In a prospective influenza-vaccination trial we show that HIV-infected individuals with CD4+ T-cell counts less than 350 μl were distinct from HIV-infected individuals with more than 350 CD4+ T-cell counts/μl, and from HIV-negative individuals, in that an influenza-specific immunoglobulin M-response was absent and expansion of interferon-γ-secreting CD4+ T cells was impaired. By contrast, immunoglobulin G-responses were induced in all study groups. These data suggest that establishing broad influenza-specific (immunoglobulin G) B-cell memory prior to severe immunodeficiency is important.
aImmunobiology Laboratory, Department of Biomedicine, Basel University, Switzerland
bDivision of Infectious Diseases and Hospital Epidemiology, Switzerland
cMedical Outpatient Department, University Hospital Basel, Switzerland
dInstitute for Medical Microbiology, Basel University, Basel, Switzerland.
*S.F. and E.M. contributed equally to this work.
Received 11 March, 2010
Revised 30 April, 2010
Accepted 24 May, 2010
Correspondence to Christoph Hess, MD, PhD, Medical Outpatient Department and Department of Biomedicine, University Hospital Basel, 4 Petersgraben, CH-4031 Basel, Switzerland. Tel: +41 61 265 44 75; e-mail: email@example.com
Antibody affinity maturation, isotype class-switch and B-cell memory formation all depend on adequate CD4+ T-cell help [1,2]. By contrast, maintenance of established B-cell memory needs little or no T-cell help in order to persist [3,4]. In HIV infection T-cell help is progressively lost as CD4+ T-cell counts decline . Seasonal influenza vaccination is advised for HIV-infected individuals . The question of whether, based on specific immunological assessments, subgroups of HIV-infected individuals should be vaccinated with priority has not been assessed.
How, in HIV-infected individuals, virosomal influenza antigen, which is known to trigger both cellular and humoral immunity , is capable of inducing influenza-specific immunoglobulin M (IgM) and immunoglobulin G (IgG), and how this antibody-production relates to total CD4+ T-cell counts and influenza-specific CD4+ T-cell function is unknown.
In a prospective observational clinical study, we characterized vaccine-specific immunity in 24 HIV-negative and in 31 HIV-infected individuals. Study participants were recruited and followed up at the University Hospital Basel during the vaccination season 2007–2008. Inclusion criteria were: age more than 18 years and in HIV-infected individuals antiretroviral therapy (ART) since more than 3 months and HIV viral load less than 200 copies/ml). Exclusion criteria were: a febrile illness, allergies to compounds of the vaccine, any vaccination within 30 days of inclusion and/or during follow-up, concomitant or planed medication with steroids/other immunosuppressive drugs, malignant disease, and pregnancy. The study was Institutional Review Board approved and written informed consent was obtained from all study participants. All participants were vaccinated with a trivalent virosomal vaccine (Inflexal V, Berna Biotech, Basel, Switzerland). Blood was collected immediately prior to vaccination and at day 7 [=follow-up 1 (FU1), 14 (=FU2) and 28 (=FU3)] post-vaccination. Influenza-specific IgM and IgG were quantified using a commercially available kit (Genzyme Virotech, Ruesselsheim, Germany), antibody-quantifications expressed as mean optical density values from duplicate measurements. The frequency of CD4+ T cells secreting interferon-γ was measured using enzyme-linked immunosorbent spot assays as previously described , using Inflexal (Berna Biotech) as the source of antigen. Peak post-vaccination frequencies are shown. Wilcoxon matched paired test was performed to compare frequencies of influenza-specific CD4+ T cells and influenza-specific IgG and IgM between differing time points.
Clinical characteristics of the study population are summarized in Supplementary Table 1, http://links.lww.com/QAD/A55. In HIV-negative individuals median levels of influenza-specific IgM (FU1–3) and IgG (FU2–3) significantly increased as compared with pre-vaccination levels (Fig. 1a, left and middle panel). The median frequency of vaccine-induced CD4+ T cells also significantly increased, with a rise observed in 22 out of 24 (92%) individuals (Fig. 1a, right panel). In HIV-infected participants with preserved CD4+ T-cell counts (>350 μl of blood), median levels of influenza-specific IgM (FU1–3) and IgG (FU1–3) also significantly increased as compared with prevaccination levels (Fig. 1b, left and middle panel). However, although the median frequency of vaccine-induced CD4+ T cells rose significantly, an increase was observed in only 14 out of 22 individuals (64%) (Fig. 1b, right panel). In contrast to HIV-negative and HIV-infected individuals with preserved CD4+ T-cell counts, in HIV-infected study participants with less than 350 CD4+ T-cell counts/μl, no significant increase in median influenza-specific IgM levels was detected at any follow-up time-point, with only two out of nine participants responding at all (Fig. 1c, left panel). However, a significant increase in median levels of postvaccination influenza-specific IgG was observed (Fig. 1c, middle panel). No significant increase in the median frequency of vaccine-induced CD4+ T cells was detected in the group of individuals with low CD4+ T-cell counts, with a response induced in only four out of nine individuals (44%) (Fig. 1c, right panel). Of note, time on ART in ‘IgM responders’ with low CD4+ T-cell counts tended to be longer, in ‘IgM nonresponders’ with high CD4+ T-cell counts shorter than among their respective intrapopulation controls. No such trend was observed comparing Centers for Disease Control disease stage, nadir CD4+ T-cell counts and virological suppression (data not shown).
Early production of IgM is independent from T-cell help . The inability of most HIV-infected individuals with low CD4+ T-cell counts to produce detectable amounts of influenza-specific IgM was thus unexpected. A possible explanation for the observed lack of IgM-production could be an intrinsic defect of B cells, which has been demonstrated in patients with viremia and, although to a much lesser extent, also in patients with undetectable viral load [10,11]. By contrast, vaccine-induced IgG increased in all study groups, including the one with low CD4+ T-cell counts. Given the lack of IgM-response in HIV-infected individuals with low CD4+ T-cell counts, increasing levels of IgG in this study group most likely reflects a memory response. This finding is important and provides an immunological rational supporting the recommendation of annual influenza vaccinations throughout the course of HIV-infection, permitting the buildup of a broad and long-lasting B-cell memory when immunological competence is still maintained. The clinical importance of such memory responses has been impressively underscored during the recent influenza H1N1 pandemic, where children, presumably owing to a lack of seroprotection from crossreactive antibodies induced by previous contact with influenza antigen, were most severely affected .
The limitations of our study are the lack of qualitative assessments of the humoral and the cellular vaccine-specific immune response, patient diversity, and the fact that the study was underpowered to evaluate clinical endpoints such as protection from influenza infection. However, the read-outs used did permit the capture of decreasing vaccine-inducible cellular responsiveness, and they uncovered a likely dependency of the vaccine-response on B-cell memory in advanced HIV infection. These preliminary data should trigger future research aiming at understanding the molecular basis of the observed lack of IgM-production, and they lend support to strictly enacting annual influenza-vaccination in all HIV-infected individuals regardless of their CD4+ T-cell count.
We thank Johannes Nemeth for discussion and critical review of the manuscript.
S.F. performed most experiments and helped write the report, E.M. initiated the study and collected patient data, B.D. and G.Z. collected patient samples and performed experiments, A.C. recruited patients, I.S. performed serological analyses, M.B. was involved in the planning and conduct of the study on the clinical side, R.N. initiated the study and collected patient data, C.H. designed the study, supervised the research, analyzed data and wrote the report. B.D. and C.H. are supported by the SNF grants (323500-119221 and PP00B 114850, respectively). The authors have no conflicts of interest to declare. S.F. and E.M contributed equally to this work.
1. Grewal IS, Flavell RA. CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol 1998; 16:111–135.
2. Parker DC. T cell-dependent B cell activation. Annu Rev Immunol 1993; 11:331–360.
3. Vieira P, Rajewsky K. Persistence of memory B cells in mice deprived of T cell help. Int Immunol 1990; 2:487–494.
4. Hebeis BJ, Klenovsek K, Rohwer P, Ritter U, Schneider A, Mach M, Winkler TH. Activation of virus-specific memory B cells in the absence of T cell help. J Exp Med 2004; 199:593–602.
5. Margolick JB, Munoz A, Donnenberg AD, Park LP, Galai N, Giorgi JV, et al
. Failure of T-cell homeostasis preceding AIDS in HIV-1 infection: the Multicenter AIDS Cohort Study. Nat Med 1995; 1:674–680.
6. Harper SA, Fukuda K, Uyeki TM, Cox NJ, Bridges CB. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2005; 54:1–40.
7. Huckriede A, Bungener L, Stegmann T, Daemen T, Medema J, Palache AM, Wilschut J. The virosome concept for influenza vaccines. Vaccine 2005; 23(Suppl 1):S26–S38.
8. Gasser O, Bihl FK, Wolbers M, Loggi E, Steffen I, Hirsch HH, et al
. HIV patients developing primary CNS lymphoma lack EBV-specific CD4+ T cell function irrespective of absolute CD4+ T cell counts. PLoS Med 2007; 4:e96.
9. Notarangelo LD, Lanzi G, Peron S, Durandy A. Defects of class-switch recombination. J Allergy Clin Immunol 2006; 117:855–864.
10. Moir S, Fauci AS. B cells in HIV infection and disease. Nat Rev Immunol 2009; 9:235–245.
11. Moir S, Ho J, Malaspina A, Wang W, DiPoto AC, O'Shea MA, et al
. Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals. J Exp Med 2008; 205:1797–1805.
12. Bansal S, Pourbohloul B, Grenfell B, Meyers LA. The shifting demographic landscape of influenza
. PLoS Curr Influenza