Prediction and Validation of Immunogenic Domains of Pneumococcal Proteins Recognized by Human CD4+ T Cells.

CD4+ T-cell mechanisms are implied in protection against pneumococcal colonization; however, their target antigens and function are not well defined. In contrast to high-throughput protein arrays for serology, basic antigen tools for CD4+ T-cell studies are lacking. Here, we evaluate the potential of a bioinformatics tool for in silico prediction of immunogenicity as a method to reveal domains of pneumococcal proteins targeted by human CD4+ T cells. For 100 pneumococcal proteins, CD4+ T-cell immunogenicity was predicted based on HLA-DRB1 binding motifs. For 20 potentially CD4+ T-cell immunogenic proteins, epitope regions were verified by testing synthetic peptides in T-cell assays using peripheral blood mononuclear cells from healthy adults. Peptide pools of 19 out of 20 proteins evoked T-cell responses. The most frequent responses (detectable in ≥20% of donors tested) were found to SP_0117 (PspA), SP_0468 (putative sortase), SP_0546 (BlpZ), SP_1650 (PsaA), SP_1923 (Ply), SP_2048 (conserved hypothetical protein), SP_2216 (PscB), and SPR_0907 (PhtD). Responding donors had diverging recognition patterns and profiles of signature cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-13 [IL-13], and/or IL-17A) against single-epitope regions. Natural HLA-DR-restricted presentation and recognition of a predicted SP_1923-derived epitope were validated through the isolation of a CD4+ T-cell clone producing IFN-γ, TNF-α, and IL-17A in response to the synthetic peptide, whole protein, and heat-inactivated pneumococcus. This proof of principle for a bioinformatics tool to identify pneumococcal protein epitopes targeted by human CD4+ T cells provides a peptide-based strategy to study cell-mediated immune mechanisms for the pneumococcal proteome, advancing the development of immunomonitoring assays and targeted vaccine approaches.

Serotype-Specific IgG Antibody Waning after Pneumococcal Conjugate Primary Series Vaccinations with either the 10-Valent or the 13-Valent Vaccine.

The two currently available ten- and thirteen-valent pneumococcal conjugate vaccines (PCV10 and PCV13) both induce serotype-specific IgG anti-polysaccharide antibodies and are effective in preventing vaccine serotype induced invasive pneumococcal disease (IPD) as well as in reducing overall vaccine-serotype carriage and transmission and thereby inducing herd protection in the whole population. IgG levels decline after vaccination and could become too low to prevent carriage acquisition and/or pneumococcal disease. We compared the levels of 10-valent (PCV10) and 13-valent (PCV13) pneumococcal vaccine induced serum IgG antibodies at multiple time points after primary vaccinations. Data from two separate studies both performed in the Netherlands in infants vaccinated at 2, 3, and 4 months of age with either PCV10 or PCV13 were compared. Antibody levels were measured at 5, 8, and 11 months of age, during the interval between the primary immunization series and the 11-months booster dose. Serotype-specific IgG levels were determined by multiplex immunoassay. Although antibody kinetics showed significant variation between serotypes and between vaccines for the majority of the 10 shared serotypes, i.e., 1, 5, 7F, 9V, 14, 18C, and 23F, antibody concentrations were sufficiently high for both vaccines, immediately after the primary series and throughout the whole period until the booster dose. In contrast, for serotypes 4 and 19F in the PCV10 group and for serotypes 4 and 6B in the PCV13 group, IgG antibody concentrations already come within reach of the frequently used seroprotection level of 0.35 µg/mL immediately after the primary series at the five month time point and/or at eight months. This paper addresses the importance of revealing differences in serotype-specific and pneumococcal vaccine-dependent IgG antibody patterns during the interval between the primary series and the booster dose, an age period with a high IPD incidence. Trial registration: www.trialregister.nl NTR3069 and NTR2316.

Immunodominance in T cell responses elicited against different domains of detoxified pneumolysin PlyD1.

Detoxified pneumolysin, PlyD1, is a protein vaccine candidate that induces protection against infections with Streptococcus pneumoniae in mouse models. Despite extensive knowledge on antibody responses against PlyD1, limited information is available about PlyD1 induced T cell recognition. Here we interrogated epitope breadth and functional characteristics of the T cell response to PlyD1 in two mouse strains. BALB/c (H-2d) and C57BL/6 (H-2b) mice were vaccinated with Al(OH)3-adjuvanted or non-adjuvanted PlyD1, or placebo, on day 0, 21 and 42 and were sacrificed at day 56 for collection of sera and spleens. Vaccination with adjuvanted and non-adjuvanted PlyD1 induced anti-pneumolysin IgG antibodies with neutralizing capacity in both mouse strains. Adjuvantation of PlyD1 enhanced the serological responses in both strains. In vitro restimulation of splenocytes with PlyD1 and 18-mer synthetic peptides derived from pneumolysin revealed specific proliferative and cytokine responses. For both mouse strains, one immunodominant and three subdominant natural epitopes were identified. Overlap between H-2d and H-2b restricted T cell epitopes was limited, yet similarities were found between epitopes processed in mice and predicted to be immunogenic in humans. H-2d restricted T cell epitopes were localized in pneumolysin domains 2 and 3, whereas H-2b epitopes were scattered over the protein. Cytokine responses show mostly a Th2 profile, with low levels of Th1 cytokines, in both mouse strains. In conclusion, PlyD1 evokes T cell responses in mice directed against multiple epitope regions, that is dependent on Major Histocompatibility Complex (MHC) background. These results are important to understand human PlyD1 T cell immunogenicity, to guide cell mediated immunity studies in the context of vaccine development.

Correction: Direct Comparison of Immunogenicity Induced by 10- or 13-Valent Pneumococcal Conjugate Vaccine around the 11-Month Booster in Dutch Infants.

[This corrects the article DOI: 10.1371/journal.pone.0144739.].

Direct Comparison of Immunogenicity Induced by 10- or 13-Valent Pneumococcal Conjugate Vaccine around the 11-Month Booster in Dutch Infants.

BACKGROUND & AIMS: Since 2009/10, a 10- and a 13-valent pneumococcal conjugate vaccine (PCV) are available, but only the 10-valent vaccine is now being used for the children in the Netherlands. As the vaccines differ in number of serotypes, antigen concentration, and carrier proteins this study was designed to directly compare quantity and quality of the antibody responses induced by PCV10 and PCV13 before and after the 11-month booster. METHODS: Dutch infants (n = 132) were immunized with either PCV10 or PCV13 and DTaP-IPV-Hib-HepB at the age of 2, 3, 4 and 11 months. Blood samples were collected pre-booster and post-booster at one week and one month post-booster for quantitative and qualitative immunogenicity against 13 pneumococcal serotypes, as well as quantitative immunogenicity against diphtheria, tetanus, pertussis and Haemophilus influenzae type b. We compared immunogenicity induced by PCV13 and PCV10 for their ten shared serotypes. RESULTS: One month post-booster, pneumococcal serotype-specific IgG geometric mean concentrations (GMCs) for the PCV13 group were higher compared with the PCV10 group for six serotypes, although avidity was lower. Serotype 19F showed the most distinct difference in IgG and, in contrast to other serotypes, its avidity was higher in the PCV13 group. One week post-booster, opsonophagocytosis for serotype 19F did not differ significantly between the PCV10- and the PCV13 group. CONCLUSION: Both PCV10 and PCV13 were immunogenic and induced a booster response. Compared to the PCV10 group, the PCV13 group showed higher levels for serotype 19F GMCs and avidity, pre- as well as post-booster, although opsonophagocytosis did not differ significantly between groups. In our study, avidity is not correlated to opsonophagocytotic activity (OPA) and correlations between IgG and OPA differ per serotype. Therefore, besides assays to determine IgG GMCs, assays to detect opsonophagocytotic activity, i.e., the actual killing of the pneumococcus, are important for PCV evaluation. How differences between the two vaccines relate to long-term protection requires further investigation. TRIAL REGISTRATION: www.trialregister.nl NTR3069.

Differential B-cell memory around the 11-month booster in children vaccinated with a 10- or 13-valent pneumococcal conjugate vaccine.

BACKGROUND: Both the 10- and 13-valent pneumococcal conjugate vaccines (PCV10 and PCV13) induce immunological memory against Streptococcus pneumoniae infections caused by vaccine serotypes. In addition to comparing serum antibody levels, we investigated frequencies of serotype-specific plasma cells (PCs) and memory B-cells (Bmems) as potential predictors of long-term immunity around the booster vaccination at 11 months of age. METHODS: Infants were immunized with PCV10 or PCV13 at 2, 3, 4, and 11 months of age. Blood was collected before the 11-month booster or 7-9 days afterward. Serotype-specific immunoglobulin G (IgG) levels were determined in serum samples by multiplex immunoassay. Circulating specific PCs and Bmems against shared serotypes 1, 6B, 7F, and 19F and against PCV13 serotypes 6A and 19A were measured in peripheral blood mononuclear cells by enzyme-linked immunospot assay. RESULTS: No major differences in IgG levels and PC frequencies between groups were found for the 4 shared serotypes. Notably, PCV13 vaccination resulted in higher frequencies of Bmems than PCV10 vaccination, both before and after the booster dose, for all 4 shared serotypes except for serotype 1 postbooster. For PCV13-specific serotypes 6A and 19A, the IgG levels and frequencies of PCs and Bmems were higher in the PCV13 group, pre- and postbooster, except for PC frequencies prebooster. CONCLUSIONS: Both PCVs are immunogenic and induce measurable IgG, PC, and Bmem booster responses at 11 months. Compared to PCV10, vaccination with PCV13 was associated with overall similar IgG levels and PC frequencies but with higher Bmem frequencies before and after the 11-month booster. The clinical implications of these results need further follow-up. CLINICAL TRIALS REGISTRATION: NTR3069.

Levels and functionality of antibodies after pneumococcal conjugate vaccine in schedules with different timing of the booster dose.

The seven-valent pneumococcal conjugate vaccine (PCV7) has been introduced in most high-income countries, although with differences in age, timing and number of primary doses before 6 months of age and presence and timing of a booster vaccination. The objective was to determine and compare the IgG antibody levels and functionality of IgG responses (avidity and opsonophagocytoses) at 1 and 2 years of age following 2 primary doses with a booster at 11 or 24 months of age. Children received PCV7 at 2 and 4 months (2-dose group), or at 2, 4 and 11 months (2+1-dose group), or no PCV7 (controls) before 1 year of age. All children received a PCV7 dose at 24 months of age. At the age of 12 months, the 2+1-dose group had higher IgG levels and functional antibody levels, compared to the 2-dose group for all serotypes, but at 25 months the difference between the 2-dose and 2+1-dose groups had disappeared for most serotypes. The kinetics of opsonophagocytic antibodies were in line with the specific IgG antibody levels for most serotypes, although differences between the 2-dose and the 2+1-dose group were more pronounced in OPA activity as compared to the IgG levels especially at the age of 24 months. Delaying the booster dose from 11 months to 24 months after 2 primary doses resulted in significantly higher OPA GMTs one month after the booster dose. This must, however, be balanced against the risk of leaving children unboosted between the age of 11 and 24 months at a time when disease risk is still high. Local decisions about the timing of a booster dose should also take into account vaccine coverage and the indirect herd effect in a well vaccinated population. Trial registration clinicaltrials.gov Identifier: NCT00189020.