vaccination is recommended for groups at increased risk of developing complications from natural infection, including the elderly, the very young, people with chronic diseases, and those who are immunosuppressed/immunodeficient [3]. HIV-infected individuals are among this last group, and, although annual vaccination is recommended for this group to prevent the severe consequences of influenza infection, few studies have addressed the immune response generated to the influenza vaccine in relation to the 2 major variables associated with HIV diseasenamely, CD4+ T cell count and plasma HIV RNA level. Studies conducted before effective antiretroviral therapy (ART) was available described an association between decreasing CD4+ T cell counts and diminished anti-influenza antibody responses to vaccination [48], without considering or controlling for the effect of plasma HIV RNA level. Numerous studies, however, have reported on the effect of influenza vaccination on CD4+ T cell count and plasma HIV RNA level (reviewed in [9]), and, since the advent of ART, a few of these studies have also considered the anti-influenza response [1014]. In 2 such studies, plasma HIV RNA level was found to impede immune responses to influenza vaccination independently of CD4+ T cell count [13, 14]. Reduction of plasma HIV RNA level by ART has also been shown to improve the immune response to influenza vaccination [15]; however, the precise relationship between plasma HIV RNA levels and immune responses to vaccination per se were not considered.
The influenza vaccine recommended for HIV-infected individuals is an inactivated formulation for parenteral administration. Although both humoral and cellular responses are elicited by influenza infection, it is the antibodies directed against hemagglutinin (HA) and neuraminidase (NA) that are thought to protect against infection [16], whereas cell-mediated immunity is thought to be more important in recovering from rather than preventing infection [17]. The antibody response to influenza vaccination is thought to be critically dependent on CD4+ T cell help [18]. The most commonly reported correlate of protection after influenza vaccination is the serum titer of hemagglutination-inhibition (HI) antibodies, which peaks 34 weeks after vaccination [19]. Recently developed assays, such as the enzyme-linked immunospot (ELISPOT) assay, aimed at detecting early antibody-secreting cells (ASCs) [20] and later memory B cell responses [21] in peripheral blood, are proving to be powerful tools in evaluating the full scope of antigen-specific B cell responses.
In an effort to increase our understanding of the effect of HIV infection on the immune response to influenza vaccination, we undertook a comprehensive analysis of influenza-specific antibody responses to the vaccine within a cohort of HIV-infected individuals with a wide spectrum of CD4+ T cell counts and plasma HIV RNA levels and compared these with responses in HIV-negative control individuals. To evaluate peak effector and memory responses to influenza vaccination, we measured both HI titers and influenza-specific ASC frequencies before and at various time points after vaccination.
PARTICIPANTS, MATERIALS, AND METHODS
Participants.
HIV-infected and HIV-negative volunteers were recruited between October 2003 and March 2004 to receive the 20032004 influenza vaccine (see below) and to donate blood at 4 time points: day 0 (D0; before vaccination), D7 (peak ASC effector response), D28 (peak HI titer), and D54 (memory ASC response). Eligibility criteria were identical for the HIV-infected and HIV-negative participants, with the exception of HIV status. We excluded participants <18 and >60 years of age, women who were pregnant, any HIV-infected individual whose antiretroviral drug regimen was recently changed or would change and would likely affect CD4+ T cell count and/or plasma HIV RNA level during the course of the study, and any HIV-infected individual who was receiving cytokine-based therapies. All procedures were approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Procedures.
Participants received a single intramuscular dose of licensed inactivated trivalent vaccine from a single lot (Fluzone; Aventis Pasteur), formulated to contain 15 g of HA of each of the strains A/Panama/2007/99 (H3N2), B/Hong Kong/1434/2002, and A/New Caledonia/20/99 (H1N1). Influenza vaccination history was documented, in addition to any event during the course of the study that would be consistent with either influenza infection, as documented by an influenza-positive nasopharyngeal wash, or influenza-like illness [22]. For HIV-infected participants, CD4+ T cell counts and plasma HIV RNA levels (bDNA with limit of detection of 50 copies/mL; Bayer) were measured at each time point (except CD4+ T cell count at D7).
Serum and whole-blood samples were collected immediately before vaccination (D0) and at D7, D28, and D54 after vaccination. HI titers were determined by a standard microtiter assay after treatment of the serum samples with neuraminidase to remove nonspecific inhibitors of HA, as described elsewhere [23]. Serum samples from all time points were tested against the 3 HA antigens of the vaccine as well as against A/Fujian/445/2003, an A/Fujian/411/2002-like strain representative of the predominant influenza A H3N2 viruses present in the United States during the 20032004 influenza season.
Whole blood was processed to obtain either peripheral blood mononuclear cells (PBMCs), by standard Ficoll density centrifugation, or B cells, by negative selection using a B cell enrichment cocktail (RosetteSep; StemCell Technologies), in accordance with the manufacturer's specifications. The percentage of B cells in each preparation was established as reported elsewhere [24], to normalize ELISPOT counts. ASC frequency was measured by an ELISPOT assay [25] adapted to measure influenza-specific responses. Briefly, nitrocellulose-bottom 96-well filtration plates (MAHA S4510; Millipore) were coated with the influenza vaccine at a concentration of 5 g/mL in PBS and were incubated overnight at 4°C. After washing with PBS containing 0.1% Tween 20 (PBS-T) and blocking with RPMI 1640 containing 5% fetal calf serum (FCS), the blocking media was replaced with 100 L of RPMI10% FCS containing 10-fold dilutions of B cells or PBMCs (D7) and was incubated for 5 h at 37°C. Plates were washed and incubated with 50 ng/mL biotinylated mouse antihuman panIgG Fc antibody (Southern Biotechnology Associates) overnight at 4°C. Plates were washed, incubated with alkaline phosphataseconjugated streptavidin, and developed with substrate (ELISpot Blue Color Module; R&D Systems). Spots were enumerated with an automated Immunospot Series 3A analyzer and software (Cellular Technology). Influenza-specific B cell memory responses were evaluated by ELISPOT assay at D54 after vaccination, after 5 days of polyclonal stimulation in vitro, similar to a recently published protocol [21]. Briefly, freshly isolated B cells were plated at 1 × 106 cells/well in 24-well plates containing RPMI10% FCS supplemented with 1/10,000 Staphylococcus aureus Cowan (SAC; EMD Biosciences) and 5 g/mL phosphorothiolated CpG oligodeoxynucleotide 2006 (Operon) [26]. After 5 days in culture, the cells were recovered, washed, and used in the ELISPOT assay described above.
Statistical analyses.
Geometric mean titers (GMTs) for the HI assays were calculated as log2-transformed reciprocal HI titers. Differences in HI titers and ASC frequencies between groups were analyzed using the Wilcoxon 2-sample test with the Bonferroni method to adjust P values for multiple testing. Correlations between variables were determined either by Spearman's rank correlation with adjustment for multiple testing or by linear regression.
RESULTS
A total of 81 participants, including 64 HIV-infected patients and 17 HIV-negative healthy donors, were enrolled in the study. To ensure sufficient heterogeneity relative to CD4+ T cell count and plasma HIV RNA level, each HIV-infected participant was recruited from 1 of 4 prespecified groups: HIV-HH (CD4+ T cell count >350 cells/L and plasma HIV RNA level >10,000 copies/mL), HIV-HL (CD4+ T cell count >350 cells/L and plasma HIV RNA level <10,000 copies/mL), HIV-LH (CD4+ T cell count <350 cells/L and plasma HIV RNA level >10,000 copies/mL), and HIV-LL (CD4+ T cell count <350 cells/L and plasma HIV RNA level <10,000 copes/mL) (table 1). The groups were defined by a CD4+ T cell count above or below 350 cells/L, on the basis of recommendations for initiation of ART [27], and by a plasma HIV RNA level above or below 10,000 copies/mL, on the basis of our previous findings ([24] and authors' unpublished data) and published literature on immunologic failure [28, 29]. Furthermore, to perform more-efficient data analyses, comparisons were made using the entire HIV cohort, in addition to the group-by-group comparisons. All analyses, with rare exceptions specifically mentioned, were performed on all participants. However, because the incidence of influenza or influenza-like symptoms during the course of the study was on the order of 15%20% in each group (table 1), analyses were also performed after excluding such cases on the basis of the time of diagnosis. These reanalyses are mentioned only if they appreciably impacted the finding reported.
An ELISPOT assay was developed to evaluate frequencies of influenza-specific ASCs in response to influenza vaccination. On the basis of preliminary experiments conducted during the 20022003 influenza season and the literature [30], D7 was chosen as the day on which to measure peak ASC frequencies in the peripheral blood. Prevaccination ASC frequency was expected to be low, because the duration of the ex vivo incubation period is too short to allow measurement of a memory response from past immunizations, and very few long-lived plasma cells circulate in the peripheral blood [20, 30]. In response to the immunization, the range of influenza-specific ASC frequencies per million B cells rose from 010 spots at D0 to 016,000 spots by D7 after vaccination, with no significant differences noted between the different groups of participants (data not shown). However, when all HIV-infected individuals were grouped together, a significant direct correlation was observed between CD4+ T cell counts and D7 ASC frequencies (r = 0.30; P < .05) (figure 1A). By D28, influenza-specific ASC frequencies had returned to baseline levels in all patients, with the exception of 1 HIV-infected patient in group HIV-LL who received a diagnosis of influenza at around D19. The D28 influenza-specific ASC frequency for this patient was >1000-fold higher than the frequency at D7, indicating that a stronger anti-influenza response was mounted against the natural infection than against the vaccine.
Whereas several studies have associated B cell defects with ongoing HIV replication [3133], studies directly addressing the impact of plasma HIV RNA level on antigen-specific B cell responses in vivo are lacking. Although, in the present study, no significant correlation was observed between plasma HIV RNA level and frequencies of either D7 effector or D54 memory ASCs when all patients with detectable plasma HIV RNA levels were considered (figure 1C and 1D), a significant inverse correlation with frequencies of D54 memory ASCs was observed when plasma HIV RNA levels >1000 copies/mL was considered (r = -0.43; P < .05), and a similar trend was observed with frequencies of D7 effector ASCs (r = -0.37; P = .07). Of note, and with the caveat that no placebo group was included in the present study, plasma HIV RNA levels in patients with >50 copies/mL were not significantly different at D0 and at D7 after vaccination (Z = -0.0125; P = .99, Wilcoxon rank sum test), suggesting that the vaccine had no measurable effect on plasma HIV RNA level.
Serum HI titers against the 3 HA antigens of the vaccine plus 1 derived from a Fujian-like strain were measured before vaccination (D0) and at 3 time points after vaccination (D7, D28, and D54) and are reported as kinetics of GMTs for each group (figure 3). The kinetics of antibody responses revealed several notable patterns: overlapping response curves for Panama and Fujian antigens, suggesting high cross-reactivity between the 2 strains; generally high D0 GMTs, followed by modest responses (see below); and peak GMTs that did not always occur at D28 in the HIV-infected groups. Because GMTs against Panama and Fujian (the strain associated with most cases of influenza during the season in question)but not against Hong Kong and New Caledoniacontinued to increase past D28 in groups HIV-HL and HIV-LH (figure 3), we suspected that cases of influenza between D28 and D54 were contributing to the increasing GMTs. When such cases were excluded from the analyses, the GMTs against the Panama and Fujian strains decreased as expected after D28 for the HIV-HL group but not for the HIV-LH group (data not shown). The continued increase or slow leveling off in GMT past D28 was a pattern consistently observed in both lowCD4+ T cell count groups (figure 3).
DISCUSSION
We investigated multiple parameters of B cell responses to influenza vaccination in a cohort of HIV-infected individuals representing a wide range of disease stages and activity and compared these responses to those in HIV-negative healthy individuals. Whereas most studies of B cell responses to influenza in immunosuppressed/immunodeficient individuals have focused primarily on HI titers [16], we used an ELISPOT assay to evaluate both the early effector and the later memory phases of B cell responses to influenza. In doing so, we have gained further insight into immune dysfunction in the setting of HIV disease. Consistent with past [48, 14] and present findings on peak antibody responses, the peak effector anti-influenza B cell response measured by ELISPOT assay at D7 was directly correlated with CD4+ T cell count. Furthermore, the memory anti-influenza B cell response measured by ELISPOT assay at D54 was also directly correlated with CD4+ T cell count, with several patients in the 2 lowCD4+ T cell count groups unable to mount a response of >10 ASCs per million B cells. Taken together, our findings indicate that the loss of CD4+ T cells in HIV-infected individuals is associated with a weak influenza-specific antibody response, as measured by HI titers and ASC frequency, as well as with a diminished memory B cell response. These observations are quite consistent with the high dependency of influenza-specific B cell responses on CD4+ T cell help [18] and with the results of numerous studies indicating that HIV infection leads to an irreversible loss in T celldependent antigen-specific B cell responses [36].
We also found evidence that plasma HIV RNA level impedes influenza-specific B cell responses at viral burdens >1000 copies/mL. These data are consistent with those from a study, conducted when the limit of detection of HIV RNA plasma levels was 10,000 copies/mL, that found an inverse correlation between plasma HIV RNA level and HI titers [14]. These data are also consistent with recent findings suggesting that ongoing HIV replication may have a dichotomous effect on CD4+ T cell responses to HIV [37]; although low plasma HIV RNA levels appear to stimulate immune responses, there is a critical level above which plasma HIV RNA level begins to have a negative impact that overrides any stimulatory effect. It remains to be determined whether such a dichotomous effect of plasma HIV RNA level on B cell responses also occurs. To fully address this possibility, a larger cohort representing a wide range of viral burdens needs to be studied.
By introducing the ELISPOT assay to measure early and memory B cell responses against influenza antigens, we have expanded the scope of assays that can be used to evaluate responsiveness to influenza vaccination. We also found a good correlation between peak frequencies of influenza-specific ASCs at D7 and peak HI titers at D28, indicating that the burst of actively secreting plasma cells detected by D7 in the peripheral blood predicts the levels of influenza-specific antibodies that will later be detected in the serum. However, as described above, these 2 assays should be viewed as complementary. Whereas the HI assay provides a well-established correlate of protection [16], the ELISPOT assay, whose readout reflects ASC responses to the entire vaccine, was not designed to measure neutralization potential. Although it remains to be determined whether the ELISPOT assay can be successfully adapted to measure frequencies of influenza-specific neutralizing ASCs, such an assay would be highly desirable, because the high prevaccination responses often detected with HI assays, which can render the interpretation of HI titers difficult, are negligible with the ELISPOT assay.
A somewhat unique and ultimately very informative feature of the ELISPOT assay is its amenability to evaluating influenza-specific memory B cell responses. Antigen-specific B cell frequencies in humans are notoriously difficult to measure by flow cytometry. Hence, the ELISPOT assay remains the assay of choice for measuring antigen-specific human memory B cell responses. By employing such an assay to measure influenza-specific memory B cell responses, we have been able to elucidate yet another defect associated with HIV infectionnamely, the perturbation of memory B cell responsesas well as the fact that perturbations are observed to varying extents in patients at all stages of disease. However, since most of the patients enrolled in the present study were chronically infected and none would be defined as acutely infected, the temporal loss of anti-influenza B cell responses after HIV infection remains to be defined.
Finally, the low and possibly delayed nature of the B cell responses to influenza vaccination observed in HIV-infected individuals with low CD4+ T cell counts and high plasma HIV RNA levels, as well as the paucity of influenza-specific memory B cells elicited by the vaccine, indicate that the recommendation to vaccinate HIV-infected individuals annually [3] may be especially important for this category of patients. The strikingly higher prevaccination HI titers that were observed irrespective of vaccination history in HIV-negative individuals, compared with those in HIV-infected individuals, suggest that healthy individuals maintain a certain level of protection that is absent in the HIV-infected population. Although it remains to be determined whether the differences in prevaccination HI titers reflect differences in frequencies of memory B cells, there are indications that memory B cells contribute to the pool of long-lived plasma cells that are responsible for maintaining serum antibody titers [38]. In light of these defects, it may also be desirable to target both effector and memory B cell responses when considering strategies to enhance anti-influenza responses in the HIV-infected population. Although booster vaccination schemes have not proven successful in immunocompromised individuals [5, 39], recent strategies that employ adjuvants may prove to be effective at enhancing influenza-specific responses in HIV-infected individuals [40, 41]. There are also indications that live attenuated influenza vaccines may elicit a broader and more potent immune response (reviewed in [42]), an advantage over the current inactivated formulas that could improve the breadth and degree of protection in high-risk populations such as HIV-infected individuals. Although the theoretical potential for adverse effects associated with the administration of a live vaccine needs to be addressed with clinical trials, findings in a small group of healthy HIV-infected individuals [43] suggest that a live attenuated influenza vaccine may be suitable for high-risk groups.
Acknowledgments
We are indebted to the patients for their willingness to participate in this study. We thank the nurses and other medical staff of the Eighth Floor Outpatient Clinic and SAIC-Frederick for their help in coordinating and processing patient samples.
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