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.

Development and Qualification of an Opsonophagocytic Killing Assay To Assess Immunogenicity of a Bioconjugated Escherichia coli Vaccine.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, -30% to 30%), and total error (total error range, -65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

A Meningococcal Outer Membrane Vesicle Vaccine Incorporating Genetically Attenuated Endotoxin Dissociates Inflammation from Immunogenicity.

BACKGROUND: Group B Neisseria meningitidis, an endotoxin-producing Gram-negative bacterium, causes the highest incidence of group B meningococcus (MenB) disease in the first year of life. The Bexsero vaccine is indicated in Europe from 8 weeks of age. Endotoxin components of outer membrane vesicles (OMVs) or soluble lipopolysaccharide (LPS) represent a potential source of inflammation and residual reactogenicity. The purpose of this study was to compare novel candidate MenB vaccine formulations with licensed vaccines, including Bexsero, using age-specific human in vitro culture systems. METHODS: OMVs from wild type- and inactivated lpxL1 gene mutant-N. meningitidis strains were characterized in human neonatal and adult in vitro whole blood assays and dendritic cell (DC) arrays. OMVs were benchmarked against licensed vaccines, including Bexsero and whole cell pertussis formulations, with respect to Th-polarizing cytokine and prostaglandin E2 production, as well as cell surface activation markers (HLA-DR, CD86, and CCR7). OMV immunogenicity was assessed in mice. RESULTS: ΔlpxLI native OMVs (nOMVs) demonstrated significantly less cytokine induction in human blood and DCs than Bexsero and most of the other pediatric vaccines (e.g., PedvaxHib, EasyFive, and bacillus Calmette-Guérin) tested. Despite a much lower inflammatory profile in vitro than Bexsero, ΔlpxLI nOMVs still had moderate DC maturing ability and induced robust anti-N. meningitidis antibody responses after murine immunization. CONCLUSION: A meningococcal vaccine comprised of attenuated LPS-based OMVs with a limited inflammatory profile in vitro induces robust antigen-specific immunogenicity in vivo.

Immunogenicity and safety of a tetravalent E. coli O-antigen bioconjugate vaccine in animal models.

BACKGROUND: Extra-intestinal pathogenic Escherichia coli (ExPEC) are major human pathogens; however, no protective vaccine is currently available. We assessed in animal models the immunogenicity and safety of a 4-valent E. coli conjugate vaccine (ExPEC-4V, serotypes O1, O2, O6 and O25 conjugated to Exotoxin A from Pseudomonas aeruginosa (EPA)) produced using a novel in vivo bioconjugation method. METHODS: Three doses of ExPEC-4V (with or without aluminum hydroxide) were administered to rabbits (2µg or 20µg per O-antigen, subcutaneously), mice (0.2µg or 2µg per O-antigen, subcutaneously) and rats (0.4µg or 4µg per O-antigen, intramuscularly). Antibody persistence and boostability were evaluated in rats using O6-EPA monovalent conjugate (0.4µg O-antigen/dose, intramuscularly). Toxicity was assessed in rats (16µg total polysaccharide, intramuscularly). Serum IgG and IgM antibodies were measured by ELISA. RESULTS: Robust antigen-specific IgG responses were observed in all animal models, with increased responses in rabbits when administered with adjuvant. O antigen-specific antibody responses persisted up to 168days post-priming. Booster immunization induced a rapid recall response. Toxicity of ExPEC-4V when administered to rats was considered to be at the no observed adverse effect level. CONCLUSIONS: ExPEC-4V conjugate vaccine showed good immunogenicity and tolerability in animal models supporting progression to clinical evaluation.

Importance of (antibody-dependent) complement-mediated serum killing in protection against Bordetella pertussis.

Pertussis is a highly contagious respiratory disease that is caused by Bordetella pertussis. Despite being vaccine preventable, pertussis rates have been rising steadily over the last decades, even in areas with high vaccine uptake. Recently, experiments with infant baboons indicated that although vaccination with acellular pertussis vaccines prevented disease, no apparent effect was observed on infection and transmission. One explanation may be that current acellular pertussis vaccines do not induce high levels of opsonophagocytic and/or bactericidal activity, implying that engineering of vaccines that promote bacterial killing may improve efficacy. Here, we discuss the importance of complement-mediated killing in vaccine-induced protection against B. pertussis. We first examine how B. pertussis may have evolved different complement evasion strategies. Second, we explore the benefits of opsonophagocytic and/or bactericidal killing in vaccine-induced protection and discuss whether or not inclusion of new opsonophagocytic or bactericidal target antigens in pertussis vaccines may benefit efficacy.

The mouse intranasal challenge model for potency testing of whole-cell pertussis vaccines.

INTRODUCTION: A mouse intracerebral challenge model is used for potency testing of whole-cell pertussis (wP) vaccines. We investigated the use of a mouse nasopharyngeal challenge model, which better reflects the clinical features of pertussis disease, to differentiate between efficacy of wP vaccines. METHODS: Efficacy of three wP vaccines (Quinvaxem(®), Easyfive(®) and Pentavac(®)) was tested in the nasopharyngeal challenge model. Mice were vaccinated at 4 and 7 weeks and challenged with Bordetella pertussis at 9 weeks. Vaccine efficacy was determined based on CFU in the lungs 5 days after challenge. RESULTS: The mouse nasopharyngeal challenge model has the capacity to differentiate between the efficacy of whole cell pertussis vaccines. CONCLUSION: The mouse nasopharyngeal challenge model could be considered as a potency and release assay for wP vaccines. Whether this model directly correlates with clinical vaccine efficacy requires further investigations. Whether this model directly correlates with clinical vaccine efficacy requires further investigations. The mouse nasopharyngeal challenge model could be considered as a potency and release assay for wP vaccines.

The history of pneumococcal conjugate vaccine development: dose selection.

Pneumococcal conjugate vaccines (PCVs) differ in polysaccharide (PS) dose, carrier protein and conjugation method. PCV development proceeded initially upon principles successfully proven in Haemophilus influenzae type b (Hib) conjugate vaccine development. However, the need to successfully incorporate multiple serotypes while minimizing the total PS dose and total carrier protein load saw some early vaccine candidates fail. Dose-range studies of individual serotypes indicated that much lower PS doses were needed compared with Hib conjugate vaccines, although subsequent studies confirmed that lower Hib PS doses were possible. Furthermore, the immune response to individual serotype doses was carrier protein dependent. A 'one-size fits most' approach has characterized PS dose selection, but peculiarities of individual serotypes are increasingly apparent, raising the question whether re-formulation of PCVs to maximize individual serotype performance is needed.

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.

Improved production process for native outer membrane vesicle vaccine against Neisseria meningitidis.

An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV) against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable aseptic equipment was implemented, replacing undesirable steps like ultracentrifugation, inactivation with phenol, and the use of preservatives. The resulting process is more consistent and gives a higher yield than published reference processes, enabling NOMV production at commercial scale. Product quality met preliminary specifications for 9 consecutive batches, and an ongoing study confirmed real-time stability up to 12 months after production. As the NOMV had low endotoxic activity and induced high bactericidal titres in mice, they are expected to be safe and effective in humans. The production process is not limited to NonaMen and may be applicable for other N. meningitidis serogroups and other gram-negative pathogens. The current results therefore facilitate the late-stage development and clinical evaluation of NOMV vaccines.

Cost-effectiveness of vaccination against meningococcal B among Dutch infants: Crucial impact of changes in incidence.

OBJECTIVE: Recently, a vaccine with the capacity to protect against serogroup B meningococcal (MenB) disease received a positive opinion of the European Medicines Agency. Previously, such a vaccine was estimated to be cost-effective. However, since then, the MenB disease incidence has declined drastically in the Netherlands. Therefore, we re-assessed the potential incremental cost-effectiveness ratio (ICER) of vaccinating infants in the Netherlands with a MenB vaccine. RESULTS: Routine infant vaccination (2, 3, 4+11 mo) could prevent 39 cases of MenB disease in a single birth cohort, corresponding to a total gain of 133 quality-adjusted life years (QALYs). However, this strategy is unlikely to be cost-effective if the vaccine costs €40 per dose (€243,778 per QALY). At a disease incidence of 5.7 per 100,000 person-years or a vaccine price of €10 per dose including administration costs, the ICER becomes more acceptable and remains below a threshold of €50,000 per QALY. METHODS: A cohort of 185,000 Dutch newborns was followed in a Markov model to compare routine vaccination against MenB disease with no vaccination. The ICER was estimated for different disease incidences. The study was performed from a societal perspective. CONCLUSIONS: At the current low level of disease incidence, introduction of routine infant vaccination, following a 2, 3, 4+11 mo schedule, against MenB disease is unlikely cost-effective in the Netherlands. If the MenB disease incidence increases or the vaccine price is substantially lower than €40, routine infant vaccination has the potential to be cost-effective.

Preclinical safety and immunogenicity evaluation of a nonavalent PorA native outer membrane vesicle vaccine against serogroup B meningococcal disease.

BACKGROUND: An improved nonavalent PorA native outer membrane vesicle vaccine was developed with intrinsic adjuvating activity due to presence of less-toxic (lpxL1) LPS. In the present study, the safety and immunogenicity of this next-generation NonaMen vaccine were evaluated following repeated vaccination in rabbits and mice. METHODS: A repeated-dose toxicology study was performed in rabbits. Immunogenicity of next-generation NonaMen was evaluated by determining the serum bactericidal antibody (SBA) titers against meningococcal serogroup B strains containing several PorA subtypes. Release of the pro-inflammatory cytokine, interleukin-6 (IL-6), by the human monocytic cell line (MM6) was measured to estimate pyrogenic activity. RESULTS: No toxicologically relevant findings were noted in vaccinated rabbits receiving plain next-generation NonaMen. In agreement, next-generation NonaMen induced reduced amounts of the pro-inflammatory cytokine, IL-6, released by human monocyte cell line. In both rabbits and mice, next-generation NonaMen induced high SBA titers against all tested MenB strains regardless of whether or not aluminium phosphate adjuvant is used. CONCLUSIONS: The data suggest that next-generation NonaMen is a safe vaccine with the potential to develop a broadly protective immune response and encourage the start of the first clinical studies.

Nonclinical vaccine safety evaluation: advantages of continuous temperature monitoring using abdominally implanted data loggers.

Fever has been reported as the most common adverse event after vaccination in infants and children. For this reason it is important that, prior to clinical testing of a new vaccine, change in body temperature following vaccination is tested carefully in nonclinical animal studies. Since both the timing and the height of the temperature peak after vaccination may differ from vaccine to vaccine, it is important that the time point for body temperature measurement should be chosen on a case-by-case basis with sufficient knowledge of the specific vaccine. In order to determine the best time point for rectal body temperature measurement after vaccination with a new vaccine candidate against N. meningitidis serogroup B, to be applied in a formal Good Laboratory Practice (GLP) toxicology study, miniature temperature data loggers were implanted into the peritoneal cavity of rabbits. The continuous body temperature monitoring appeared to give a complete picture of the entire body temperature kinetics after vaccination. The body temperature peaked at 4 h after vaccination, and this time point was subsequently applied in the toxicology study. Measured body temperature values at the selected time point of 4 h after vaccination were comparable in the continuous temperature setting and in the formal toxicology study, i.e. rectal temperature measurement at one time point. In the present study implanted temperature loggers were successfully used to define an adequate time point to be applied in determining rectal body temperature in a formal GLP toxicology study with a new vaccine candidate.

Salivary immune responses to the 7-valent pneumococcal conjugate vaccine in the first 2 years of life.

BACKGROUND: The CRM197-conjugated 7-valent pneumococcal vaccine (PCV7) is protective against vaccine serotype disease and nasopharyngeal carriage. Data on PCV7-induced mucosal antibodies in relation to systemic or natural anticapsular antibodies are scarce. METHODS: In a randomized controlled setting, children received PCV7 at age 2 and 4 months (2-dose group), at age 2, 4 and 11 months (2+1-dose group) or no PCV7 (control group). From 188 children paired saliva samples were collected at 12 and 24 months of age. From a subgroup of 15 immunized children also serum samples were collected. IgG and IgA antibody-levels were measured by multiplex immunoassay. RESULTS: At 12 months, both vaccine groups showed higher serum and saliva IgG-levels against vaccine serotypes compared with controls which sustained until 24 months for most serotypes. Salivary IgG-levels were 10-20-fold lower compared to serum IgG, however, serum and saliva IgG-levels were highly correlated. Serum and salivary IgA-levels were higher in both vaccine groups at 12 months compared with controls, except for serotype 19F. Higher salivary IgA levels remained present for most serotypes in the 2+1-dose group until 24 months, but not in the 2-dose group. Salivary IgA more than IgG, increased after documented carriage of serotypes 6B, 19F and 23F In contrast to IgG, salivary IgA-levels were comparable with serum, suggesting local IgA-production. CONCLUSIONS: PCV7 vaccination results in significant increases in salivary IgG and IgA-levels, which are more pronounced for IgG when compared to controls. In contrast, salivary anticapsular IgA-levels seemed to respond more to natural boosting. Salivary IgG and IgA-levels correlate well with systemic antibodies, suggesting saliva might be useful as potential future surveillance tool.

Long-term effects of pneumococcal conjugate vaccine on nasopharyngeal carriage of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis.

BACKGROUND: Shifts in pneumococcal serotypes following introduction of 7-valent pneumococcal conjugate vaccine (PCV-7) may alter the presence of other bacterial pathogens co-inhabiting the same nasopharyngeal niche. METHODOLOGY/PRINCIPAL FINDINGS: Nasopharyngeal prevalence rates of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis were investigated before, 3 and 4.5 years after introduction of PCV-7 in the national immunisation program in children at 11 and 24 months of age, and parents of 24-month-old children (n≈330/group) using conventional culture methods. Despite a virtual disappearance of PCV-7 serotypes over time, similar overall pneumococcal rates were observed in all age groups, except for a significant reduction in the 11-month-old group (adjusted Odds Ratio after 4.5 years 0.48, 95% Confidence Interval 0.34-0.67). Before, 3 and 4.5 years after PCV-7 implementation, prevalence rates of S. aureus were 5%, 9% and 14% at 11 months of age (3.59, 1.90-6.79) and 20%, 32% and 34% in parents (1.96, 1.36-2.83), but remained similar at 24 months of age, respectively. Prevalence rates of H. influenzae were 46%, 65% and 65% at 11 months (2.22, 1.58-3.13), 52%, 73% and 76% at 24 months of age (2.68, 1.88-3.82) and 23%, 30% and 40% in parents (2.26, 1.58-3.33), respectively. No consistent changes in M. catarrhalis carriage rates were observed over time. CONCLUSIONS/SIGNIFICANCE: In addition to large shifts in pneumococcal serotypes, persistently higher nasopharyngeal prevalence rates of S. aureus and H. influenzae were observed among young children and their parents after PCV-7 implementation. These findings may have implications for disease incidence and antibiotic treatment in the post-PCV era.

Is a single dose of meningococcal serogroup C conjugate vaccine sufficient for protection? experience from the Netherlands.

BACKGROUND: The first meningococcal serogroup C (MenC) conjugate vaccine was licensed in 1999 and introduced in the United Kingdom. Countries that have implemented the MenC vaccine since then in their national immunisation programmes use different schedules. Nevertheless, all involved countries seem to experience substantial declines in the incidence of MenC disease. DISCUSSION: Since 2001, the MenC conjugate vaccine has been implemented in the Netherlands by offering a single dose to all children aged 14 months. Prior to the introduction of the vaccine into the national immunisation programme, a catch-up vaccination campaign was initiated in which a single dose of the MenC conjugate vaccine was offered to all children aged from 14 months up to and including 18 years. Since then, there has been no report of any case of MenC disease among immunocompetent vaccinees. Administration of a single dose of MenC conjugate vaccine after infancy could be beneficial considering the already complex immunisation schedules with large numbers of vaccinations in the first year of life. The present paper deals with the advantages and critical aspects of a single dose of the MenC conjugate vaccine. SUMMARY: A single dose of MenC conjugate vaccine at the age of 14 months in combination with a catch up vaccine campaign appeared to be a successful strategy to prevent MenC disease in the Netherlands, thereby confirming that a single dose of the vaccine could sufficiently protect against disease. Nevertheless, this approach can only be justified in countries with a relatively low incidence of serogroup C meningococcal disease in the first year of life. Furthermore, a good surveillance programme is recommended for timely detection of vaccine breakthroughs and outbreaks among non-vaccinees, since long-term protection after a single dose in the second year of life cannot currently be guaranteed.

Lower immunoglobulin G antibody responses to pneumococcal conjugate vaccination at the age of 2 years after previous nasopharyngeal carriage of Streptococcus pneumoniae.

OBJECTIVE: To determine whether nasopharyngeal pneumococcal carriage with serotypes 6B, 19F, or 23F interferes with immunoglobulin G (IgG) antibody responses to vaccination with 7-valent pneumococcal conjugate vaccine (PCV7) at the age 24 months. STUDY DESIGN: Blood samples were collected before and after a PCV7 challenge vaccination at age 24 months from subsets of children participating in a randomized controlled trial. Children previously had received two doses of PCV7 at 2 and 4 months, two plus one doses of PCV7 at 2, 4, and 11 months, or no dosage until 24 months. Nasopharyngeal swabs were cultured at for Streptococcus pneumoniae at age 6 weeks and at 6, 12, 18, and 24 months. IgG responses were determined with enzyme immunoassay. RESULTS: Lower IgG responses against serotypes 6B, 19F, and 23F were observed on PCV7 challenge vaccination at 24 months in children who had received earlier PCV7 vaccinations and had been found positive for homologous carriage compared with non-carriers of these serotypes. Lower non-homologous IgG responses were observed after the PCV7 challenge at 24 months against serotype 6B after earlier 19F carriage and against serotype 19F after earlier 23F carriage compared with children who had not been positive for carriage of these serotypes. CONCLUSIONS: Pneumococcal colonization with serotypes 6B, 19F, and 23F is associated with diminished immune responses against these serotypes on PCV7 vaccination at 2 years of age. Underlying mechanisms deserve further investigation.

A cyclic undecamer peptide mimics a turn in folded Alzheimer amyloid ß and elicits antibodies against oligomeric and fibrillar amyloid and plaques.

The 39- to 42-residue amyloid ß (Aß) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aß immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aß, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aß-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aß(22-28)-YNGK' elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound--although more weakly--to Aß(1-42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aß deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice.

Carriage of Streptococcus pneumoniae 3 years after start of vaccination program, the Netherlands.

To evaluate the effectiveness of the 7-valent pneumococcal conjugate vaccine (PCV7) program, we conducted a cross-sectional observational study on nasopharyngeal carriage of Streptococcus pneumoniae 3 years after implementation of the program in the Netherlands. We compared pneumococcal serotypes in 329 prebooster 11-month-old children, 330 fully vaccinated 24-month-old children, and 324 parents with age-matched pre-PCV7 (unvaccinated) controls (ages 12 and 24 months, n = 319 and n = 321, respectively) and 296 of their parents. PCV7 serotype prevalences before and after PCV7 implementation, respectively, were 38% and 8% among 11-month-old children, 36% and 4% among 24-month-old children, and 8% and 1% among parents. Non-PCV7 serotype prevalences were 29% and 39% among 11-month-old children, 30% and 45% among 24-month-old children, and 8% and 15% among parents, respectively; serotypes 11A and 19A were most frequently isolated. PCV7 serotypes were largely replaced by non-PCV7 serotypes. Disappearance of PCV7 serotypes in parents suggests strong transmission reduction through vaccination.

The structure of Neisseria meningitidis lipid A determines outcome in experimental meningococcal disease.

Lipopolysaccharide (LPS), a major component of the meningococcal outer membrane, is sensed by the host through activation of Toll-like receptor 4 (TLR4). Recently, we demonstrated that a surprisingly large fraction of Neisseria meningitidis disease isolates are lipid A mutants, due to inactivating mutations in the lpxL1 gene. The lpxL1 mutants activate human TLR4 much less efficiently than wild-type bacteria, which may be advantageous by allowing them to escape from the innate immune system. Here we investigated the influence of lipid A structure on virulence in a mouse model of meningococcal sepsis. One limitation, however, is that murine TLR4 recognizes lpxL1 mutant bacteria much better than human TLR4. We show that an lpxL2 mutant, another lipid A mutant lacking an acyl chain at a different position, activates murine TLR4 less efficiently than the lpxL1 mutant. Therefore, the lpxL2 mutant in mice might be a better model for infections with lpxL1 mutants in humans. Interestingly, we found that the lpxL2 mutant is more virulent in mice than the wild-type strain, whereas the lpxL1 mutant is actually much less virulent than the wild-type strain. These results demonstrate the crucial role of N. meningitidis lipid A structure in virulence.

Hyperproliferation of B cells specific for a weakly immunogenic PorA in a meningococcal vaccine model.

Highly homologous meningococcal porin A (PorA) proteins induce protective humoral immunity against Neisseria meningitidis group B infection but with large and consistent differences in the levels of serum bactericidal activity achieved. We investigated whether a poor PorA-specific serological outcome is associated with a limited size of the specific B-cell subpopulation involved. The numbers of PorA-specific splenic plasma cells, bone marrow (BM) plasma cells, and splenic memory B cells were compared between mice that received priming and boosting with the weakly immunogenic PorA (P1.7-2,4) protein and those that received priming and boosting with the highly immunogenic PorA (P1.5-1,2-2) protein. Immunoglobulin G (IgG) titers (except at day 42), bactericidal activity, and the avidity of IgG produced against P1.7-2,4 were significantly lower at all time points after priming and boosting than against P1.5-1,2-2. These differences, however, were not associated with a lack of P1.7-2,4-specific plasma cells. Instead, priming with both of the PorAs resulted in the initial expansion of comparable numbers of splenic and BM plasma cells. Moreover, P1.7-2,4-specific BM plasma cells, but not P1.5-1,2-2-specific plasma cells, expanded significantly further after boosting. Likewise, after a relative delay during the priming phase, the splenic P1.7-2,4-specific memory B cells largely outnumbered those specific for P1.5-1,2-2, upon boosting. These trends were observed with different vaccine formulations of the porins. Our results show for the first time that B-cell subpopulations involved in a successfully maturated antibody response against a clinically relevant vaccine antigen are maintained at smaller population sizes than those associated with poor affinity maturation. This bears consequences for the interpretation of immunological memory data in clinical vaccine trials.