Advertisement

The controversial impact of B cells subsets on immune response to pneumococcal vaccine in HIV-1 patients

Open AccessPublished:July 17, 2015DOI:https://doi.org/10.1016/j.ijid.2015.07.008

      Highlights

      • The vast majority of vaccine non-responders were on HAART.
      • Most B cell subsets were lower among ART-naïve patients compared to treated patients at baseline.
      • Memory B cells and IgM memory B cells demonstrated no correlation with the vaccine IgG response.
      • The total B cell count and exhausted memory B cells predicted the antibody response to the vaccine.
      • Activated B cells at baseline were associated with the baseline viral load, and resting memory B cells moderately correlated negatively with viral load at baseline.

      Summary

      Background

      Chronic HIV infection leads to severe perturbations of the B cell populations and hypo-responsiveness to vaccines. The associations between circulating B cell subpopulations and the antibody response to pneumococcal polysaccharide vaccine in antiretroviral-naïve and treated patients were studied.

      Methods

      Sixty-six HIV-infected adults were grouped according to antiretroviral therapy (ART) and CD4+ cell count; 31 were ART-naïve and 35 were ART-treated, and they were matched for age, CD4 cell count, and duration of HIV infection. All subjects were immunized with the 23-valent polysaccharide vaccine against Streptococcus pneumoniae. Pre- and post-vaccination B cell subpopulations were assessed by flow cytometry. Serum IgG concentrations for vaccine serotypes were quantified by ELISA at baseline and at 4 and 48 weeks post-vaccination.

      Results

      Patients under highly active antiretroviral therapy (HAART) had significantly higher antibody levels against pneumococcal vaccine antigens, while an adequate number of patients responded to vaccination. Memory B cells were diminished over time, although treated patients maintained higher levels of all subsets studied, with the exception of activated memory and isotype-switched memory B cells.

      Conclusions

      Low concentrations of total B cells and exhausted memory B cells was the strongest independent predictor of poor pneumococcal vaccine responsiveness, emphasizing that B cell subset disturbances are associated with a poor vaccine response among HIV-infected patients.

      Keywords

      1. Introduction

      Streptococcus pneumoniae infections are the most common cause of bacterial pneumonia among HIV-infected patients and account for high morbidity and mortality.
      • Feikin D.R.
      • Feldman C.
      • Schuchat A.
      • Janoff E.N.
      Global strategies to prevent bacterial pneumonia in adults with HIV disease.
      The introduction of highly active antiretroviral therapy (HAART) has led to a decline in the incidence of invasive pneumococcal disease, although it still affects HIV-infected more often than healthy individuals, even those with preserved CD4 cell counts.
      • Kohli R.
      • Lo Y.
      • Homel P.
      • Flanigan T.P.
      • Gardner L.I.
      • Howard A.A.
      • et al.
      Bacterial pneumonia, HIV therapy, and disease progression among HIV-infected women in the HIV epidemiologic research (HER) study.
      • Sogaard O.S.
      • Lohse N.
      • Gerstoft J.
      • Kronborg G.
      • Ostergaard L.
      • Pedersen C.
      • et al.
      Hospitalization for pneumonia among individuals with and without HIV infection, 1995–2007: a Danish population-based, nationwide cohort study.
      • Barry P.M.
      • Zetola N.
      • Keruly J.C.
      • Moore R.D.
      • Gebo K.A.
      • Lucas G.M.
      Invasive pneumococcal disease in a cohort of HIV-infected adults: incidence and risk factors, 1990–2003.
      • Foster D.
      • Knox K.
      • Walker A.S.
      • Griffiths D.T.
      • Moore H.
      • Haworth E.
      • et al.
      Oxford Invasive Pneumococcal Surveillance Group. Invasive pneumococcal disease: epidemiology in children and adults prior to implementation of the conjugate vaccine in the Oxfordshire region, England.
      • Grau I.
      • Pallares R.
      • Tubau F.
      • Schulze M.H.
      • Llopis F.
      • Podzamczer D.
      • et al.
      Spanish Pneumococcal Infection Study Network (G03/103). Epidemiologic changes in bacteremic pneumococcal disease in patients with human immunodeficiency virus in the era of highly active antiretroviral therapy.
      • Pedersen R.H.
      • Lohse N.
      • Østergaard L.
      • Søgaard O.S.
      The effectiveness of pneumococcal polysaccharide vaccination in HIV-infected adults: a systematic review.
      The guidelines still recommend vaccination of HIV-infected people with the 23-valent polysaccharide vaccine (PPV-23)
      • Kaplan J.E.
      • Benson C.
      • Holmes K.K.
      • Brooks J.T.
      • Pau A.
      • Masur H.
      Centers for Disease Control and Prevention (CDC)National Institutes of HealthHIV Medicine Association of the Infectious Diseases Society of America
      Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America.
      despite its challenge from the conjugate polysaccharide vaccine. Despite scheduled immunization and the introduction of HAART,
      • Falcó V.
      • Jordano Q.
      • Cruz M.J.
      • Len O.
      • Ribera E.
      • Campins M.
      • et al.
      Serological response to pneumococcal vaccination in HAART-treated HIV-infected patients: one year follow-up study.
      • Søgaard O.S.
      • Schønheyder H.C.
      • Bukh A.R.
      • Harboe Z.B.
      • Rasmussen T.A.
      • Ostergaard L.
      • et al.
      Pneumococcal conjugate vaccination in persons with HIV: the effect of highly active antiretroviral therapy.
      • Crum-Cianflone N.F.
      • Huppler Hullsiek K.
      • Roediger M.
      • Ganesan A.
      • Patel S.
      • Landrum M.L.
      • et al.
      A randomized clinical trial comparing revaccination with pneumococcal conjugate vaccine to polysaccharide vaccine among HIV-infected adults.
      • Madhi S.A.
      • Klugman K.P.
      • Kuwanda L.
      • Cutland C.
      • Käyhty H.
      • Adrian P.
      Quantitative and qualitative anamnestic immune responses to pneumococcal conjugate vaccine in HIV-infected and HIV-uninfected children 5 years after vaccination.
      the risk of pneumococcal infection among these immunocompromised hosts remains high.
      • Barry P.M.
      • Zetola N.
      • Keruly J.C.
      • Moore R.D.
      • Gebo K.A.
      • Lucas G.M.
      Invasive pneumococcal disease in a cohort of HIV-infected adults: incidence and risk factors, 1990–2003.
      • Dworkin M.S.
      • Ward J.W.
      • Hanson D.L.
      • Jones J.L.
      • Kaplan J.E.
      Pneumococcal disease among human immunodeficiency virus-infected persons: incidence, risk factors, and impact of vaccination.
      • Yin Z.
      • Rice B.D.
      • Waight P.
      • Miller E.
      • George R.
      • Brown A.E.
      • et al.
      Invasive pneumococcal disease among HIV-positive individuals, 2000–2009.
      B cells comprise one the most dysfunctional lymphocyte populations in patients with HIV infection.
      • Lane H.C.
      • Masur H.
      • Edgar L.C.
      • Whalen G.
      • Rook A.H.
      • Fauci A.S.
      Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome.
      During chronic viral replication, functional perturbations of B cells occur, including polyclonal activation,
      • Morris L.
      • Binley J.M.
      • Clas B.A.
      • Bonhoeffer S.
      • Astill T.P.
      • Kost R.
      • et al.
      HIV-1 antigen-specific and -nonspecific B cell responses are sensitive to combination antiretroviral therapy.
      hypergammaglobulinemia,
      • Lane H.C.
      • Masur H.
      • Edgar L.C.
      • Whalen G.
      • Rook A.H.
      • Fauci A.S.
      Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome.
      dysregulation of isotype switching, variation in the proportions and absolute numbers of circulating B cells, and impaired immune responses to immunization.
      • Hart M.
      • Steel A.
      • Clark S.A.
      • Moyle G.
      • Nelson M.
      • Henderson D.C.
      • et al.
      Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease.
      • Shen X.
      • Tomaras G.D.
      Alterations of the B-cell response by HIV-1 replication.
      • Moir S.
      • Buckner C.M.
      • Ho J.
      • Wang W.
      • Chen J.
      • Waldner A.J.
      • et al.
      B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy.
      Significant heterogeneity is also obvious among memory B cells, and despite their ambiguous functional heterogeneity, many phenotypic subpopulations have been recognized.
      • Sanza I.
      • Weia C.
      • Leeb F.E.
      • Anolika J.
      Phenotypic and functional heterogeneity of human memory B cells.
      The loss of memory B cells is one aspect of dysfunction in HIV infection.
      • Moir S.
      • Fauci A.S.
      Pathogenic mechanisms of B lymphocyte dysfunction in HIV disease.
      Moreover, there is evidence of alterations in B cell populations against T cell-independent antigens such as pneumococcal polysaccharides.
      • Hart M.
      • Steel A.
      • Clark S.A.
      • Moyle G.
      • Nelson M.
      • Henderson D.C.
      • et al.
      Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease.
      A reduced frequency of IgM+ memory B cells, which mediate memory responses against pneumococcal infection, is also observed.
      • Kruetzmann S.
      • Rosado M.M.
      • Weber H.
      • Germing U.
      • Tournilhac O.
      • Peter H.H.
      • et al.
      Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen.
      Most activated B cells undergo apoptosis after infections lapse, except for a few cells comprising the resting memory B cell compartment, necessary for a rapid secondary immune response.
      During the natural course of HIV-1 infection, the B cell subsets are altered, including resting memory B cells, which are severely depleted.
      • De Milito A.
      • Morch C.
      • Sonnerborg A.
      • Chiodi F.
      Loss of memory (CD27) B lymphocytes in HIV-1 infection.
      Additionally, defective B cell populations, including activated memory and exhausted memory B cells, rise in HIV-1-infected individuals, while they circulate at very low levels in healthy individuals.
      • Moir S.
      • Ho J.
      • Malaspina A.
      • Wang W.
      • DiPoto A.C.
      • O'Shea M.A.
      • et al.
      Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals.
      These aberrant cells express a low level of CD21 on their surface and present features of immune activation
      • Moir S.
      • Malaspina A.
      • Ogwaro K.M.
      • Donoghue E.T.
      • Hallahan C.W.
      • Ehler L.A.
      • et al.
      HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals.
      and cellular exhaustion.
      • Moir S.
      • Ho J.
      • Malaspina A.
      • Wang W.
      • DiPoto A.C.
      • O'Shea M.A.
      • et al.
      Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals.
      HAART initiation reduces polyclonal B cell activation
      • Moir S.
      • Buckner C.M.
      • Ho J.
      • Wang W.
      • Chen J.
      • Waldner A.J.
      • et al.
      B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy.
      • Moir S.
      • Ho J.
      • Malaspina A.
      • Wang W.
      • DiPoto A.C.
      • O'Shea M.A.
      • et al.
      Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals.
      and normalizes limited numbers of naïve and memory B cell subsets.
      • Hart M.
      • Steel A.
      • Clark S.A.
      • Moyle G.
      • Nelson M.
      • Henderson D.C.
      • et al.
      Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease.
      • Shen X.
      • Tomaras G.D.
      Alterations of the B-cell response by HIV-1 replication.
      • Moir S.
      • Buckner C.M.
      • Ho J.
      • Wang W.
      • Chen J.
      • Waldner A.J.
      • et al.
      B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy.
      • Chong Y.
      • Ikematsu H.
      • Kikuchi K.
      • Yamamoto M.
      • Murata M.
      • Nishimura M.
      • et al.
      Selective CD27+ (memory) B cell reduction and characteristic B cell alteration in drug-naive and HAART-treated HIV type 1-infected patients.
      However, neither the amount of circulating isotype-switched memory B cells nor their functional activity are restored by HAART,
      • Moir S.
      • Malaspina A.
      • Ho J.
      • Wang W.
      • Dipoto A.C.
      • O'Shea M.A.
      • et al.
      Normalization of B cell counts and subpopulations after antiretroviral therapy in chronic HIV disease.
      • Moir S.
      • Fauci A.S.
      B cells in HIV infection and disease.
      which might lead to impaired immunity, even in treated patients.
      • Crum-Cianflone N.F.
      • Huppler Hullsiek K.
      • Roediger M.
      • Ganesan A.
      • Patel S.
      • Landrum M.L.
      • et al.
      A randomized clinical trial comparing revaccination with pneumococcal conjugate vaccine to polysaccharide vaccine among HIV-infected adults.
      • Madhi S.A.
      • Klugman K.P.
      • Kuwanda L.
      • Cutland C.
      • Käyhty H.
      • Adrian P.
      Quantitative and qualitative anamnestic immune responses to pneumococcal conjugate vaccine in HIV-infected and HIV-uninfected children 5 years after vaccination.
      The loss of memory is reflected in the decline in antigen-specific memory B cells after vaccination, which are not restored by HAART.
      • Amu S.
      • Ruffin N.
      • Rethi B.
      • Chiodi F.
      Impairment of B-cell functions during HIV-1 infection.
      • Titanji K.
      • De Milito A.
      • Cagigi A.
      • Thorstensson R.
      • Grutzmeier S.
      • Atlas A.
      • et al.
      Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection.
      HAART has only a limited effect on the normalization of the B cell compartment. Resting memory B cells are maintained if HAART is initiated early post primary HIV infection.
      • Pensieroso S.
      • Galli L.
      • Nozza S.
      • Ruffin N.
      • Castagna A.
      • Tambussi G.
      • et al.
      B-cell subset alterations and correlated factors in HIV-1 infection.
      It remains, though, to be elucidated whether HAART restores certain B cell defects. Furthermore, viremia has been associated with certain B cell defects.
      • Amu S.
      • Ruffin N.
      • Rethi B.
      • Chiodi F.
      Impairment of B-cell functions during HIV-1 infection.
      However, the impacts of HIV viremia and the level of nadir CD4 cell counts have not been fully clarified, except in a few studies.
      • Pensieroso S.
      • Galli L.
      • Nozza S.
      • Ruffin N.
      • Castagna A.
      • Tambussi G.
      • et al.
      B-cell subset alterations and correlated factors in HIV-1 infection.
      Although antibody levels are useful as a surrogate marker of protection and have been used during recent decades as the gold standard assay, they have limitations and cannot fully describe the immune response to vaccines; consequently other markers have been established to assess immunogenicity and protection of immunization more precisely.
      • Song J.Y.
      • Moseley M.A.
      • Burton R.L.
      • Nahm M.H.
      Pneumococcal vaccine and opsonic pneumococcal antibody.
      • Park S.
      • Nahm M.H.
      Older adults have a low capacity to opsonize pneumococci due to low IgM antibody response to pneumococcal vaccinations.
      • Parkkali T.
      • Väkeväinen M.
      • Käyhty H.
      • Ruutu T.
      • Ruutu P.
      Opsonophagocytic activity against Streptococcus pneumoniae type 19F in allogeneic BMT recipients before and after vaccination with pneumococcal polysaccharide vaccine.
      • Shatz D.V.
      • Schinsky M.F.
      • Pais L.B.
      • Romero-Steiner S.
      • Kirton O.C.
      • Carlone G.M.
      Immune responses of splenectomized trauma patients to the 23-valent pneumococcal polysaccharide vaccine at 1 versus 7 versus 14 days after splenectomy.
      • Nunes M.C.
      • Madhi S.A.
      Safety, immunogenicity and efficacy of pneumococcal conjugate vaccine in HIV-infected individuals.
      • Feikin D.R.
      • Elie C.M.
      • Goetz M.B.
      • Lennox J.L.
      • Carlone G.M.
      • Romero-Steiner S.
      • et al.
      Randomized trial of the quantitative and functional antibody responses to a 7-valent pneumococcal conjugate vaccine and/or 23-valent polysaccharide vaccine among HIV-infected adults.
      • Siber G.R.
      • Chang I.
      • Baker S.
      • Fernsten P.
      • O’Brien K.L.
      • Santosham M.
      • et al.
      Estimating the protective concentration of anti-pneumococcal capsular polysaccharide antibodies.
      • Wernette C.M.
      • Frasch C.E.
      • Madore D.
      • Carlone G.
      • Goldblatt D.
      • Plikaytis B.
      • et al.
      Enzyme-linked immunosorbent assay for quantitation of human antibodies to pneumococcal polysaccharides.
      The aim of this study was to fully assess the implication of memory B cells in the antibody response to the 23-PPV in HIV patients. Specifically, it was sought to record and evaluate associations between circulating B cell subpopulations in the peripheral blood and the antibody response during a 48-week period, pre and post immunological stimulation with the recommended vaccine against S. pneumoniae in HIV-1 patients. Two patient groups of distinct infection status were studied, those treated successfully with HAART and those who were HAART-naïve.

      2. Materials and methods

      2.1 Study participants and ethics approval

      This was a longitudinal study involving 66 HIV-1 patients, of whom 31 were antiretroviral-naïve and had preserved CD4 cells (CD4 cell count above 500 copies/mm3) and 35 were on successful HAART, with satisfactory viral suppression (HIV-1 viral load below 50 copies/ml); these patients were matched for age, CD4 cell count, and duration of HIV infection. All clinical, epidemiological, and laboratory data, including age, gender, HIV-1 transmission route, co-morbidities, HIV-1 viral load, current CD4 T cell count, and nadir CD4 cell count, were recorded or measured.
      All patients were immunized for the first time against S. pneumoniae. Individuals who had been on HAART for less than 6 months or who had an HIV RNA above 50 copies/ml while being under HAART, those who had a CD4 T cell count below 200 cells/ml, and those who missed the first visit (4th week) post-immunization were excluded.
      All patients were followed-up in the Infectious Diseases Unit of AHEPA University Hospital in Thessaloniki Greece. The ethics committee of the study institution approved the study protocol and all participants submitted a written informed consent.

      2.2 Immunization and blood sample collection

      All patients underwent blood sampling and then received 0.5 ml of the 23-valent polysaccharide vaccine Pneumovax 23 (Merck and Co., Inc.) via intramuscular administration. Patients returned 4 and 48 weeks post vaccination for further blood sampling. Fifteen millilitres of fresh whole blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes for B cell staining; 10 ml more of whole blood was collected into heparin-coated, pyrogen-free tubes for phagocytosis assessment and 15 ml in tubes without anti-clot agent for antibody measurement via ELISA. Blood samples were analysed within 2 h of collection.

      2.3 B cell immunophenotyping

      The following mouse anti-human fluorochrome-conjugated monoclonal antibodies of Immunostep Company were used: CD19-PerCP, CD27-PE, IgM-FITC, IgD-FITC, and CD21-FITC. Results were expressed as the subset percentage of the total B cell fraction. Sample processing and analysis of results were performed in the XL Epics cytometer (Beckman Coulter Company, Miami, FL, USA). Upon addition of 10 μl of the above combined monoclonal antibodies, 100 μl of each blood sample was incubated in the dark for 10 min. Subsequently, red blood cells were thawed upon ingestion of 2 ml of Lysis Buffer (Becton Dickinson Biosciences, San Jose, CA, USA) and incubated for another 20 min at room temperature. The cell staining, input capture, and flow analysis were performed promptly in a blinded pattern. B cells were assessed prior to vaccination. The different B cell subsets were characterized at baseline as follows: total B cells (CD19+), memory B cells (CD19+CD27+, BMC), resting memory B cells (CD19+CD27+CD21high, RM), exhausted memory B cells (CD19+CD21lowCD27−, EM), IgM memory B cells (CD19+CD27+IgMhigh), isotype-switched memory B cells (CD19+CD27+IgM−, ITS), and activated memory B cells (CD19+CD21low+CD27+, AM). Results were expressed as B cell concentrations or as a percentage of the total B cell fraction. The cell staining, data capture, and flow analysis were performed in a blinded fashion.

      2.4 ELISA

      Prior to vaccination and at weeks 4 and 48, blood samples were collected, allowed to clot naturally, and the serum separated; ELISA was then performed immediately. Microwells were pre-coated with the Pneumocystis jirovecii Pneumonia (PCP) antigen (1–5, 6B, 7F, 8, 9N, 9 V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, 33F). Calibrators and controls were pre-absorbed against capsular polysaccharide (CPS) and samples were diluted in diluents containing CPS. The calibrators, controls, and diluted patient samples were then added to the wells and antibodies recognizing the PCP antigen bound, during the first incubation. Samples were run in duplicate.
      The rest of the procedure was executed according to manufacturer's instructions.

      ELISA Manufacturers Manual. Birmingham, UK: The Binding Site Ltd, 2011. http://www.quimiolab.com/userfiles/Docuemtnos%20Anexos/Binding%20Site/MK012.pdf/ [accessed 13.06.12].

      In order to interpret the immune response to the vaccine, several criteria are used: the post-vaccination antibody titre, the fold-increase, and the frequencies of serotypes with a satisfactory response. Currently there is no global definition for the adequate response to pneumococcal polysaccharide vaccine. In this study a satisfactory antibody response was considered a two-fold antibody increase from the initial level.
      • Kernéis S.
      • Launay O.
      • Turbelin C.
      • Batteux F.
      • Hanslik T.
      • Boëlle P.Y.
      Long-term immune responses to vaccination in HIV-infected patients: a systematic review and meta-analysis.

      2.5 Statistical analysis

      Data were expressed as the mean ± standard deviation (SD) or median (interquartile range (IQR)) (in the case of violation of normality) for continuous variables, and as counts and percentages for categorical data. The Kolmogorov–Smirnov test was used for normality analysis of the parameters.
      The one-factor repeated measures analysis of variance (ANOVA) model was used to compare the different time measurements of variables for each group. Pair-wise multiple comparisons were performed using the critical difference method of Tukey.
      The vaccine antibody responses were calculated as the relative vaccine-specific IgG increase from pre-vaccination baseline to 4 and 48 weeks, thus providing a correlate for total vaccine response adjusted for the patient's IgG concentration of the 23 vaccine serotypes at the time of enrolment. The number of B cells and B memory cell subpopulations were compared between the two patient groups by Student's t-test, or the Mann–Whitney test in the case of violation of normality.
      This was supplemented with multivariable regression analyses with adjustments for patient group (ART-naïve and treated patients) and current smoking status (yes/no). Logarithmic transformation was used when appropriate to obtain normality.
      All tests are two-sided, and a p-value of <0.05 was used to denote statistical significance. All analyses were carried out using the statistical package SPSS version 16.00 (SPSS Inc., Chicago, IL, USA).

      3. Results

      The demographic, clinical, and laboratory data of the 66 HIV-1 patients are summarized in Table 1.
      Table 1Baseline characteristics of the study participants at inclusion
      HAART-naïve patients (n = 31)Treated patients (n = 35)p-Value
      Age, years, mean ± SD30.46 ± 7.1233.15 ± 7.90.178
      Gender, male/female, n (%)31 (100.0%)/0 (0.0%)31 (85.7%)/4 (14.3%)0.065
      Years of HIV infection, mean ± SD3.84 ± 1.854.68 ± 4.440.345
      CD4 cell count before vaccination, mean ± SD (copies/mm3)637.9 ± 270.4708.7 ± 296.10.316
      Nadir CD4 cell count, mean ± SD (copies/mm3)596.6 ± 239.1315 ± 192.10.0005
      VL before vaccination, median (IQR)36 503 (165 080)47 (0)0.0005
      HAART duration in months, mean ± SDNA35.6 ± 14.35NA
      Body weight, kg, mean ± SD76.1 ± 10.2273.0 ± 10.870.239
      HCV infection, n (%)1 (3.2%)3 (8.6%)0.616
      HBV infection, n (%)6 (19.4%)3 (8.6%)0.287
      Current smoker, n (%)21 (67.7%)12 (34.3%)0.013
      HAART, highly active antiretroviral therapy; SD, standard deviation; VL, HIV RNA viral load; IQR, interquartile range; NA, not applicable; HCV, hepatitis C virus; HBV, hepatitis B virus.
      CD4 cell counts were higher in the treated group and their progress was different compared to naïve patients (p = 0.024), who had a downward trend in their CD4 cell counts. Concerning each post-vaccination time point separately, there was a significant variation at week 4 (p = 0.029) and week 48 (p = 0.001) in CD4 frequencies (data not shown). The two groups differed significantly concerning smoking habit (p = 0.013) and nadir CD4 cell count (p < 0.0005) (Table 1).

      3.1 Antibody response to 23-PPV

      Patients under HAART had significantly higher antibody levels at all three time points: at baseline (p = 0.0005) and at 4 weeks (p = 0.006) and 48 weeks (p = 0.001) post-vaccination. Fifty patients doubled their initial IgG level (responders), while 16 were non-responders (Table 2). Interestingly, the vast majority of non-responders were under HAART treatment. No correlation between an adequate response and current CD4 or nadir CD4 cell count was detected.
      Table 2Antibody response to 23-PPV in naïve and treated HIV patients
      Treated patients maintained higher levels of antibodies against Streptococcus pneumoniae pre- and post-immunization. Most patients responded successfully to the vaccine, irrelevant of HAART intake. However, there were twice as many treated non-responders as naïve non-responders.
      HAART-naïve patients (n = 31)Treated patients (n = 35)p-Value
      Antibodies (log) pre-immunization, mean ± SD3.16 ± 0.984.4 ± 1.430.0005
      Antibodies (log) 4th week, mean ± SD4.62 ± 1.385.46 ± 1.040.006
      Antibodies (log) 48th week, mean ± SD0.29 ± 0.50.65 ± 0.330.001
      Responders26 (83.8%)24 (68.5%)0.047
      Non-responders5 (16.1%)11 (31.4%)0.047
      Fold increase, mean ± SD6.72 ± 6.88.18 ± 13.41NS
      23-PPV, 23-valent pneumococcal polysaccharide vaccine; HAART, highly active antiretroviral therapy; SD, standard deviation; NS, not significant.
      a Treated patients maintained higher levels of antibodies against Streptococcus pneumoniae pre- and post-immunization. Most patients responded successfully to the vaccine, irrelevant of HAART intake. However, there were twice as many treated non-responders as naïve non-responders.

      3.2 Most B cell subsets were lower among ART-naïve patients compared to treated patients at baseline

      The frequency of total B cells, BMC, RM, and EM was higher in treated patients compared to naïve patients; in contrast, IgM memory B cells and ITS were higher in the naïve group. Furthermore, ART-naïve patients also preserved higher fractions of AM pre vaccination (Figure 1). These differences were not statistically significant. Memory B cells were diminished over time in both groups, although treated patients maintained higher levels of all subsets studied, with the exception of AM and ITS. There was no significant relationship between CD4 cell counts and the studied B cell subsets pre vaccination. In contrast, on multivariate analysis, low levels of nadir CD4 correlated with the diminished levels of resting (p < 0.01) and activated memory B cells (p = 0.03) (data not shown).
      Figure thumbnail gr1
      Figure 1Distinct frequencies of total B cells and other B memory subpopulations in the peripheral blood of HIV viremic and aviremic (treated) individuals before immunization with the PPV-23 vaccine against Streptococcus pneumoniae. No significant differences in the levels of total B cells and certain memory B cell subsets were detected among antiretroviral-naïve and treated patients at baseline. HAART had no effect on the fluctuation of the initial B lymphocyte levels in HIV-1 infection.

      3.3 Memory B cells and IgM memory B cells demonstrated no correlation with vaccine IgG response

      In unadjusted linear regression analyses, the levels of memory B cell and IgM memory B cell counts did not predict the vaccine response at all time points. Similarly, regression analyses adjusted for patient group showed no effect of memory B cell and IgM memory B cell concentrations on IgG vaccine response.

      3.4 Total B cell count and exhausted memory B cells predicted the antibody response to the vaccine

      The vaccine-specific IgG concentration measured 4 weeks post vaccination correlated positively with total baseline B cell (p = 0.02) and exhausted memory B cell subsets (p = 0.07) in the unadjusted (Figure 2) and adjusted linear regression analyses. No other baseline B cell compartment was associated with the antibody response and preservation of the antibody concentrations post vaccination over the 48-week study period (data not shown).
      Figure thumbnail gr2
      Figure 2Linear regression plots: pre-vaccination baseline B cell subpopulations as predictors of the IgG antibody response. Scatter plots with the best fitted line. Association between the two B cell subsets (log cells/μl) plotted against the IgG vaccine-specific antibody concentration (log IgG μg/ml) at 4 weeks post-immunization, which were found to be significant, implying a positive predictive correlation of the initial levels of total B cells and the exhausted memory B cell subpopulation and the antibody response to PPV-23.

      3.5 Contribution of viremia and nadir CD4 cell count

      In order to investigate the role of different factors associated with B cell alterations, demographic, virological, and immunological factors at baseline were evaluated by multivariate analysis. Age correlated with activated and resting memory B cells, while gender did not seem to affect these populations. HIV RNA at baseline showed no correlation with AM, RM, or IgM memory B cell counts at baseline. The nadir CD4 cell count correlated with low levels of resting memory and activated memory B cells (data not shown).
      To evaluate more precisely the accumulated results from the previous analysis, the possibility of a linear association of B cell subpopulation frequencies with HIV RNA viral load and CD4 T cell counts at all three time points were further investigated separately (before vaccination and at week 4 and week 48 post-vaccination). Activated B cells at baseline were associated with the baseline viral load (r = 0.54, p = 0.01), and resting memory B cells were moderately negatively correlated with viral load at baseline (r = −0.45, p = 0.026) (data not shown). No association was observed between CD4 cell counts and AM or RM cells at any of the investigated time points (data not shown).
      The effect of HAART on distinct B cell subpopulations, along with the vaccine effect during the study period, is summarized in Table 3. Moreover, certain differences in B cell frequencies are illustrated compared to healthy individuals. There were significant variations in distinct memory B cell subsets in HIV-1 patients. Vaccination with the 23-PPV had an immunological effect on certain B cell subsets like AM, EM, RM, and ITS memory B cells, triggering alterations in their counts in the peripheral blood of HIV-infected patients. Moreover, HAART intake appears to have controversial implications in a few of these memory B cell populations.
      Table 3Effect of HAART and polysaccharide vaccine administration on different B cell subpopulations at weeks 4 and 48, compared to the frequencies of B cells in healthy adults
      Activated memory B cells increase in HIV-infected individuals, with conflicting HAART impact on normalization of their frequencies and activity. It is regarded to restore their expansion, especially if the initiation of HAART is prompt. The fact that activated memory B cells are expanded in chronic viral replication is in line with the data of the present study, which show that naïve patients maintain higher levels of this subset compared to treated patients, confirming the normalizing effect of HAART. These cells are prone to extrinsic apoptosis during the natural course of HIV infection. Due to the decline in their frequencies post vaccination in this study, irrespective of HAART, it is speculated that this is attributed to PPV-23 administration. Resting memory B cells are depleted during HIV infection, whereas their levels are high in healthy adults. It has been confirmed that HAART has a restoring effect on their population, triggering their increase, which is in line with the results of the present study, in which treated patients had higher levels of RM B cells compared to naïve patients. Regarding the subset of exhausted memory B cells, it is known that they accumulate in HIV-infected patients irrespective of viral control and HAART intake, and this has been attributed to high plasma viral loads, chronic immune activation, or disease progression. Additionally, their levels may change independent of immune activation or viral replication. Moreover, similar to healthy individuals, HIV controllers have been detected to have a high frequency of exhausted, tissue-like B cells, despite having low to undetectable viral loads. This suggests that the accumulation of dysfunctional exhausted memory B cells may be linked to intrinsic infection-induced alterations in the B cell compartment, which in the case of the present study could be attributed to the initial impairment of the CD4 T cell counts of the treated patients group (low nadir CD4 cell counts), despite current HAART intake. Isotype-switched memory B cells are known to depend on T cell help, which has been thought to be defective in ART-treated adults compared to naïve, so it is believed that the results of this study may reflect the better immunological status of naïve patients, imprinted on higher nadir CD4 cell levels compared to treated patients. However, these differences are minor.
      Viral load before vaccination in the treated group: <47 copies/mm3 (undetectable)

      Viral load before vaccination in the naïve group: 36 503 (165 080) copies/mm3
      Baseline B cell subpopulationsTreatment effectB cell subpopulations 4 weeksVaccination effect from baselineB cell subpopulations 48 weeksVaccination effect from baseline
      Total B cells (NR 4.9–18.4%)
       Treated8.25Increase8.49No change7.64No change
       Naïve6.62NA6.36No change5.92No change
      Memory B cells (NR 7.2–18.9%)
       Treated2.16Increase2.28Increase1.91Decrease
       Naïve1.98NA2.07Increase1.76Decrease
      Resting memory B cells (NR 44–78.3%)
       Treated40.89No change60.63Increase59.61Decrease
       Naïve40.66NA44.62Increase46.55Decrease
      Exhausted memory B cells (NR 7.2–11.2%)
       Treated55.19Increase46.36Decrease45.28Decrease
       Naïve47.49NA37.99Decrease39.55Decrease
      IgM memory B cells (NR 7.3–32.5%)
       Treated20.02Increase24.64No change28.73No change
       Naïve23.99NA24.44Increase28.24Increase
      Isotype- switched memory B cells (NR 6.5–29.1%)
       Treated22.29No change26.11Increase38.73Increase
       Naïve22.42NA25.03Increase37.28Increase
      Activated memory B cells (NR 1.0–3.6%)
       Treated58.87Decrease39.48Decrease40.68Decrease
       Naïve61.62NA54.79Decrease52.78Decrease
      HAART, highly active antiretroviral therapy; NR, normal range; NA, not applicable.
      a Activated memory B cells increase in HIV-infected individuals, with conflicting HAART impact on normalization of their frequencies and activity. It is regarded to restore their expansion, especially if the initiation of HAART is prompt. The fact that activated memory B cells are expanded in chronic viral replication is in line with the data of the present study, which show that naïve patients maintain higher levels of this subset compared to treated patients, confirming the normalizing effect of HAART. These cells are prone to extrinsic apoptosis during the natural course of HIV infection. Due to the decline in their frequencies post vaccination in this study, irrespective of HAART, it is speculated that this is attributed to PPV-23 administration. Resting memory B cells are depleted during HIV infection, whereas their levels are high in healthy adults. It has been confirmed that HAART has a restoring effect on their population, triggering their increase, which is in line with the results of the present study, in which treated patients had higher levels of RM B cells compared to naïve patients. Regarding the subset of exhausted memory B cells, it is known that they accumulate in HIV-infected patients irrespective of viral control and HAART intake, and this has been attributed to high plasma viral loads, chronic immune activation, or disease progression. Additionally, their levels may change independent of immune activation or viral replication. Moreover, similar to healthy individuals, HIV controllers have been detected to have a high frequency of exhausted, tissue-like B cells, despite having low to undetectable viral loads. This suggests that the accumulation of dysfunctional exhausted memory B cells may be linked to intrinsic infection-induced alterations in the B cell compartment, which in the case of the present study could be attributed to the initial impairment of the CD4 T cell counts of the treated patients group (low nadir CD4 cell counts), despite current HAART intake. Isotype-switched memory B cells are known to depend on T cell help, which has been thought to be defective in ART-treated adults compared to naïve, so it is believed that the results of this study may reflect the better immunological status of naïve patients, imprinted on higher nadir CD4 cell levels compared to treated patients. However, these differences are minor.

      4. Discussion

      HIV-1 infection in its natural course leads to significant B cell defects, including hyperactivation, cell switch, and finally the onset of cell subsets that are normally lacking or appear at low levels in healthy individuals, and HIV patients present poor antibody responses to polysaccharide vaccines. The introduction of HAART triggered further study of these populations, concluding that these alterations are reversible with successful antiviral treatment, suggesting a causal relationship with viremia. The effect of viremia in B cell divergences has not been assessed thoroughly except in a few studies,
      • Pensieroso S.
      • Galli L.
      • Nozza S.
      • Ruffin N.
      • Castagna A.
      • Tambussi G.
      • et al.
      B-cell subset alterations and correlated factors in HIV-1 infection.
      and consequently several differences have been recorded among ART-naïve and treated patients. This study, in line with those of other authors, showed that viremia is correlated with specific B cell populations. Patients with a viral load >50 copies/ml had elevated activated B cells and a decline in resting memory B cells.
      • Pensieroso S.
      • Galli L.
      • Nozza S.
      • Ruffin N.
      • Castagna A.
      • Tambussi G.
      • et al.
      B-cell subset alterations and correlated factors in HIV-1 infection.
      It was found that ART-treated patients had higher levels of several B cell subsets before vaccine administration. Moreover, it was determined that naïve patients with continuous viral replication preserved increased AM and ITS memory B cell counts, while nadir CD4 predicted low RM and ITS over time. It has been confirmed that HAART has a restoring effect on their population, triggering their increase, which is in line with the present results, in which treated patients had higher levels of RM B cells compared to naïve patients.
      • Moir S.
      • Buckner C.M.
      • Ho J.
      • Wang W.
      • Chen J.
      • Waldner A.J.
      • et al.
      B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy.
      Similarly, activated memory B cells are expanded in chronic viral replication, which is in line with the present data; these showed that naïve patients maintained higher levels of this subset compared to the treated group, confirming the normalizing effect of HAART. With regard to isotype-switched memory B cells, it is known that this subset is dependent on T cell help, which has been thought to be defective in ART-treated adults compared to naïve; hence it is believed that the present results may reflect the better immunological status of naïve patients, reflected in higher nadir CD4 cell levels compared to treated patients. However, it should be noted that these differences are minor.
      • Glennie S.J.
      • Banda D.
      • Gould K.
      • Hinds J.
      • Kamngona A.
      • Everett D.D.
      • et al.
      Defective pneumococcal-specific Th1 responses in HIV-infected adults precedes a loss of control of pneumococcal colonization.
      Furthermore, it was observed that total B cells, memory B cells, and ITS memory B cells correlated with IgG responses at baseline. Finally, ART was associated with increased total B cells and the EM B cell compartment, which seemed to predict IgG responses.
      HIV-1 infection impairs memory B cells, and seropositive patients display a low antibody response in T cell-independent (TI) antigens, such as those included in the 23-valent polysaccharide vaccine, irrespective of HAART, which further declines over time. These data were also confirmed in the present study, along with the fact that the majority of non-vaccine responders were ART-treated; the data further enhance the aspect that the antibody response alone does not constitute an efficient protection index for pneumococcal infection in immunocompromised individuals.
      • Kernéis S.
      • Launay O.
      • Turbelin C.
      • Batteux F.
      • Hanslik T.
      • Boëlle P.Y.
      Long-term immune responses to vaccination in HIV-infected patients: a systematic review and meta-analysis.
      • Johannesson T.G.
      • Søgaard O.S.
      • Tolstrup M.
      • Petersen M.S.
      • Bernth-Jensen J.M.
      • Østergaard L.
      • et al.
      The impact of B-cell perturbations on pneumococcal conjugate vaccine response in HIV-infected adults.
      In this study, vaccination with the 23-PPV elicited satisfactory antibody responses in both groups, in accordance with previous studies.
      • Wernette C.M.
      • Frasch C.E.
      • Madore D.
      • Carlone G.
      • Goldblatt D.
      • Plikaytis B.
      • et al.
      Enzyme-linked immunosorbent assay for quantitation of human antibodies to pneumococcal polysaccharides.
      • Kernéis S.
      • Launay O.
      • Turbelin C.
      • Batteux F.
      • Hanslik T.
      • Boëlle P.Y.
      Long-term immune responses to vaccination in HIV-infected patients: a systematic review and meta-analysis.
      Studies investigating long-term serological responses to 23-valent PPV in HIV patients have produced inconsistent results.
      • Amendola A.
      • Tanzi E.
      • Zappa A.
      • Colzani D.
      • Boschini A.
      • Musher D.M.
      • et al.
      Safety and immunogenicity of 23-valent pneumococcal polysaccharide vaccine in HIV-1-infected former drug users.
      • Rodriguez-Barradas M.C.
      • Alexandraki I.
      • Nazir T.
      • Foltzer M.
      • Musher D.M.
      • Brown S.
      • et al.
      Response of human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy to vaccination with 23-valent pneumococcal polysaccharide vaccine.
      • Falcó V.
      • Jordano Q.
      • Cruz M.J.
      • Len O.
      • Ribera E.
      • Campins M.
      • et al.
      Serological response to pneumococcal vaccination in HAART-treated HIV-infected patients: one year follow-up study.
      • Subramaniam K.S.
      • Segal R.
      • Lyles R.H.
      • Rodriguez-Barradas M.C.
      • Pirofski L.A.
      Qualitative change in antibody responses of human immunodeficiency virus-infected individuals to pneumococcal capsular polysaccharide vaccination associated with highly active antiretroviral therapy.
      Treated patients had higher levels of antibodies throughout the study period, although their progress over time was similar, irrespective of HAART intake. It was found that HIV viral suppression was associated with higher rates of antibody response.
      • Kernéis S.
      • Launay O.
      • Turbelin C.
      • Batteux F.
      • Hanslik T.
      • Boëlle P.Y.
      Long-term immune responses to vaccination in HIV-infected patients: a systematic review and meta-analysis.
      This is consistent with other reports, which have also implied a negative correlation between plasma HIV RNA load and serological responses to PPV-23 that could be restored by HAART.
      • Falcó V.
      • Jordano Q.
      • Cruz M.J.
      • Len O.
      • Ribera E.
      • Campins M.
      • et al.
      Serological response to pneumococcal vaccination in HAART-treated HIV-infected patients: one year follow-up study.
      • Subramaniam K.S.
      • Segal R.
      • Lyles R.H.
      • Rodriguez-Barradas M.C.
      • Pirofski L.A.
      Qualitative change in antibody responses of human immunodeficiency virus-infected individuals to pneumococcal capsular polysaccharide vaccination associated with highly active antiretroviral therapy.
      A moderate response to vaccine in treated patients probably reflects the exhaustion of the immune system along with the natural course of HIV infection.
      • Kernéis S.
      • Launay O.
      • Turbelin C.
      • Batteux F.
      • Hanslik T.
      • Boëlle P.Y.
      Long-term immune responses to vaccination in HIV-infected patients: a systematic review and meta-analysis.
      A recent study assessing the effectiveness of PPV-23 also suggested that, irrespective of CD4 cell count at immunization, the vaccine gave no benefit when it was administered to patients with high viremia.
      • Teshale E.H.
      • Hanson D.
      • Flannery B.
      • Phares C.
      • Wolfe M.
      • Schuchat A.
      • et al.
      Effectiveness of 23-valent polysaccharide pneumococcal vaccine on pneumonia in HIV-infected adults in the United States, 1998–2003.
      This may be related to continuous HIV replication, which probably perturbs B cell function, or may be correlated to the premature exhaustion of B cells, which results in ineffective humoral responses to antigen stimulation.
      • Moir S.
      • Ho J.
      • Malaspina A.
      • Wang W.
      • DiPoto A.C.
      • O'Shea M.A.
      • et al.
      Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals.
      • Moir S.
      • Ogwaro K.M.
      • Malaspina A.
      • Vasquez J.
      • Donoghue E.T.
      • Hallahan C.W.
      • et al.
      Perturbations in B cell responsiveness to CD4 T cell help in HIV-infected individuals.
      The present study also suggests that CD4 cell restoration upon HAART improved the antibody responses of HIV-infected patients receiving 23-valent PPV during the study period.
      No significant correlation between baseline CD4 cell counts and the studied B cell subsets was identified, which is in line with previous data.
      • Amu S.
      • Ruffin N.
      • Rethi B.
      • Chiodi F.
      Impairment of B-cell functions during HIV-1 infection.
      In contrast, on multivariate analysis, low levels of nadir CD4 correlated with the diminished levels of resting and activated memory B cells. This implies an intense effect of initial CD4 cell count levels, despite the subsequent immune restoration by following HAART, in the preservation and functionality of vital B cell populations.
      The phenotype of the B cells responsible for immune responses to the polysaccharide pneumococcal vaccine has been controversial, and this was the reason for the present investigation into most of the correlated B cell subsets. More precisely, IgM and isotype-switched memory B cells have a significant role in the immune response to 23-PPV. The present study is in line with those reported by other authors,
      • Glennie S.J.
      • Banda D.
      • Gould K.
      • Hinds J.
      • Kamngona A.
      • Everett D.D.
      • et al.
      Defective pneumococcal-specific Th1 responses in HIV-infected adults precedes a loss of control of pneumococcal colonization.
      who tried to investigate the effect of B cell subsets on the final response to 23-PPV. Total B cells and BMCs were elevated in treated patients compared to naïve patients both pre and post vaccination, as shown in other studies as well.
      • Shen X.
      • Tomaras G.D.
      Alterations of the B-cell response by HIV-1 replication.
      • Moir S.
      • Buckner C.M.
      • Ho J.
      • Wang W.
      • Chen J.
      • Waldner A.J.
      • et al.
      B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy.
      • Sanza I.
      • Weia C.
      • Leeb F.E.
      • Anolika J.
      Phenotypic and functional heterogeneity of human memory B cells.
      It has been confirmed that patients with diminished or a lack of IgM memory B cells, respond modestly to polysaccharide vaccine and are susceptible to invasive pneumococcal disease.
      • Weller S.
      • Braun M.C.
      • Tan B.K.
      • Rosenwald A.
      • Cordier C.
      • Conley M.E.
      • et al.
      Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire.
      • Shi Y.
      • Yamazaki T.
      • Okubo Y.
      • Uehara Y.
      • Sugane K.
      • Agematsu K.
      Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell.
      Several studies have demonstrated the loss of IgM and/or switched memory B cells in HIV patients, which were also confirmed in the present data. However, IgM memory B cells are not solely responsible for anti-polysaccharide antibody formation. Isotype-switched memory B cells have also been shown to produce effective antibodies post vaccination in vitro.
      • Takizawa M.
      • Sugane K.
      • Agematsu K.
      Role of tonsillar IgD+CD27+ memory B cells in humoral immunity against pneumococcal infection.
      • Khaskhely N.
      • Mosakowski J.
      • Thompson R.S.
      • Khuder S.
      • Smithson S.L.
      • Westerink M.A.
      Phenotypic analysis of pneumococcal polysaccharide-specific B cells.
      In agreement with other authors, higher levels of isotype-switched memory B cells were seen in treated patients compared to naïve patients,
      • Song J.Y.
      • Moseley M.A.
      • Burton R.L.
      • Nahm M.H.
      Pneumococcal vaccine and opsonic pneumococcal antibody.
      which correlated with the baseline levels of IgG against S. pneumoniae.
      Moreover, it is speculated that the association of the IgG response with total B cells and exhausted memory B cells reflects that these two B cell compartments participate in mounting an adequate response to PPV-23. Exhausted memory B cells comprise a rare population expanded in chronic HIV infection irrespective of HAART and viral replication.
      • Sciaranghella G.
      • Tong N.
      • Mahan A.E.
      • Suscovich T.J.
      • Alter G.
      Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection.
      It is known that EM accumulate in HIV-infected patients irrespective of viral control and HAART intake, and this has been attributed to high plasma viral loads, chronic immune activation, or disease progression. Additionally, their levels may change independent of immune activation or viral replication. Moreover, similar to healthy individuals, HIV controllers have been detected to have a high frequency of exhausted B cells despite having low to undetectable viral loads. This suggests that the accumulation of dysfunctional exhausted memory B cells may be linked to intrinsic infection-induced alterations in the B cell compartment, which in the case of the present study could be attributed to the initial impairment of the CD4 T cell counts of the treated patients group (low nadir CD4 cell counts), despite current HAART intake.
      • Sciaranghella G.
      • Tong N.
      • Mahan A.E.
      • Suscovich T.J.
      • Alter G.
      Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection.
      This study has some limitations. The peripheral blood B cell population was studied since this is by far the easiest accessible compartment and the most widely investigated, and the peripheral blood B cell subsets are only representative of cells participating in the response. The data indicate that total B cells and the exhausted memory B cell compartment are the only peripheral populations that can be used to predict the IgG response to PPV-23, although other significant associations are reported, which may lead to further investigations on the role of B cells in effective vaccine responses.
      This study emphasizes the fact that the disturbance of certain B cell subsets is associated with a poor vaccine response among HIV-infected patients and supports the idea that B cell defects play an important and independent role in the antibody response over time. Moreover, antibody assessment cannot comprise an index of effective protection against S. pneumoniae in immunocompromised hosts. In conclusion, an integrated study of all implicated aspects of the immune response may be a better approach to the evaluation of immunogenicity of the 23-PPV in HIV-infected patients.

      Acknowledgements

      We want to thank the staff of the National AIDS Reference Centre for their valuable contribution in processing the samples.
      Conflict of interest: None of the authors has any potential financial conflict of interest related to this manuscript.
      Authors’ contributions: O.T. participated in recruiting patients, immunizing, organizing the study and in the writing of the article. L.S., P.Z. and M.D. have organized the study and edited the text. A.M. and N.M. have processed the samples and executed the flow cytometry and extracted the results. A.G. has performed the statistical analysis of the study. M.S. participated in recruiting patients and writing/editing of the article.

      References

        • Feikin D.R.
        • Feldman C.
        • Schuchat A.
        • Janoff E.N.
        Global strategies to prevent bacterial pneumonia in adults with HIV disease.
        Lancet Infect Dis. 2004; 4: 445-455
        • Kohli R.
        • Lo Y.
        • Homel P.
        • Flanigan T.P.
        • Gardner L.I.
        • Howard A.A.
        • et al.
        Bacterial pneumonia, HIV therapy, and disease progression among HIV-infected women in the HIV epidemiologic research (HER) study.
        Clin Infect Dis. 2006; 43: 90-98
        • Sogaard O.S.
        • Lohse N.
        • Gerstoft J.
        • Kronborg G.
        • Ostergaard L.
        • Pedersen C.
        • et al.
        Hospitalization for pneumonia among individuals with and without HIV infection, 1995–2007: a Danish population-based, nationwide cohort study.
        Clin Infect Dis. 2008; 47: 1345-1353
        • Barry P.M.
        • Zetola N.
        • Keruly J.C.
        • Moore R.D.
        • Gebo K.A.
        • Lucas G.M.
        Invasive pneumococcal disease in a cohort of HIV-infected adults: incidence and risk factors, 1990–2003.
        AIDS. 2006; 20: 437-444
        • Foster D.
        • Knox K.
        • Walker A.S.
        • Griffiths D.T.
        • Moore H.
        • Haworth E.
        • et al.
        Oxford Invasive Pneumococcal Surveillance Group. Invasive pneumococcal disease: epidemiology in children and adults prior to implementation of the conjugate vaccine in the Oxfordshire region, England.
        J Med Microbiol. 2008; 57: 480-487
        • Grau I.
        • Pallares R.
        • Tubau F.
        • Schulze M.H.
        • Llopis F.
        • Podzamczer D.
        • et al.
        Spanish Pneumococcal Infection Study Network (G03/103). Epidemiologic changes in bacteremic pneumococcal disease in patients with human immunodeficiency virus in the era of highly active antiretroviral therapy.
        Arch Intern Med. 2005; 165: 1533-1540
        • Pedersen R.H.
        • Lohse N.
        • Østergaard L.
        • Søgaard O.S.
        The effectiveness of pneumococcal polysaccharide vaccination in HIV-infected adults: a systematic review.
        HIV Med. 2011; 12: 323-333
        • Kaplan J.E.
        • Benson C.
        • Holmes K.K.
        • Brooks J.T.
        • Pau A.
        • Masur H.
        • Centers for Disease Control and Prevention (CDC)
        • National Institutes of Health
        • HIV Medicine Association of the Infectious Diseases Society of America
        Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America.
        MMWR Recomm Rep. 2009; 58 (quiz CE201–4): 1-207
        • Falcó V.
        • Jordano Q.
        • Cruz M.J.
        • Len O.
        • Ribera E.
        • Campins M.
        • et al.
        Serological response to pneumococcal vaccination in HAART-treated HIV-infected patients: one year follow-up study.
        Vaccine. 2006; 24: 2567-2574
        • Søgaard O.S.
        • Schønheyder H.C.
        • Bukh A.R.
        • Harboe Z.B.
        • Rasmussen T.A.
        • Ostergaard L.
        • et al.
        Pneumococcal conjugate vaccination in persons with HIV: the effect of highly active antiretroviral therapy.
        AIDS. 2010; 24: 1315-1322
        • Crum-Cianflone N.F.
        • Huppler Hullsiek K.
        • Roediger M.
        • Ganesan A.
        • Patel S.
        • Landrum M.L.
        • et al.
        A randomized clinical trial comparing revaccination with pneumococcal conjugate vaccine to polysaccharide vaccine among HIV-infected adults.
        J Infect Dis. 2010; 202: 1114-1125
        • Madhi S.A.
        • Klugman K.P.
        • Kuwanda L.
        • Cutland C.
        • Käyhty H.
        • Adrian P.
        Quantitative and qualitative anamnestic immune responses to pneumococcal conjugate vaccine in HIV-infected and HIV-uninfected children 5 years after vaccination.
        J Infect Dis. 2009; 199: 1168-1176
        • Barry P.M.
        • Zetola N.
        • Keruly J.C.
        • Moore R.D.
        • Gebo K.A.
        • Lucas G.M.
        Invasive pneumococcal disease in a cohort of HIV-infected adults: incidence and risk factors, 1990–2003.
        AIDS. 2006; 20: 437-444
        • Dworkin M.S.
        • Ward J.W.
        • Hanson D.L.
        • Jones J.L.
        • Kaplan J.E.
        Pneumococcal disease among human immunodeficiency virus-infected persons: incidence, risk factors, and impact of vaccination.
        Clin Infect Dis. 2001; 32: 794-800
        • Yin Z.
        • Rice B.D.
        • Waight P.
        • Miller E.
        • George R.
        • Brown A.E.
        • et al.
        Invasive pneumococcal disease among HIV-positive individuals, 2000–2009.
        AIDS. 2012; 26: 87-94https://doi.org/10.1097/QAD.0b013e32834dcf27
        • Lane H.C.
        • Masur H.
        • Edgar L.C.
        • Whalen G.
        • Rook A.H.
        • Fauci A.S.
        Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome.
        N Engl J Med. 1983; 309: 453-458
        • Morris L.
        • Binley J.M.
        • Clas B.A.
        • Bonhoeffer S.
        • Astill T.P.
        • Kost R.
        • et al.
        HIV-1 antigen-specific and -nonspecific B cell responses are sensitive to combination antiretroviral therapy.
        J Exp Med. 1998; 188: 233-245
        • Hart M.
        • Steel A.
        • Clark S.A.
        • Moyle G.
        • Nelson M.
        • Henderson D.C.
        • et al.
        Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease.
        J Immunol. 2007; 178: 8212-8220
        • Shen X.
        • Tomaras G.D.
        Alterations of the B-cell response by HIV-1 replication.
        Curr HIV/AIDS Rep. 2011; 8 (73, http://dx.doi.org/10.1007/s11904-010-0064-2): 23-30
        • Moir S.
        • Buckner C.M.
        • Ho J.
        • Wang W.
        • Chen J.
        • Waldner A.J.
        • et al.
        B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy.
        Blood. 2010; 116: 5571-5579
        • Sanza I.
        • Weia C.
        • Leeb F.E.
        • Anolika J.
        Phenotypic and functional heterogeneity of human memory B cells.
        Semin Immunol. 2008; 20: 67-82https://doi.org/10.1016/j.smim.2007.12.006
        • Moir S.
        • Fauci A.S.
        Pathogenic mechanisms of B lymphocyte dysfunction in HIV disease.
        J Allergy Clin Immunol. 2008; 122: 12-21https://doi.org/10.1016/j.jaci.2008.04.034
        • Kruetzmann S.
        • Rosado M.M.
        • Weber H.
        • Germing U.
        • Tournilhac O.
        • Peter H.H.
        • et al.
        Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen.
        J Exp Med. 2003; 197: 939-945
        • De Milito A.
        • Morch C.
        • Sonnerborg A.
        • Chiodi F.
        Loss of memory (CD27) B lymphocytes in HIV-1 infection.
        AIDS. 2001; 15: 957-964
        • Moir S.
        • Ho J.
        • Malaspina A.
        • Wang W.
        • DiPoto A.C.
        • O'Shea M.A.
        • 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
        • Moir S.
        • Malaspina A.
        • Ogwaro K.M.
        • Donoghue E.T.
        • Hallahan C.W.
        • Ehler L.A.
        • et al.
        HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals.
        Proc Natl Acad Sci U S A. 2001; 98: 10362-10367
        • Chong Y.
        • Ikematsu H.
        • Kikuchi K.
        • Yamamoto M.
        • Murata M.
        • Nishimura M.
        • et al.
        Selective CD27+ (memory) B cell reduction and characteristic B cell alteration in drug-naive and HAART-treated HIV type 1-infected patients.
        AIDS Res Hum Retroviruses. 2004; 20: 219-226
        • Moir S.
        • Malaspina A.
        • Ho J.
        • Wang W.
        • Dipoto A.C.
        • O'Shea M.A.
        • et al.
        Normalization of B cell counts and subpopulations after antiretroviral therapy in chronic HIV disease.
        J Infect Dis. 2008; 197: 572-579
        • Moir S.
        • Fauci A.S.
        B cells in HIV infection and disease.
        Nat Rev Immunol. 2009; 9: 235-245
        • Amu S.
        • Ruffin N.
        • Rethi B.
        • Chiodi F.
        Impairment of B-cell functions during HIV-1 infection.
        AIDS. 2013; 27: 2323-2334
        • Titanji K.
        • De Milito A.
        • Cagigi A.
        • Thorstensson R.
        • Grutzmeier S.
        • Atlas A.
        • et al.
        Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection.
        Blood. 2006; 108: 1580-1587
        • Pensieroso S.
        • Galli L.
        • Nozza S.
        • Ruffin N.
        • Castagna A.
        • Tambussi G.
        • et al.
        B-cell subset alterations and correlated factors in HIV-1 infection.
        AIDS. 2013; 27: 1209-1217
        • Song J.Y.
        • Moseley M.A.
        • Burton R.L.
        • Nahm M.H.
        Pneumococcal vaccine and opsonic pneumococcal antibody.
        J Infect Chemother. 2013; 19: 412-425
        • Park S.
        • Nahm M.H.
        Older adults have a low capacity to opsonize pneumococci due to low IgM antibody response to pneumococcal vaccinations.
        Infect Immun. 2011; 79: 314-320
        • Parkkali T.
        • Väkeväinen M.
        • Käyhty H.
        • Ruutu T.
        • Ruutu P.
        Opsonophagocytic activity against Streptococcus pneumoniae type 19F in allogeneic BMT recipients before and after vaccination with pneumococcal polysaccharide vaccine.
        Bone Marrow Transplant. 2001; 27: 207-211
        • Shatz D.V.
        • Schinsky M.F.
        • Pais L.B.
        • Romero-Steiner S.
        • Kirton O.C.
        • Carlone G.M.
        Immune responses of splenectomized trauma patients to the 23-valent pneumococcal polysaccharide vaccine at 1 versus 7 versus 14 days after splenectomy.
        J Trauma. 1998; 44: 760-765
        • Nunes M.C.
        • Madhi S.A.
        Safety, immunogenicity and efficacy of pneumococcal conjugate vaccine in HIV-infected individuals.
        Hum Vaccines Immunother. 2012; 8: 161-173
        • Feikin D.R.
        • Elie C.M.
        • Goetz M.B.
        • Lennox J.L.
        • Carlone G.M.
        • Romero-Steiner S.
        • et al.
        Randomized trial of the quantitative and functional antibody responses to a 7-valent pneumococcal conjugate vaccine and/or 23-valent polysaccharide vaccine among HIV-infected adults.
        Vaccine. 2001; 20: 545-553
        • Siber G.R.
        • Chang I.
        • Baker S.
        • Fernsten P.
        • O’Brien K.L.
        • Santosham M.
        • et al.
        Estimating the protective concentration of anti-pneumococcal capsular polysaccharide antibodies.
        Vaccine. 2007; 25: 3816-3826
        • Wernette C.M.
        • Frasch C.E.
        • Madore D.
        • Carlone G.
        • Goldblatt D.
        • Plikaytis B.
        • et al.
        Enzyme-linked immunosorbent assay for quantitation of human antibodies to pneumococcal polysaccharides.
        Clin Diagn Lab Immunol. 2003; 10: 514-519
      1. ELISA Manufacturers Manual. Birmingham, UK: The Binding Site Ltd, 2011. http://www.quimiolab.com/userfiles/Docuemtnos%20Anexos/Binding%20Site/MK012.pdf/ [accessed 13.06.12].

        • Kernéis S.
        • Launay O.
        • Turbelin C.
        • Batteux F.
        • Hanslik T.
        • Boëlle P.Y.
        Long-term immune responses to vaccination in HIV-infected patients: a systematic review and meta-analysis.
        Clin Infect Dis. 2014; 58: 1130-1139https://doi.org/10.1093/cid/cit937
        • Glennie S.J.
        • Banda D.
        • Gould K.
        • Hinds J.
        • Kamngona A.
        • Everett D.D.
        • et al.
        Defective pneumococcal-specific Th1 responses in HIV-infected adults precedes a loss of control of pneumococcal colonization.
        Clin Infect Dis. 2013; 56: 291-299
        • Johannesson T.G.
        • Søgaard O.S.
        • Tolstrup M.
        • Petersen M.S.
        • Bernth-Jensen J.M.
        • Østergaard L.
        • et al.
        The impact of B-cell perturbations on pneumococcal conjugate vaccine response in HIV-infected adults.
        PLoS One. 2012; 7: e42307
        • Amendola A.
        • Tanzi E.
        • Zappa A.
        • Colzani D.
        • Boschini A.
        • Musher D.M.
        • et al.
        Safety and immunogenicity of 23-valent pneumococcal polysaccharide vaccine in HIV-1-infected former drug users.
        Vaccine. 2002; 20: 3720-3724
        • Rodriguez-Barradas M.C.
        • Alexandraki I.
        • Nazir T.
        • Foltzer M.
        • Musher D.M.
        • Brown S.
        • et al.
        Response of human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy to vaccination with 23-valent pneumococcal polysaccharide vaccine.
        Clin Infect Dis. 2003; 37: 438-447
        • Falcó V.
        • Jordano Q.
        • Cruz M.J.
        • Len O.
        • Ribera E.
        • Campins M.
        • et al.
        Serological response to pneumococcal vaccination in HAART-treated HIV-infected patients: one year follow-up study.
        Vaccine. 2006; 24: 2567-2574
        • Subramaniam K.S.
        • Segal R.
        • Lyles R.H.
        • Rodriguez-Barradas M.C.
        • Pirofski L.A.
        Qualitative change in antibody responses of human immunodeficiency virus-infected individuals to pneumococcal capsular polysaccharide vaccination associated with highly active antiretroviral therapy.
        J Infect Dis. 2003; 187: 758-768
        • Teshale E.H.
        • Hanson D.
        • Flannery B.
        • Phares C.
        • Wolfe M.
        • Schuchat A.
        • et al.
        Effectiveness of 23-valent polysaccharide pneumococcal vaccine on pneumonia in HIV-infected adults in the United States, 1998–2003.
        Vaccine. 2008; 26: 5830-5834
        • Moir S.
        • Ogwaro K.M.
        • Malaspina A.
        • Vasquez J.
        • Donoghue E.T.
        • Hallahan C.W.
        • et al.
        Perturbations in B cell responsiveness to CD4 T cell help in HIV-infected individuals.
        Proc Natl Acad Sci U S A. 2003; 100: 6057-6062
        • Weller S.
        • Braun M.C.
        • Tan B.K.
        • Rosenwald A.
        • Cordier C.
        • Conley M.E.
        • et al.
        Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire.
        Blood. 2004; 104: 3647-3654
        • Shi Y.
        • Yamazaki T.
        • Okubo Y.
        • Uehara Y.
        • Sugane K.
        • Agematsu K.
        Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell.
        J Immunol. 2005; 175: 3262-3267
        • Takizawa M.
        • Sugane K.
        • Agematsu K.
        Role of tonsillar IgD+CD27+ memory B cells in humoral immunity against pneumococcal infection.
        Hum Immunol. 2006; 67: 966-975
        • Khaskhely N.
        • Mosakowski J.
        • Thompson R.S.
        • Khuder S.
        • Smithson S.L.
        • Westerink M.A.
        Phenotypic analysis of pneumococcal polysaccharide-specific B cells.
        J Immunol. 2012; 188: 2455-2463https://doi.org/10.4049/jimmunol.1102809
        • Sciaranghella G.
        • Tong N.
        • Mahan A.E.
        • Suscovich T.J.
        • Alter G.
        Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection.
        AIDS. 2013; 27: 175-180