Immunogenicity and Immunological Memory Induced by the 13-Valent Pneumococcal Conjugate Followed by the 23-Valent Polysaccharide Vaccine in HIV-Infected Adults

Immunogenicity and Immunological Memory Induced by the 13-Valent Pneumococcal Conjugate Followed... Abstract Background Vaccine-induced memory B-cell (MBC) subsets have distinct roles in the establishment of protective immunity; MBCs expressing nonswitched immunoglobulin M (IgM+ MBCs) replenish the MBC pool, whereas MBCs expressing isotype-switched immunoglobulin (sIg+ MBCs) differentiate into plasma cells upon antigen reencounter. We investigated immunogenicity and MBCs induced by combined 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPV23) in human immunodeficiency virus (HIV)–infected adults. Methods Forty HIV-seropositive adults receiving ART with undetectable viral loads were enrolled. Seventeen had a CD4+ T-cell count of ≥400 cells/μL (group A), and 23 had a CD4+ T-cell count of 200–399 cells/μL (group B). All adults received PCV13 and, 1 year later, PPV23. Levels of IgM+ MBCs (defined as polysaccharide [PS]–specific CD19+CD10−CD27+CD21++IgM+ MBCs) and sIg+ MBCs (defined as PS-specific CD19+CD10−CD27+CD21++IgM− MBCs) and antibodies against PS14 and PS3 were measured prior and 1 month after each vaccination. Results Immunization caused a significant increase in PS antibodies, compared with levels at baseline (P < .001). Group B achieved significantly lower titers than group A (P < .05 for both PS14 and PS3). After receipt of PCV13, levels of IgM+ MBCs were unchanged, whereas levels of sIg+ MBCs increased significantly (P < .05 for PS14 and P < .001 for PS3). In contrast, following PPV23 receipt, levels of IgM+ MBCs were significantly reduced, and levels of sIg+ MBCs remained stable. A positive correlation was observed between baseline IgM+ and sIg+ MBC counts 1 month after PCV13 receipt but not after PPV23 receipt. Conclusions PPV23 receipt 12 months after PCV13 receipt improved PCV13 immunogenicity. The reduction in the IgM+ MBC count observed after PPV23 receipt suggests that PPV23 has a depleting effect on PCV13-associated immunological memory. Clinical Trials Registration NCT03041051. Immunological memory, HIV, pneumococcal vaccination, memory B cells Streptococcus pneumoniae remains a leading cause of serious bacterial infections in human immunodeficiency virus (HIV)–infected individuals, even in the era of effective antiretroviral therapy (ART) [1]. Two types of pneumococcal vaccines, with different immunological characteristics and numbers of pneumococcal serotypes targeted, are currently in use [2]. The 23-valent pneumococcal polysaccharide vaccine (PPV23) induces a T-cell–independent, exclusively humoral immune response resulting in PS-specific antibody formation. On the other hand, the 13-valent conjugate pneumococcal vaccine (PCV13) induces a T-cell–dependent immune response resulting in the formation of both pneumococcal serotype(PS)–specific antibodies and memory B cells (MBCs), thus establishing immunological memory, which is considered as an important correlate of vaccine effectiveness and long-lasting protection [3, 4]. Long-term protection, which is the goal of successful immunization, is thought to rely both on protective serum antibody levels and immunological memory [5, 6]. Although the mechanisms responsible for the induction and establishment of immunological memory are not fully understood, it has been postulated that memory responses stimulate and produce different MBC subpopulations, depending on the type of vaccine [4, 7]. Evidence coming from murine studies identifies 2 MBC subsets, MBCs expressing nonswitched immunoglobulin M (IgM+ MBCs) and those expressing isotype–switched immunoglobulin (sIg+ MBCs), with distinct roles in the induction of long-lasting immunity [8]. More specifically, sIg+ MBCs seem to differentiate rapidly into antibody-secreting plasma cells upon encountering a T-cell–dependent or –independent antigen, while IgM+ MBCs follow different pathways, depending on the type of antigen. Specifically, T-cell–independent antigens lead IgM+ MBCs to terminal differentiation, whereas T-cell–dependent antigens compel IgM+ MBCs to reenter germinal centers and enrich the MBC pool by producing new MBCs [6, 8, 9]. Guidelines regarding the optimal vaccination schedule for PPV23 in HIV-seropositive individuals are still under debate [10, 11]. The rationale of a combined PCV13/PPV23 vaccination schedule is based on the potential benefit of broad immunological memory with maximum serotype coverage. However, despite its extensive use, PPV23 has been associated with impaired responses to subsequent vaccinations, a phenomenon known as “hyporesponsiveness,” suggesting a negative effect on PS-specific immunological memory [3, 12, 13]. The investigation of vaccine-induced immunological memory, although important for optimization of immunization schedules, has not been studied in HIV-positive subjects. In the present study, we investigated the effect of 1 dose of PPV23 on PCV13-induced immunogenicity and immunological memory in a cohort of HIV-infected adults receiving ART, to evaluate whether the combined schedule PCV13/PPV23 could confer the best possible protection in this high-risk population. METHODS Participants and Study Design Forty HIV-infected adults (of whom 35 were men) who were receiving ART and had no history of previous PCV13 immunization were enrolled while undergoing follow-up at the Department of Infectious Diseases, Korgialeneio-Benakeio Hospital (Athens, Greece). Patients were excluded from the study if they had a CD4+ T-cell counts of <200 cells/μL, an HIV load of >50 copies/mL, and/or other causes of immunosuppression, such as coinfections and cancer, or if they had received PPV23 within the last year. Ethical approval for the study was obtained from the hospital’s ethics committee, and written informed consent was obtained from all participants prior to study enrollment. The study was registered at Clinicaltrials.gov (registration NCT03041051). Participants were stratified in 2 groups according to CD4+ T-cell count at baseline: group A comprised 17 participants with a CD4+ T-cell count of ≥400 cells/μL, and group B comprised 23 participants with a CD4+ T-cell count of 200–399 cells/μL. Twelve patients had never received PPV23, while 28 had received up to 3 doses of PPV23. The interval from receipt of the most recent PPV23 dose to enrollment ranged from 1 to 5 years. All patients received 1 dose of PCV13 (Prevenar 13; Pfizer Inc., Pearl River), followed 12 months later by 1 dose of PPV23 (Pneumovax 23, MSD, Dublin, Ireland). Vaccination was administrated by intramuscular injection into the deltoid muscle of the nondominant arm. Blood samples were obtained before and 1 month after each vaccination. Preparation of Peripheral Blood Mononuclear Cells (PBMCs) and Serum A maximum volume of 12 mL of heparinized blood was available for the isolation of PBMCs by density gradient centrifugation with Lymphosep lymphocyte separation medium (Biosera Nualle, France). An additional blood specimen (volume, 2 mL) was collected, and serum was separated, frozen, and stored at −20oC until tested. Flow Cytometry and Pneumococcal Polysaccharide Enzyme-Linked Immunosorbent Analysis (ELISA) PS-specific MBCs in a population of fresh PBMCs were identified using biotinylated PS antigens loaded on anti–biotin-coated beads, as previously described [3]. Briefly, anti–biotin-coated beads (MACSIbead, Miltenyi Biotec, Bergisch Gladbach, Germany) were incubated with biotinylated PS antigens (either PS14 or PS3) for 2 hours at room temperature. PS-loaded beads were subsequently washed and, after incubation for 5 minutes at 4°C–8°C, were labeled with anti–biotin-FITC conjugate. Based on side-scatter and forward-scatter measurements, gating was performed to include all viable cells in the cell population. The specificity of the PS-bound B cells was validated by use of appropriate controls. Negative controls of single anti–biotin-coated beads conjugated with FITC or APC antibody were used in the assay. Analyses of anti–biotin-coated beads coated with biotinylated antigens and then tagged with either anti–biotin-FITC or anti–biotin-APC antibody were performed and compared to negative controls. Freshly collected PBMCs were then incubated for 10 minutes at room temperature with either FITC-labeled PS-coated beads or unlabelled beads as a negative control, as well as with a combination of 21-PE (clone BL13), CD10-PC5.5 (cloneALB1), CD19-PC7 (cloneJ3.119), anti-IgM-APC (clone SA-DA4), and CD27-APC Alexafluor750 (clone1A4CD27; all from Beckman Coulter, Immunotech, Marseille, France). Ammonium chloride was used to lyse red blood cells, after which remaining cells were washed and analyzed by flow cytometry, using a 2-laser Navios instrument (Beckman Coulter). Analysis of FCS files was performed with Navios or Kaluza1.2 software. B-cell subsets were defined as follows: naive B cells, D19+CD10−CD21++CD27−; sIg+ MBCs, CD19+CD10−CD21++ CD27+IgM−; IgM+ MBCs, CD19+CD10−CD21++CD27+IgM+; and exhausted B cells, CD19+CD10−CD21low. Subset analysis was performed both on the total population of B cells and on PS-specific B cells, with regions defined for the former group used to define regions of the latter group (Figure 1). Figure 1. View largeDownload slide Basic gating strategy for the identification of polysaccharide (PS)–specific B-cell subsets. A, B cells were initially identified as low CD19+ side-scatter (SS) signals. B and C, PS positivity cutoff for B cells (B) was determined with the help of the simultaneously analyzed unstained bead control (C). D and E, After gating out CD10+ signals (D), naive, memory, and exhausted PS+ B-cell subsets were defined on the basis of their CD21 vs CD27 expression pattern (E). F, Further segregation of memory PS+ B cells in switched and nonswitched conditions was based on surface immunoglobulin M (IgM) expression. G, Analysis of the above markers on total B cells was performed to help define the respective regions for the PS-specific B cells. Figure 1. View largeDownload slide Basic gating strategy for the identification of polysaccharide (PS)–specific B-cell subsets. A, B cells were initially identified as low CD19+ side-scatter (SS) signals. B and C, PS positivity cutoff for B cells (B) was determined with the help of the simultaneously analyzed unstained bead control (C). D and E, After gating out CD10+ signals (D), naive, memory, and exhausted PS+ B-cell subsets were defined on the basis of their CD21 vs CD27 expression pattern (E). F, Further segregation of memory PS+ B cells in switched and nonswitched conditions was based on surface immunoglobulin M (IgM) expression. G, Analysis of the above markers on total B cells was performed to help define the respective regions for the PS-specific B cells. The World Health Organization ELISA protocol was used for the detection of anti-PS immunoglobulin G (IgG) antibodies in serum samples. Statistical Analysis Quantitative variables were expressed as mean values (±SD). Repeated-measures analysis of variance was adopted to evaluate the changes observed in IgM+ MBCs, sIg+ MBCs, and IgG antibody values over the follow-up period for the total sample and by baseline CD4+ T-cell count (ie, between groups A and B). Pearson correlation coefficients were used to explore associations between 2 continuous variables. Correlation coefficients between 0.1 and 0.3 were considered low, those between 0.31 and 0.5 were considered moderate, and those >0.5 were considered high. Univariate and multiple linear regression analyses were used, with antibody concentrations at 13 months serving as the outcome variable. The regression equation included terms for CD4+ T cell counts and IgG titers at baseline. Adjusted regression coefficients (β), with standard errors (SEs), and standardized regression coefficients (b) were computed from the results of linear regression analyses. All reported P values are 2-tailed. Statistical significance was set at a P value of <.05, and analyses were conducted using SPSS statistical software (version 19.0). RESULTS Kinetics of PS-Specific IgG Antibodies Following PCV13 and PPV23 Receipt The characteristics of study participants are shown in Table 1. We evaluated the kinetics of antibodies against PS14 and PS3, which are common to both vaccines but differ in immunogenicity, with PS3 being the least immunogenic serotype [14]. Overall, the combined vaccination schedule with PCV13 and PPV23 induced a significant increase in PS-specific IgG antibody concentrations for both serotypes, compared with baseline (P < .001). One month following PCV13 receipt, a 2-fold rise in IgG antibody levels was observed (P < .001), with levels increased further after PPV23, albeit to a lower degree (P = .02). Group B patients had up to 40% lower antibody concentrations than group A patients for both serotypes at all time points (P < .001) and did not achieve the protective threshold of 0.35 μg/mL for PS3 following PCV13 receipt (Table 2). In addition, a low baseline CD4+ T-cell count had a significant negative effect on the magnitude of the change in antibody kinetics through the study period (P < .001). No statistically significant correlation was found between immune response to either vaccine and other demographic characteristics. Table 1. Demographic Characteristics of Human Immunodeficiency Virus–Infected Adults, Overall and by Study Group Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Abbreviation: PPV23, 23-valent pneumococcal polysaccharide vaccine. aGroup A comprised 17 participants with a CD4+ T-cell count of ≥400 cells/μL, and group B comprised 23 participants with a CD4+ T-cell count of 200–399 cells/μL. View Large Table 1. Demographic Characteristics of Human Immunodeficiency Virus–Infected Adults, Overall and by Study Group Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Abbreviation: PPV23, 23-valent pneumococcal polysaccharide vaccine. aGroup A comprised 17 participants with a CD4+ T-cell count of ≥400 cells/μL, and group B comprised 23 participants with a CD4+ T-cell count of 200–399 cells/μL. View Large Table 2. Kinetics of (a) PS-specific IgG antibody concentrations (μg/ml) and (b) IgM and sIg+ MBC (cells/106 PBMC) in HIV-infected adults vaccinated with PCV13/PPV23   Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001          Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001        aP-value for time effect. bRepeated measurements ANOVA. Effects reported include differences between the groups in the degree of change over the follow-up period. cIndicates significant difference from prior measurement. dP-value for group effect. View Large Table 2. Kinetics of (a) PS-specific IgG antibody concentrations (μg/ml) and (b) IgM and sIg+ MBC (cells/106 PBMC) in HIV-infected adults vaccinated with PCV13/PPV23   Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001          Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001        aP-value for time effect. bRepeated measurements ANOVA. Effects reported include differences between the groups in the degree of change over the follow-up period. cIndicates significant difference from prior measurement. dP-value for group effect. View Large MBC Responses Following PCV13 and PPV23 Before vaccination, all study participants had detectable PS-specific IgM+ MBCs and sIg+ MBCs regardless of previous pneumococcal vaccination history. The PS-specific IgM+ MBC count was notably reduced for both serotypes, compared with baseline, after completion of the vaccination schedule (P < .001). More specifically, although their levels were not affected by PCV13 receipt and remained stable during the following 12 months, PPV23 caused a significant reduction in the IgM+ MBC pool (P < .001; Table 2). In contrast, combined vaccination enriched the PS-specific sIg+ MBC pool (P < .001), mainly because of a significant increase following PCV13 receipt. However, after PPV23 receipt there were no significant changes, and at the end of follow-up period sIg+ MBC counts remained above baseline levels for both serotypes. Group A patients had significantly higher MBC counts than group B patients at baseline (MBC count, 42 574 cells/μL vs 39 609 cells/μL; IgM+ MBC count, 5624 cells/μL vs 4132 cells/μL; sIg+ MBC count, 4527 cells/μL vs 2529 cells/μL) and at all points at which blood specimens were obtained (P < .05 for all comparisons; Table 2). Moreover, the magnitude of the increase in the sIg+ MBC count was positively correlated with baseline CD4+ T-cell levels for both serotypes (r = 0.38 and P = .016 for PS14; r = 0.67 and P < .001 for PS3; Table 2). Correlations between MBC responses at baseline and 1 month after vaccination were different for each vaccine formulation. More specifically, while baseline PS-specific IgM+ MBC counts were significantly correlated with sIg+ MBC counts after PCV13 receipt (r = 0.78 and P < .001 for PS14; r = 0.57 and P < .001 for PS3), such correlation was not observed following PPV23 receipt (r = −0.10 and P = .7 for PS14; r = 0.2 and P = .7 for PS3; Figure 2). Figure 2. View largeDownload slide Pearson correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). Circles denote values for PS14, and diamonds denote values for PS3. Figure 2. View largeDownload slide Pearson correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). Circles denote values for PS14, and diamonds denote values for PS3. In contrast, correlations between baseline MBC subset counts and humoral responses were not vaccine dependent. All study subjects had baseline IgM+ and sIg+ MBC counts that were positively correlated with antibody levels at 1 month, induced by either vaccine and for both serotypes. Baseline IgΜ+ MBC counts and sIg+ MBC counts were significantly correlated with IgG antibody levels 1 month after PCV13 receipt (P < .001 for responses to PS14 and PS3). Similarly after PPV23 receipt, baseline IgΜ+ MBC counts and sIg+ MBC counts were significantly correlated with IgG antibody titers at 1 month (P < .001 for responses to PS14 and PS3; Figure 3). However, such correlations failed to reach statistical significance when the analysis was conducted separately for each group, probably because of the small number of patients. Figure 3. View largeDownload slide Spearman correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). A, For analysis of the IgM+ MBC count before PCV13 receipt vs the IgG antibody level 1 month after receipt: r = 0.62 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PCV13 receipt vs the IgG antibody 1 month after PCV13 receipt, r = 0.60 and P < .001 for PS14 and r = 0.63, P < .001 for PS3. B, For analysis of the IgM+ MBC count before PPV23 receipt the IgG antibody level 1 month after PPV23 receipt, r = 0.56 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PPV23 receipt vs the IgG antibody titer 1 month after PPV23 receipt, r = 0.64 and P < .001 for PS14, and r = 0.60 and P < .001 for PS3. Circles denote values for PS3, and diamonds denote values for P14. Figure 3. View largeDownload slide Spearman correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). A, For analysis of the IgM+ MBC count before PCV13 receipt vs the IgG antibody level 1 month after receipt: r = 0.62 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PCV13 receipt vs the IgG antibody 1 month after PCV13 receipt, r = 0.60 and P < .001 for PS14 and r = 0.63, P < .001 for PS3. B, For analysis of the IgM+ MBC count before PPV23 receipt the IgG antibody level 1 month after PPV23 receipt, r = 0.56 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PPV23 receipt vs the IgG antibody titer 1 month after PPV23 receipt, r = 0.64 and P < .001 for PS14, and r = 0.60 and P < .001 for PS3. Circles denote values for PS3, and diamonds denote values for P14. DISCUSSION This is the first study to investigate the effect of a combined vaccination schedule of PCV13 followed 1 year later by PPV23 on the immunogenicity and PS-specific immunological memory induced by PCV13 in HIV-infected adults receiving ART. Despite the relatively small study size, our findings indicate that PPV23 enhanced PCV13 immunogenicity but had a negative effect on PCV13-induced immunological memory by reducing the IgM+ MBC subpopulation. One month after receipt of 1 dose of PPV23, there was a significant increase in circulating PS-specific IgG antibody concentrations. The added value of additional doses of pneumococcal vaccines in HIV-infected subjects has not been consistently demonstrated in previous studies [15–21]. Abzug et al reported that a combined schedule of 2 doses of PCV7 followed 8 weeks later by 1 dose PPV23 induced only a slight increase in antibody levels [22]. Similar findings were reported even when an additional dose of PPV23 was given 6 months after the first PPV23 dose, which could be attributed to the increased levels of circulating plasma cells and antibodies that can block the formation of germinal centers when a subsequent dose is given at very short intervals [23, 24]. Our study provides further evidence that longer intervals between pneumococcal vaccinations are beneficial for vaccine immunogenicity [25, 26]. Similar to previous studies, we have shown that the magnitude of the immune response to both vaccines was associated with the degree of immunodeficiency, expressed as the baseline CD4+ T-cell count, in seropositive patients [27]. Especially for PS3, which is a common cause of PCV13 failure in healthy subjects [28], patients with low baseline CD4+ T-cell counts did not achieve even the protective threshold of 0.35 μg/mL, a finding suggesting that seropositive patients vaccinated with 1 dose of PCV13 are probably not protected against PS3. The generation of long-lived antigen-specific memory is regarded as the cornerstone of successful immunization [4, 6]. Our study provides important new understanding regarding the phenotype of PS-specific MBCs after receipt of conjugate and plain-polysaccharide pneumococcal vaccines. Immunization with PCV13 induced new PS-specific sIg+ MBCs, whereas the IgM+ MBC count remained unchanged. Similar findings were reported by Papadatou et al in a study of 35 asplenic adults with β-thalassemia major, in which kinetics were evaluated after participants were vaccinated with PCV13 [29]. In contrast, PPV23 did not induce any changes in sIg+ MBC counts but caused a significant reduction in IgM+ MBC counts (by 20% and 40% for PS14-specific and PS3-specific IgM+ MBCs, respectively). The divergent effects of conjugate and polysaccharide pneumococcal vaccines on antigen-specific MBCs have also been reported mainly in adults aged >65 years [3, 30]. The detection of new sIg+ MBCs 1 month after PCV13 receipt is in line with the T-cell–dependent nature of the immune response to the conjugate vaccine, which has the ability to enrich the polysaccharide-specific MBC pool through germinal cell formation [8]. Similar results have been previously reported not only following receipt of PCV13 but also after receipt of meningococcal C conjugate vaccine [29, 31]. Interestingly, the IgM+ MBC count remained stable following PCV13 receipt, while vaccination with PPV23 led to a significant decrease in this subpopulation. Since both MBC subsets are expressed as absolute cell counts at all time points, we believe that the decrease in the IgM+ MBC count following PPV23 is true and does not reflect an increase in the relative proportion of sIg+ MBCs. Our findings are in keeping with the hypothesis that T-cell–independent antigens drive preexisting MBCs into terminal differentiation without replenishment of the MBC pool, owing to the lack of germinal center formation [8]. Moreover, the positive correlation between the baseline IgM+ MBC count and the sIg+ MBC count 1 month following PCV13 receipt suggests that a part of the preexisting IgM+ MBC population reenters germinal centers and differentiates into sIg+ MBCs, providing further support for germinal center formation following T-cell–dependent stimulation [7]. Taking into account that IgM+ MBCs survive for longer periods than sIg+ MBCs and are thought to mediate late memory responses when levels of sIg+ MBCs and antibodies are reduced [24, 32], the observed reduction of this subpopulation could have biological significance for subjects with advanced HIV disease, as shown in an early study performed in HIV-infected adults not receiving ART, in which a surprising excess of PPV23 recipients developed pneumococcal disease as compared to placebo recipients [33]. Additional studies investigating the association between PPV23-induced IgM+ MBC depletion and differences in Ig class and subclass antibody responses and serum bactericidal activity, as well as the long-term kinetics of IgM or IgG antibody levels, will provide further evidence for the biological significance of our finding to HIV infection. Whether baseline memory can predict the magnitude of the antibody response after vaccination remains debatable, and results from relevant studies of healthy and immunocompromised populations are conflicting [29, 34–36]. We found a consistent, positive correlation between baseline MBC subsets and antibody levels 1 month after receipt of either PCV13 or PPV23. The correlation, however, did not reach statistical significance when the analysis was conducted separately for each group of patients, probably because of the small study size. It is possible that differences between healthy and immunocompromised subjects could be attributed to the existence of additional cell populations in healthy individuals that are also involved in the mechanisms of long-lasting immunological memory, such as long-lived IgM+ plasma cells, which could be impaired in immunocompromised subjects [37]. Although we did not include a control group to compare the effect of the polysaccharide vaccine in healthy subjects, similar findings in healthy adults were reported recently by O’Connor et al [38]. They compared B-cell responses to conjugate and polysaccharide meningococcal vaccines by means of a number of different methods, including gene expression microarray, and demonstrated distinct plain-polysaccharide gene signatures, possibly linked to hyporesponsiveness, associated with depletion of switched B memory cells. In conclusion, our study highlights the divergent effects of conjugate and polysaccharide vaccines in the establishment of immunological memory in HIV-infected patients and supports BHIVA, EACS, and PENTA guidelines, which recommend PCV13 instead of PPV23 for HIV-seropositive subjects [11, 39–41]. Most seropositive4 subjects now receive ART, which can reverse most B-cell defects, with the exception of IgM+ MBC loss [42]; therefore, questions may arise regarding the use of PPV23 in seropositive subjects with decreased IgM+ MBC counts. Further evidence of the significance of the decline of IgM+ MBC counts, provided by studies of the long-term kinetics of IgM and IgG antibody levels following PPV23 vaccination, will be useful for reevaluation of current recommendations of pneumococcal vaccination in these high-risk individuals. Notes Acknowledgments.  We thank Dr Elizabeth Clutterbuck, Oxford Vaccine Group, Department of Paediatrics, University of Oxford, and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom, for providing us the pneumococcal serotype biotinylation protocol. All authors have substantially contributed to the submitted work. P. F. codesigned and performed the research; collected, analyzed, and interpreted the data; and wrote the manuscript. M. T. and C. P. provided expertise and supervision in the performance of the study and edited the manuscript. M. C., N. M., and M. L. codesigned the study, collected data, and followed up the study participants. V. S. codesigned the research and edited the manuscript. Financial support. This work was supported by the Hellenic Center for Disease Control and Prevention (grant 51657) and the State Scholarship Foundation of Greece–Siemens Program (Research Projects for Excellence grant 254845). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Presented in part: 35th Annual Meeting of the European Society for Paediatric Infectious Diseases, Madrid, Spain, 23–27 May 2017 (abstract ESP17-1122); Meeting of the European Society for Clinical Cell Analysis, Thessaloniki, Greece, 24–27 September 2017 (abstract 99). References 1. 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Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Immunogenicity and Immunological Memory Induced by the 13-Valent Pneumococcal Conjugate Followed by the 23-Valent Polysaccharide Vaccine in HIV-Infected Adults

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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1537-6613
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10.1093/infdis/jiy135
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Abstract

Abstract Background Vaccine-induced memory B-cell (MBC) subsets have distinct roles in the establishment of protective immunity; MBCs expressing nonswitched immunoglobulin M (IgM+ MBCs) replenish the MBC pool, whereas MBCs expressing isotype-switched immunoglobulin (sIg+ MBCs) differentiate into plasma cells upon antigen reencounter. We investigated immunogenicity and MBCs induced by combined 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPV23) in human immunodeficiency virus (HIV)–infected adults. Methods Forty HIV-seropositive adults receiving ART with undetectable viral loads were enrolled. Seventeen had a CD4+ T-cell count of ≥400 cells/μL (group A), and 23 had a CD4+ T-cell count of 200–399 cells/μL (group B). All adults received PCV13 and, 1 year later, PPV23. Levels of IgM+ MBCs (defined as polysaccharide [PS]–specific CD19+CD10−CD27+CD21++IgM+ MBCs) and sIg+ MBCs (defined as PS-specific CD19+CD10−CD27+CD21++IgM− MBCs) and antibodies against PS14 and PS3 were measured prior and 1 month after each vaccination. Results Immunization caused a significant increase in PS antibodies, compared with levels at baseline (P < .001). Group B achieved significantly lower titers than group A (P < .05 for both PS14 and PS3). After receipt of PCV13, levels of IgM+ MBCs were unchanged, whereas levels of sIg+ MBCs increased significantly (P < .05 for PS14 and P < .001 for PS3). In contrast, following PPV23 receipt, levels of IgM+ MBCs were significantly reduced, and levels of sIg+ MBCs remained stable. A positive correlation was observed between baseline IgM+ and sIg+ MBC counts 1 month after PCV13 receipt but not after PPV23 receipt. Conclusions PPV23 receipt 12 months after PCV13 receipt improved PCV13 immunogenicity. The reduction in the IgM+ MBC count observed after PPV23 receipt suggests that PPV23 has a depleting effect on PCV13-associated immunological memory. Clinical Trials Registration NCT03041051. Immunological memory, HIV, pneumococcal vaccination, memory B cells Streptococcus pneumoniae remains a leading cause of serious bacterial infections in human immunodeficiency virus (HIV)–infected individuals, even in the era of effective antiretroviral therapy (ART) [1]. Two types of pneumococcal vaccines, with different immunological characteristics and numbers of pneumococcal serotypes targeted, are currently in use [2]. The 23-valent pneumococcal polysaccharide vaccine (PPV23) induces a T-cell–independent, exclusively humoral immune response resulting in PS-specific antibody formation. On the other hand, the 13-valent conjugate pneumococcal vaccine (PCV13) induces a T-cell–dependent immune response resulting in the formation of both pneumococcal serotype(PS)–specific antibodies and memory B cells (MBCs), thus establishing immunological memory, which is considered as an important correlate of vaccine effectiveness and long-lasting protection [3, 4]. Long-term protection, which is the goal of successful immunization, is thought to rely both on protective serum antibody levels and immunological memory [5, 6]. Although the mechanisms responsible for the induction and establishment of immunological memory are not fully understood, it has been postulated that memory responses stimulate and produce different MBC subpopulations, depending on the type of vaccine [4, 7]. Evidence coming from murine studies identifies 2 MBC subsets, MBCs expressing nonswitched immunoglobulin M (IgM+ MBCs) and those expressing isotype–switched immunoglobulin (sIg+ MBCs), with distinct roles in the induction of long-lasting immunity [8]. More specifically, sIg+ MBCs seem to differentiate rapidly into antibody-secreting plasma cells upon encountering a T-cell–dependent or –independent antigen, while IgM+ MBCs follow different pathways, depending on the type of antigen. Specifically, T-cell–independent antigens lead IgM+ MBCs to terminal differentiation, whereas T-cell–dependent antigens compel IgM+ MBCs to reenter germinal centers and enrich the MBC pool by producing new MBCs [6, 8, 9]. Guidelines regarding the optimal vaccination schedule for PPV23 in HIV-seropositive individuals are still under debate [10, 11]. The rationale of a combined PCV13/PPV23 vaccination schedule is based on the potential benefit of broad immunological memory with maximum serotype coverage. However, despite its extensive use, PPV23 has been associated with impaired responses to subsequent vaccinations, a phenomenon known as “hyporesponsiveness,” suggesting a negative effect on PS-specific immunological memory [3, 12, 13]. The investigation of vaccine-induced immunological memory, although important for optimization of immunization schedules, has not been studied in HIV-positive subjects. In the present study, we investigated the effect of 1 dose of PPV23 on PCV13-induced immunogenicity and immunological memory in a cohort of HIV-infected adults receiving ART, to evaluate whether the combined schedule PCV13/PPV23 could confer the best possible protection in this high-risk population. METHODS Participants and Study Design Forty HIV-infected adults (of whom 35 were men) who were receiving ART and had no history of previous PCV13 immunization were enrolled while undergoing follow-up at the Department of Infectious Diseases, Korgialeneio-Benakeio Hospital (Athens, Greece). Patients were excluded from the study if they had a CD4+ T-cell counts of <200 cells/μL, an HIV load of >50 copies/mL, and/or other causes of immunosuppression, such as coinfections and cancer, or if they had received PPV23 within the last year. Ethical approval for the study was obtained from the hospital’s ethics committee, and written informed consent was obtained from all participants prior to study enrollment. The study was registered at Clinicaltrials.gov (registration NCT03041051). Participants were stratified in 2 groups according to CD4+ T-cell count at baseline: group A comprised 17 participants with a CD4+ T-cell count of ≥400 cells/μL, and group B comprised 23 participants with a CD4+ T-cell count of 200–399 cells/μL. Twelve patients had never received PPV23, while 28 had received up to 3 doses of PPV23. The interval from receipt of the most recent PPV23 dose to enrollment ranged from 1 to 5 years. All patients received 1 dose of PCV13 (Prevenar 13; Pfizer Inc., Pearl River), followed 12 months later by 1 dose of PPV23 (Pneumovax 23, MSD, Dublin, Ireland). Vaccination was administrated by intramuscular injection into the deltoid muscle of the nondominant arm. Blood samples were obtained before and 1 month after each vaccination. Preparation of Peripheral Blood Mononuclear Cells (PBMCs) and Serum A maximum volume of 12 mL of heparinized blood was available for the isolation of PBMCs by density gradient centrifugation with Lymphosep lymphocyte separation medium (Biosera Nualle, France). An additional blood specimen (volume, 2 mL) was collected, and serum was separated, frozen, and stored at −20oC until tested. Flow Cytometry and Pneumococcal Polysaccharide Enzyme-Linked Immunosorbent Analysis (ELISA) PS-specific MBCs in a population of fresh PBMCs were identified using biotinylated PS antigens loaded on anti–biotin-coated beads, as previously described [3]. Briefly, anti–biotin-coated beads (MACSIbead, Miltenyi Biotec, Bergisch Gladbach, Germany) were incubated with biotinylated PS antigens (either PS14 or PS3) for 2 hours at room temperature. PS-loaded beads were subsequently washed and, after incubation for 5 minutes at 4°C–8°C, were labeled with anti–biotin-FITC conjugate. Based on side-scatter and forward-scatter measurements, gating was performed to include all viable cells in the cell population. The specificity of the PS-bound B cells was validated by use of appropriate controls. Negative controls of single anti–biotin-coated beads conjugated with FITC or APC antibody were used in the assay. Analyses of anti–biotin-coated beads coated with biotinylated antigens and then tagged with either anti–biotin-FITC or anti–biotin-APC antibody were performed and compared to negative controls. Freshly collected PBMCs were then incubated for 10 minutes at room temperature with either FITC-labeled PS-coated beads or unlabelled beads as a negative control, as well as with a combination of 21-PE (clone BL13), CD10-PC5.5 (cloneALB1), CD19-PC7 (cloneJ3.119), anti-IgM-APC (clone SA-DA4), and CD27-APC Alexafluor750 (clone1A4CD27; all from Beckman Coulter, Immunotech, Marseille, France). Ammonium chloride was used to lyse red blood cells, after which remaining cells were washed and analyzed by flow cytometry, using a 2-laser Navios instrument (Beckman Coulter). Analysis of FCS files was performed with Navios or Kaluza1.2 software. B-cell subsets were defined as follows: naive B cells, D19+CD10−CD21++CD27−; sIg+ MBCs, CD19+CD10−CD21++ CD27+IgM−; IgM+ MBCs, CD19+CD10−CD21++CD27+IgM+; and exhausted B cells, CD19+CD10−CD21low. Subset analysis was performed both on the total population of B cells and on PS-specific B cells, with regions defined for the former group used to define regions of the latter group (Figure 1). Figure 1. View largeDownload slide Basic gating strategy for the identification of polysaccharide (PS)–specific B-cell subsets. A, B cells were initially identified as low CD19+ side-scatter (SS) signals. B and C, PS positivity cutoff for B cells (B) was determined with the help of the simultaneously analyzed unstained bead control (C). D and E, After gating out CD10+ signals (D), naive, memory, and exhausted PS+ B-cell subsets were defined on the basis of their CD21 vs CD27 expression pattern (E). F, Further segregation of memory PS+ B cells in switched and nonswitched conditions was based on surface immunoglobulin M (IgM) expression. G, Analysis of the above markers on total B cells was performed to help define the respective regions for the PS-specific B cells. Figure 1. View largeDownload slide Basic gating strategy for the identification of polysaccharide (PS)–specific B-cell subsets. A, B cells were initially identified as low CD19+ side-scatter (SS) signals. B and C, PS positivity cutoff for B cells (B) was determined with the help of the simultaneously analyzed unstained bead control (C). D and E, After gating out CD10+ signals (D), naive, memory, and exhausted PS+ B-cell subsets were defined on the basis of their CD21 vs CD27 expression pattern (E). F, Further segregation of memory PS+ B cells in switched and nonswitched conditions was based on surface immunoglobulin M (IgM) expression. G, Analysis of the above markers on total B cells was performed to help define the respective regions for the PS-specific B cells. The World Health Organization ELISA protocol was used for the detection of anti-PS immunoglobulin G (IgG) antibodies in serum samples. Statistical Analysis Quantitative variables were expressed as mean values (±SD). Repeated-measures analysis of variance was adopted to evaluate the changes observed in IgM+ MBCs, sIg+ MBCs, and IgG antibody values over the follow-up period for the total sample and by baseline CD4+ T-cell count (ie, between groups A and B). Pearson correlation coefficients were used to explore associations between 2 continuous variables. Correlation coefficients between 0.1 and 0.3 were considered low, those between 0.31 and 0.5 were considered moderate, and those >0.5 were considered high. Univariate and multiple linear regression analyses were used, with antibody concentrations at 13 months serving as the outcome variable. The regression equation included terms for CD4+ T cell counts and IgG titers at baseline. Adjusted regression coefficients (β), with standard errors (SEs), and standardized regression coefficients (b) were computed from the results of linear regression analyses. All reported P values are 2-tailed. Statistical significance was set at a P value of <.05, and analyses were conducted using SPSS statistical software (version 19.0). RESULTS Kinetics of PS-Specific IgG Antibodies Following PCV13 and PPV23 Receipt The characteristics of study participants are shown in Table 1. We evaluated the kinetics of antibodies against PS14 and PS3, which are common to both vaccines but differ in immunogenicity, with PS3 being the least immunogenic serotype [14]. Overall, the combined vaccination schedule with PCV13 and PPV23 induced a significant increase in PS-specific IgG antibody concentrations for both serotypes, compared with baseline (P < .001). One month following PCV13 receipt, a 2-fold rise in IgG antibody levels was observed (P < .001), with levels increased further after PPV23, albeit to a lower degree (P = .02). Group B patients had up to 40% lower antibody concentrations than group A patients for both serotypes at all time points (P < .001) and did not achieve the protective threshold of 0.35 μg/mL for PS3 following PCV13 receipt (Table 2). In addition, a low baseline CD4+ T-cell count had a significant negative effect on the magnitude of the change in antibody kinetics through the study period (P < .001). No statistically significant correlation was found between immune response to either vaccine and other demographic characteristics. Table 1. Demographic Characteristics of Human Immunodeficiency Virus–Infected Adults, Overall and by Study Group Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Abbreviation: PPV23, 23-valent pneumococcal polysaccharide vaccine. aGroup A comprised 17 participants with a CD4+ T-cell count of ≥400 cells/μL, and group B comprised 23 participants with a CD4+ T-cell count of 200–399 cells/μL. View Large Table 1. Demographic Characteristics of Human Immunodeficiency Virus–Infected Adults, Overall and by Study Group Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Characteristics  Overall (n = 40)  Group Aa (n = 17)  Group Ba (n = 23)  Age, y         Mean ± SD  50.6 ± 8.6  48.94 ± 8.934  51.87 ± 8.412   Range  32–63  33–62  32–63  Male sex, no. (%)  35 (87.5)  16 (94.1)  19 (82.6)  Viral load, copies/mL  <50  <50  <50  CD4+ T-cell count, cells/mL         Mean ± SD  529.8 ± 266.4  790.4 ± 212.6  337.2 ± 40.91   Range  234–1135  443–1135  234–394  Previous PPV23 doses, no.  28  14  14  Time since last PPV23 dose, y         <3  15  8  7   3–5  13  7  6  Abbreviation: PPV23, 23-valent pneumococcal polysaccharide vaccine. aGroup A comprised 17 participants with a CD4+ T-cell count of ≥400 cells/μL, and group B comprised 23 participants with a CD4+ T-cell count of 200–399 cells/μL. View Large Table 2. Kinetics of (a) PS-specific IgG antibody concentrations (μg/ml) and (b) IgM and sIg+ MBC (cells/106 PBMC) in HIV-infected adults vaccinated with PCV13/PPV23   Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001          Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001        aP-value for time effect. bRepeated measurements ANOVA. Effects reported include differences between the groups in the degree of change over the follow-up period. cIndicates significant difference from prior measurement. dP-value for group effect. View Large Table 2. Kinetics of (a) PS-specific IgG antibody concentrations (μg/ml) and (b) IgM and sIg+ MBC (cells/106 PBMC) in HIV-infected adults vaccinated with PCV13/PPV23   Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001          Baseline  1 Month  12 Months  13 Months  Change from baseline to 13 months      (a)                IgG antibodies Mean (SD)            Pa  Pb  PS14                All patients  1.21 (0.55)  2.97 (1.22)c  2.95 (1.22)  4.27 (1.38)c  3.06 (0.97)  <.001    Group A  1.63 (0.45)  3.95 (0.9)c  3.94 (0.91)  5.37 (0.91)c  3.74 (0.67)  <.001  <.001  Group B  0.9 (0.39)  2.24 (0.85)c  2.21 (0.85)  3.46 (1.08)c  2.55 (0.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  0.25 (0.14)  0.49 (0.21)c  0.48 (0.21)  0.64 (0.28)c  0.39 (0.18)  <.001    Group A  0.37 (0.13)  0.69 (0.16)c  0.68 (0.16)  0.9 (0.22)c  0.54 (0.17)  <.001  <.001  Group B  0.16 (0.06)  0.34 (0.09)c  0.34 (0.09)  0.44 (0.11)c  0.28 (0.08)  <.001    Pd  <.001  <.001  <.001  <.001        (b)                IgM MBCMean (SD)            Pa  Pb  PS14                All patients  12.1 (3.33)  10.8 (3.24)  9.18 (3.12)  7.38 (3.03)c  –4.73 (1.11)  <.001    Group A  15.41 (0.87)  14.00 (1.00)  12.29 (1.05)  10.47 (0.72)c  –4.94 (0.9)  <.001  .290  Group B  9.65 (2.08)  8.43 (2.04)  6.87 (1.84)  5.09 (1.73)c  –4.57 (1.24)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  9.83 (2.67)  8.73 (2.57)  7.53 (2.4)  5.53 (2.16)c  –4.3 (1.11)  <.001    Group A  12.29 (1.4)  11.18 (1.24)  9.82 (1.33)  7.53 (1.33)c  –4.76 (1.09)  <.001  .002  Group B  8 (1.73)  6.91 (1.59)  5.83 (1.34)  4.04 (1.26)c  –3.96 (1.02)  <.001    Pd  <.001  <.001  <.001  <.001        sIg+ MBC Mean (SD)                PS14                All patients  13.2 (2.78)  20.88 (3.94)c  18.58 (3.84)c  17.33 (3.87)  4.13 (2.08)  <.001    Group A  16.06 (1.25)  25.00 (1.73)c  22.59 (1.70)c  21.35 (1.62)  5.29 (1.83)  <.001  .023  Group B  11.09 (1.24)  17.83 (1.56)c  15.61 (1.53)c  14.35 (1.67)  3.26 (1.84)  <.001    Pd  <.001  <.001  <.001  <.001        PS3                All patients  10.25 (1.55)  18 (3.63)c  15.55 (3.48)c  14.43 (3.4)  4.18 (2.62)  <.001    Group A  11.29 (1.31)  20.76 (3.91)c  18.18 (3.75)c  17 (3.61)  5.71 (3.22)  <.001  .001  Group B  9.48 (1.24)  15.96 (1.4)c  13.61 (1.41)c  12.52 (1.44)  3.04 (1.22)  <.001    Pd  <.001  <.001  <.001  <.001        aP-value for time effect. bRepeated measurements ANOVA. Effects reported include differences between the groups in the degree of change over the follow-up period. cIndicates significant difference from prior measurement. dP-value for group effect. View Large MBC Responses Following PCV13 and PPV23 Before vaccination, all study participants had detectable PS-specific IgM+ MBCs and sIg+ MBCs regardless of previous pneumococcal vaccination history. The PS-specific IgM+ MBC count was notably reduced for both serotypes, compared with baseline, after completion of the vaccination schedule (P < .001). More specifically, although their levels were not affected by PCV13 receipt and remained stable during the following 12 months, PPV23 caused a significant reduction in the IgM+ MBC pool (P < .001; Table 2). In contrast, combined vaccination enriched the PS-specific sIg+ MBC pool (P < .001), mainly because of a significant increase following PCV13 receipt. However, after PPV23 receipt there were no significant changes, and at the end of follow-up period sIg+ MBC counts remained above baseline levels for both serotypes. Group A patients had significantly higher MBC counts than group B patients at baseline (MBC count, 42 574 cells/μL vs 39 609 cells/μL; IgM+ MBC count, 5624 cells/μL vs 4132 cells/μL; sIg+ MBC count, 4527 cells/μL vs 2529 cells/μL) and at all points at which blood specimens were obtained (P < .05 for all comparisons; Table 2). Moreover, the magnitude of the increase in the sIg+ MBC count was positively correlated with baseline CD4+ T-cell levels for both serotypes (r = 0.38 and P = .016 for PS14; r = 0.67 and P < .001 for PS3; Table 2). Correlations between MBC responses at baseline and 1 month after vaccination were different for each vaccine formulation. More specifically, while baseline PS-specific IgM+ MBC counts were significantly correlated with sIg+ MBC counts after PCV13 receipt (r = 0.78 and P < .001 for PS14; r = 0.57 and P < .001 for PS3), such correlation was not observed following PPV23 receipt (r = −0.10 and P = .7 for PS14; r = 0.2 and P = .7 for PS3; Figure 2). Figure 2. View largeDownload slide Pearson correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). Circles denote values for PS14, and diamonds denote values for PS3. Figure 2. View largeDownload slide Pearson correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). Circles denote values for PS14, and diamonds denote values for PS3. In contrast, correlations between baseline MBC subset counts and humoral responses were not vaccine dependent. All study subjects had baseline IgM+ and sIg+ MBC counts that were positively correlated with antibody levels at 1 month, induced by either vaccine and for both serotypes. Baseline IgΜ+ MBC counts and sIg+ MBC counts were significantly correlated with IgG antibody levels 1 month after PCV13 receipt (P < .001 for responses to PS14 and PS3). Similarly after PPV23 receipt, baseline IgΜ+ MBC counts and sIg+ MBC counts were significantly correlated with IgG antibody titers at 1 month (P < .001 for responses to PS14 and PS3; Figure 3). However, such correlations failed to reach statistical significance when the analysis was conducted separately for each group, probably because of the small number of patients. Figure 3. View largeDownload slide Spearman correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). A, For analysis of the IgM+ MBC count before PCV13 receipt vs the IgG antibody level 1 month after receipt: r = 0.62 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PCV13 receipt vs the IgG antibody 1 month after PCV13 receipt, r = 0.60 and P < .001 for PS14 and r = 0.63, P < .001 for PS3. B, For analysis of the IgM+ MBC count before PPV23 receipt the IgG antibody level 1 month after PPV23 receipt, r = 0.56 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PPV23 receipt vs the IgG antibody titer 1 month after PPV23 receipt, r = 0.64 and P < .001 for PS14, and r = 0.60 and P < .001 for PS3. Circles denote values for PS3, and diamonds denote values for P14. Figure 3. View largeDownload slide Spearman correlation coefficient analysis of the correlation between numbers of memory B cells expressing polysaccharide (PS)–specific nonswitched immunoglobulin M (IgM+ MBCs) or isotype-switched immunoglobulin (sIg+ MBCs; in cells/106 peripheral blood mononuclear cells) at baseline and PS-specific immunoglobulin G (IgG) antibody levels (in μg/mL) 1 month after receipt of 13-valent pneumococcal conjugate vaccine (PVC13; A) and 23-valent pneumococcal polysaccharide vaccine (PPV23; B). A, For analysis of the IgM+ MBC count before PCV13 receipt vs the IgG antibody level 1 month after receipt: r = 0.62 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PCV13 receipt vs the IgG antibody 1 month after PCV13 receipt, r = 0.60 and P < .001 for PS14 and r = 0.63, P < .001 for PS3. B, For analysis of the IgM+ MBC count before PPV23 receipt the IgG antibody level 1 month after PPV23 receipt, r = 0.56 and P < .001 for PS14 and r = 0.66 and P < .001 for PS3. For analysis of the slg+ MBC count before PPV23 receipt vs the IgG antibody titer 1 month after PPV23 receipt, r = 0.64 and P < .001 for PS14, and r = 0.60 and P < .001 for PS3. Circles denote values for PS3, and diamonds denote values for P14. DISCUSSION This is the first study to investigate the effect of a combined vaccination schedule of PCV13 followed 1 year later by PPV23 on the immunogenicity and PS-specific immunological memory induced by PCV13 in HIV-infected adults receiving ART. Despite the relatively small study size, our findings indicate that PPV23 enhanced PCV13 immunogenicity but had a negative effect on PCV13-induced immunological memory by reducing the IgM+ MBC subpopulation. One month after receipt of 1 dose of PPV23, there was a significant increase in circulating PS-specific IgG antibody concentrations. The added value of additional doses of pneumococcal vaccines in HIV-infected subjects has not been consistently demonstrated in previous studies [15–21]. Abzug et al reported that a combined schedule of 2 doses of PCV7 followed 8 weeks later by 1 dose PPV23 induced only a slight increase in antibody levels [22]. Similar findings were reported even when an additional dose of PPV23 was given 6 months after the first PPV23 dose, which could be attributed to the increased levels of circulating plasma cells and antibodies that can block the formation of germinal centers when a subsequent dose is given at very short intervals [23, 24]. Our study provides further evidence that longer intervals between pneumococcal vaccinations are beneficial for vaccine immunogenicity [25, 26]. Similar to previous studies, we have shown that the magnitude of the immune response to both vaccines was associated with the degree of immunodeficiency, expressed as the baseline CD4+ T-cell count, in seropositive patients [27]. Especially for PS3, which is a common cause of PCV13 failure in healthy subjects [28], patients with low baseline CD4+ T-cell counts did not achieve even the protective threshold of 0.35 μg/mL, a finding suggesting that seropositive patients vaccinated with 1 dose of PCV13 are probably not protected against PS3. The generation of long-lived antigen-specific memory is regarded as the cornerstone of successful immunization [4, 6]. Our study provides important new understanding regarding the phenotype of PS-specific MBCs after receipt of conjugate and plain-polysaccharide pneumococcal vaccines. Immunization with PCV13 induced new PS-specific sIg+ MBCs, whereas the IgM+ MBC count remained unchanged. Similar findings were reported by Papadatou et al in a study of 35 asplenic adults with β-thalassemia major, in which kinetics were evaluated after participants were vaccinated with PCV13 [29]. In contrast, PPV23 did not induce any changes in sIg+ MBC counts but caused a significant reduction in IgM+ MBC counts (by 20% and 40% for PS14-specific and PS3-specific IgM+ MBCs, respectively). The divergent effects of conjugate and polysaccharide pneumococcal vaccines on antigen-specific MBCs have also been reported mainly in adults aged >65 years [3, 30]. The detection of new sIg+ MBCs 1 month after PCV13 receipt is in line with the T-cell–dependent nature of the immune response to the conjugate vaccine, which has the ability to enrich the polysaccharide-specific MBC pool through germinal cell formation [8]. Similar results have been previously reported not only following receipt of PCV13 but also after receipt of meningococcal C conjugate vaccine [29, 31]. Interestingly, the IgM+ MBC count remained stable following PCV13 receipt, while vaccination with PPV23 led to a significant decrease in this subpopulation. Since both MBC subsets are expressed as absolute cell counts at all time points, we believe that the decrease in the IgM+ MBC count following PPV23 is true and does not reflect an increase in the relative proportion of sIg+ MBCs. Our findings are in keeping with the hypothesis that T-cell–independent antigens drive preexisting MBCs into terminal differentiation without replenishment of the MBC pool, owing to the lack of germinal center formation [8]. Moreover, the positive correlation between the baseline IgM+ MBC count and the sIg+ MBC count 1 month following PCV13 receipt suggests that a part of the preexisting IgM+ MBC population reenters germinal centers and differentiates into sIg+ MBCs, providing further support for germinal center formation following T-cell–dependent stimulation [7]. Taking into account that IgM+ MBCs survive for longer periods than sIg+ MBCs and are thought to mediate late memory responses when levels of sIg+ MBCs and antibodies are reduced [24, 32], the observed reduction of this subpopulation could have biological significance for subjects with advanced HIV disease, as shown in an early study performed in HIV-infected adults not receiving ART, in which a surprising excess of PPV23 recipients developed pneumococcal disease as compared to placebo recipients [33]. Additional studies investigating the association between PPV23-induced IgM+ MBC depletion and differences in Ig class and subclass antibody responses and serum bactericidal activity, as well as the long-term kinetics of IgM or IgG antibody levels, will provide further evidence for the biological significance of our finding to HIV infection. Whether baseline memory can predict the magnitude of the antibody response after vaccination remains debatable, and results from relevant studies of healthy and immunocompromised populations are conflicting [29, 34–36]. We found a consistent, positive correlation between baseline MBC subsets and antibody levels 1 month after receipt of either PCV13 or PPV23. The correlation, however, did not reach statistical significance when the analysis was conducted separately for each group of patients, probably because of the small study size. It is possible that differences between healthy and immunocompromised subjects could be attributed to the existence of additional cell populations in healthy individuals that are also involved in the mechanisms of long-lasting immunological memory, such as long-lived IgM+ plasma cells, which could be impaired in immunocompromised subjects [37]. Although we did not include a control group to compare the effect of the polysaccharide vaccine in healthy subjects, similar findings in healthy adults were reported recently by O’Connor et al [38]. They compared B-cell responses to conjugate and polysaccharide meningococcal vaccines by means of a number of different methods, including gene expression microarray, and demonstrated distinct plain-polysaccharide gene signatures, possibly linked to hyporesponsiveness, associated with depletion of switched B memory cells. In conclusion, our study highlights the divergent effects of conjugate and polysaccharide vaccines in the establishment of immunological memory in HIV-infected patients and supports BHIVA, EACS, and PENTA guidelines, which recommend PCV13 instead of PPV23 for HIV-seropositive subjects [11, 39–41]. Most seropositive4 subjects now receive ART, which can reverse most B-cell defects, with the exception of IgM+ MBC loss [42]; therefore, questions may arise regarding the use of PPV23 in seropositive subjects with decreased IgM+ MBC counts. Further evidence of the significance of the decline of IgM+ MBC counts, provided by studies of the long-term kinetics of IgM and IgG antibody levels following PPV23 vaccination, will be useful for reevaluation of current recommendations of pneumococcal vaccination in these high-risk individuals. Notes Acknowledgments.  We thank Dr Elizabeth Clutterbuck, Oxford Vaccine Group, Department of Paediatrics, University of Oxford, and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom, for providing us the pneumococcal serotype biotinylation protocol. All authors have substantially contributed to the submitted work. P. F. codesigned and performed the research; collected, analyzed, and interpreted the data; and wrote the manuscript. M. 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Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of Infectious DiseasesOxford University Press

Published: May 2, 2018

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