Combined deficient response to polysaccharide-based and protein-based vaccines predicts a severe clinical phenotype.

OBJECTIVES: Antibody response on polysaccharide- and protein-based vaccines is useful to test B cell functionality. As only few studies have explored the value of studying immune response to both vaccines, we evaluated the clinical value of anti-polysaccharide and anti-protein Luminex-based multiplex assays in context of primary immunodeficiency (PID) diagnosis. METHODS: A 10-plex Luminex-based assay detecting antibodies to ten pneumococcal polysaccharide (PnPS) serotypes [present in unconjugated Pneumovax, not in 13-valent pneumococcal conjugated vaccine (PCV)] and a 5-plex assay detecting antibodies to five protein antigens (present in DTap/Tdap) were clinically validated in healthy individuals (n=99) and in retrospective (n=399) and prospective (n=108) patient cohorts. Clinical features of individuals with impaired response to PnPS and/or proteins were compared to those with normal response. RESULTS: Antigen-specific antibody thresholds were determined in healthy individuals. Individuals with impaired anti-PnPS responses and deficient immunoglobulin levels suffered more from autoimmune diseases and had lower B cell levels compared to individuals with impaired anti-PnPS response with normal immunoglobulin levels. Individuals with combined impaired response to PnPS and proteins showed more severe clinical manifestations compared to individuals with isolated impaired response to PnPS or proteins. Eight of the 11 individuals with severely impaired responses to both PnPS and proteins had common variable immunodeficiency. Evaluation of the anti-PnPS response to four serotypes not contained in 20-valent PCV was comparable to evaluation to ten serotypes not contained in 13-valent PCV. CONCLUSIONS: Multiplexed assessment of anti-PnPS and anti-protein responses combined with immunoglobulin quantification provides useful clinical information to support PID diagnosis.

ANTIGENIC STIMULATION DURING PREGNANCY MODIFIES SPECIFIC IGA1 AND IGA2 SUBCLASSES IN HUMAN COLOSTRUM ACCORDING TO THE CHEMICAL COMPOSITION OF THE ANTIGEN.

BACKGROUND: Several studies have evaluated the effect of infectious diseases and vaccine protocols during pregnancy on maternal milk immunoglobulin (Ig) levels, to understand the protection conferred by lactation on newborns. Colostrum is the primary source of maternal IgA for the newborn. IgA participates in protection mechanisms in the neonate's mucosa. In humans, IgA has two subclasses with differential anatomical distribution among mucosal compartments. Total IgA levels in maternal milk vary after antigen stimulation and have differential affinities in function of the chemical composition of the antigens. We studied the effect of antigenic stimulation during pregnancy on the concentrations of specific IgA1 and IgA2 subclasses in human colostrum. METHODS: We analyzed data from 113 women in Mexico City and compared the amount of IgA subclasses in colostrum against three antigens: two from vaccine protocols (tetanus toxoid and pneumococcal polysaccharides) and lipopolysaccharide, a ubiquitous antigen in the gastrointestinal tract. RESULTS: In agreement with the previous reports, we showed that IgA1 from colostrum mainly recognized protein antigens; in sharp contrast, IgA2 was mostly directed against polysaccharide antigens. These levels increased in women who had previous contacts through vaccination or infections during pregnancy. CONCLUSIONS: Antigen interaction during pregnancy increased the amount of specific IgA subclasses, depending on the chemical composition of the antigen.

SLy2 controls the antibody response to pneumococcal vaccine through an IL-5Rα-dependent mechanism in B-1 cells.

The adaptor protein SLy2 (Src homology domain 3 lymphocyte protein 2) is located on human chromosome 21 and was reported to be among a group of genes amplified in Down's syndrome (DS) patients. DS patients characteristically show an impaired immunity to pneumococcal infections. However, molecular mechanisms linking gene amplifications with specific DS phenotypes remain elusive. To investigate the effect of SLy2 gene amplification on the mammalian immune system, we studied SLy2 overexpressing transgenic-SLy2 (TG) mice. We found that baseline immunoglobulin M (IgM) levels as well as IgM responses following Pneumovax immunizations were reduced in TG mice. Moreover, B-1 cells, the major natural IgM-producing population in mice, were reduced in the peritoneal cavity of TG mice, while other immune cell compartments were unaltered. Mechanistically, SLy2 overexpression attenuated the expression of the IL-5 receptor α chain on B-1 cells, resulting in decreased B-1 cell numbers and decreased differentiation into Ab-secreting cells. Since B-1 cells essentially contribute to immunity against Streptococcus pneumoniae, the present study provides a novel molecular link between SLy2 expression and pneumococcal-specific IgM responses in vivo. These studies suggest that the adaptor protein SLy2 is a potential future target for immunomodulatory strategies for pneumococcal infections.

Development and application of HPLC-RI and HPLC-MS/MS based methods for quantification of residual deoxycholate levels in pneumococcal polysaccharides.

The analysis of residual sodium deoxycholate (DOC); a detergent of biological origin used in manufacturing of polysaccharide vaccines is challenging due to complex sample matrices and the lack of suitable methods. Here we report, rapid and sensitive high-performance liquid chromatography-refractive index (HPLC-RI) and tandem mass spectrometry (HPLC-MS/MS) methods for estimation of residual DOC in pneumococcal polysaccharides. For HPLC-RI method, separation was achieved using Luna C18 column and mobile phase compositions of acetonitrile: methanol: 20 mM sodium acetate (60:05:35% v/v). For HPLC-MS/MS method, separation was achieved using a Hypersil BDS C18 column with gradient elution of methanol and water (0.1% formic acid). MS/MS method showed linearity (r2 = 0.997) over the range of 10-320 ng/mL with limits of detection (LOD) and lower limit of quantitation (LOQ) of 3 and 10 ng/mL respectively. Precision (% RSD) and accuracy (% recovery) for both methods were in the range of 0.74-8.29% and 82.33-117.86% respectively. Sample matrices interferences were addressed following novel sample clean-up method based on liquid-liquid extraction. Both methods enabled traceable quantitation of DOC in intermediate and purified pneumococcal polysaccharides of serotypes: 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F.

Real-time in situ monitoring of CRM-197 and polysaccharide conjugation reaction by fluorescence spectroscopy.

Aims: Process analytical technology (PAT) is increasingly being adopted within the pharmaceutical industry to build quality into a process. Development of PAT that provides real-time in situ analysis of critical quality attributes are highly desirable for rapid, improved process development. Conjugation of CRM-197 with pneumococcal polysaccharides to produce a desired pneumococcal conjugate vaccine is a significantly intricate process that can tremendously benefit from real-time process monitoring. Methods: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time. Results & conclusion: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time.

Identification and quantitation of isomeric pneumococcal polysaccharides by partial chemical degradation followed by mass spectrometry.

Quantitation of isomeric pneumococcal polysaccharides in vaccines is a challenging task due to mixture complexity, their low quantities, and identical monosaccharide compositions. Differentiation and quantitation of isomeric pneumococcal polysaccharides were investigated here based on a partial chemical degradation mass spectrometry approach to generate an oligosaccharide marker for one isomer, and not the other. Mild base conditions were successful at generating unique ions for the isomers with the weakest glycosidic bonds, while strong base and acid conditions were successful at generating unique ions for the more stable isomers. Linear relationships between the ion abundance of the oligosaccharide marker and the starting pneumococcal polysaccharides concentration were established for all isomers. Furthermore, precision measurements for each method were below 12% demonstrating good robustness. Therefore, partial chemical degradation followed by mass spectrometry was successful at differentiating and quantifying isomeric pneumococcal polysaccharides and may be adopted for other bacterial types.

IGA Antibody Induced by Immunization With Pneumococcal Polysaccharides Is a Prognostic Tool in Common Variable Immune Deficiencies.

The evaluation of the response to vaccination in patients with inborn errors of immunity is a tool to evaluate T-dependent and T-independent antibody residual function of B lymphocytes and it is part of the diagnostic definition for Common Variable Immune Deficiencies. Currently used classifications for Common Variable Immune Deficiencies patients are based on the frequency of B cell subsets, and have been proven as a valid instrument for identification of patients at higher risk of infectious and non-infectious complications. This 6-years period observational study delineated the measurement of specific IgA antibodies induced by a 23-valent pneumococcal polysaccharides vaccine by a standardized ELISA for the quantification of IgA antibodies to all 23 pneumococcal serotypes as an additional prognostic marker in 74 CVID patients. The inability to mount an IgA-mediated response against the pneumococcal polysaccharide antigens or the inability to maintain the antibody response over time identified poor IgA CVID responders with severe immunological impairment, great risk of co-morbidities, and poor prognosis. The division of CVID patient into specific IgA-non responders and IgA-responders discriminated better than other CVID classifications for infectious risk, while it overlapped for non-infectious complications. Our study suggested to add the evaluation of the antibody response by the 23-valent IgA assay in the clinical monitoring of CVID patients.

Comparison between a new multiplex electrochemiluminescence assay and the WHO reference enzyme-linked immunosorbent assay to measure serum antibodies against pneumococcal serotype-specific polysaccharides.

BACKGROUND: Two electrochemiluminescence (ECL) assays were developed which, together, can simultaneously measure serum antibodies against pneumococcal capsular polysaccharides (PnPS) for 17 serotypes. The assays were validated for the 13 PnPS included in the 13-valent pneumococcal conjugate vaccine (PCV13). As recommended by the World Health Organization (WHO), we compared the ECL assays with the WHO reference enzyme-linked immunosorbent assay (ELISA) and derived a threshold corresponding to the 0.35 µg/mL threshold established for the WHO reference ELISA for the non-inferiority comparison and licensure of new PCVs against invasive pneumococcal disease. METHODS: A panel of 452 serum samples from children vaccinated with one of the three licensed PCVs was assessed with the ECL assays and the WHO reference ELISA. The ECL assay threshold for the aggregated seven PnPS included in the 7-valent PCV (PCV7) and serotype-specific thresholds were determined using a receiver operating characteristics (ROC) curve-based approach and Deming regression. To evaluate concordance between the ECL assays and the WHO reference ELISA, serostatus agreement rates between both assays and geometric means of the ratios (GMRs) of concentrations obtained with both assays were calculated. RESULTS: The thresholds for the seven aggregated PCV7 serotypes obtained with the ROC curve-based approach and Deming regression approximated 0.35 µg/mL (0.38 and 0.34 µg/mL, respectively). Individual thresholds for the PCV13 serotypes ranged between 0.24 and 0.51 µg/mL across both approaches. Serostatus agreement rates using a 0.35 µg/mL threshold for both assays were ≥86.9% for all PCV13 serotypes. GMRs ranged between 0.85 and 1.25 for 11/13 serotypes and were <1.29 for the two remaining serotypes. CONCLUSION: The ECL assays were comparable to the WHO reference ELISA and offer a sensitive, time- and serum volume-saving method to quantify serotype-specific anti-PnPS antibodies in pediatric sera. A 0.35 µg/mL threshold will be used for each PCV13 serotype to assess PCV immunogenicity in clinical trials.

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides.

The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children.

Response to Pneumococcal Polysaccharide Vaccination in HIV-Positive Individuals on Long Term Highly Active Antiretroviral Therapy.

BACKGROUND AND OBJECTIVES: Streptococcus pneumoniae continues to cause serious infections in HIV-positive individuals in the era of highly active anti-retroviral therapy. This led to the recommendation to revaccinate HIV-positive individuals with PPV23 five years after primary vaccination. The benefits of revaccination and the impact of long term highly active anti-retroviral therapy (HAART) on antigen-specific B cell reconstitution have remained unclear thus far and were investigated. DESIGN AND METHODS: We assessed antibody levels, opsonophagocytic activity and phenotype of pneumococcal polysaccharide (PPS) specific-B cells post-revaccination in long term HAART cohorts stratified according to CD4 count as group A (CD4>200) and group B (CD4<200). Anti-PPS IgG, IgM and functional antibody response against vaccine serotypes 14 and 23F were measured by ELISA and opsonophagocytic assay followed by phenotypic analysis of PPS14 and 23F-specific B cells using fluorescently labeled PPS. RESULTS: Significant increases in total and functional antibody titers were noted in groups A and B post-vaccination concomitant with significant rise in PPS-specific IgM memory B cells, a critical B cell subset required for protection against PPS although the overall response remained significantly diminished compared to HIV-negative volunteers. CONCLUSION: Comparable increases in opsonophagocytic titers between study groups A and B concomitant with a comparable rise in PPS-specific IgM memory B cells indicate revaccination to be beneficial regardless of the degree of CD4 T cell reconstitution. These findings emphasize the importance of defining effective vaccination practices amongst high-risk individuals.

Antigenic stimulation during pregnancy modifies specific iga1 and iga2 subclasses in human colostrum according to the chemical composition of the antigen

ABSTRACT Background: Several studies have evaluated the effect of infectious diseases and vaccine protocols during pregnancy on maternal milk immunoglobulin (Ig) levels, to understand the protection conferred by lactation on newborns. Colostrum is the primary source of maternal IgA for the newborn. IgA participates in protection mechanisms in the neonate's mucosa. In humans, IgA has two subclasses with differential anatomical distribution among mucosal compartments. Total IgA levels in maternal milk vary after antigen stimulation and have differential affinities in function of the chemical composition of the antigens. We studied the effect of antigenic stimulation during pregnancy on the concentrations of specific IgA1 and IgA2 subclasses in human colostrum. Methods: We analyzed data from 113 women in Mexico City and compared the amount of IgA subclasses in colostrum against three antigens: two from vaccine protocols (tetanus toxoid and pneumococcal polysaccharides) and lipopolysaccharide, a ubiquitous antigen in the gastrointestinal tract. Results: In agreement with the previous reports, we showed that IgA1 from colostrum mainly recognized protein antigens; in sharp contrast, IgA2 was mostly directed against polysaccharide antigens. These levels increased in women who had previous contacts through vaccination or infections during pregnancy. Conclusions: Antigen interaction during pregnancy increased the amount of specific IgA subclasses, depending on the chemical composition of the antigen.

The host immune dynamics of pneumococcal colonization: implications for novel vaccine development.

The human nasopharynx (NP) microbiota is complex and diverse and Streptococcus pneumoniae (pneumococcus) is a frequent member. In the first few years of life, children experience maturation of their immune system thereby conferring homeostatic balance in which pneumococci are typically rendered as harmless colonizers in the upper respiratory environment. Pneumococcal carriage declines in many children before they acquire capsular-specific antibodies, suggesting a capsule antibody-independent mechanism of natural protection against pneumococcal carriage in early childhood. A child's immune system in the first few years of life is Th2-skewed so as to avoid inflammation-induced immunopathology. Understanding Th1/Th2 and Th17 ontogeny in early life and how adjuvant vaccine formulations shift the balance of T helper-cell differentiation, may facilitate the development of new protein-based pneumococcal vaccines. This article will discuss the immune dynamics of pneumococcal colonization in infants. The discussion aims to benefit the design and improvement of protein subunit-based next-generation pneumococcal vaccines.

Genetic variants within the MHC region are associated with immune responsiveness to childhood vaccinations.

The influence of genetic variability within the major histocompatibility complex (MHC) region on variations in immune responses to childhood vaccination was investigated. The study group consisted of 135 healthy infants who had been immunized with hepatitis B (HBV), 7-valent pneumococcal conjugate (PCV7), and diphtheria, tetanus, acellular pertussis (DTaP) vaccines according to standard childhood immunization schedules. Genotype analysis was performed on genomic DNA using Illumina Goldengate MHC panels (Mapping and Exon Centric). At the 1 year post vaccination check-up total, isotypic, and antigen-specific serum antibody levels were measured using multiplex immunoassays. A number of single nucleotide polymorphisms (SNPs) within MHC Class I and II genes were found to be associated with variations in the vaccine specific antibody responses and serum levels of immunoglobulins (IgG, IgM) and IgG isotypes (IgG1, IgG4) (all at p<0.001). Linkage disequilibrium patterns and functional annotations showed that significant SNPs were strongly correlated with other functional regulatory SNPs. These SNPs were found to regulate the expression of a group of genes involved in antigen processing and presentation including HLA-A, HLA-C, HLA-G, HLA-H, HLA-DRA, HLA-DRB1, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOB, and TAP-2. The results suggest that genetic variations within particular MHC genes can influence immune response to common childhood vaccinations, which in turn may influence vaccine efficacy.