The Effect of Human Factor H on Immunogenicity of Meningococcal Native Outer Membrane Vesicle Vaccines with Over-Expressed Factor H Binding Protein

The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH), is fH-binding protein (fHbp), which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV) from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (P = 0.001) and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (P = 0.003). By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (P = 0.002), and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (P = 0.001). Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans.     

Process development and immunogenicity studies on a serogroup ‘X’ Meningococcal polysaccharide conjugate vaccine

Meningococcal group X (MenX) is responsible for recent outbreaks of meningitis reported in sub-Saharan region of Africa. Although protective vaccines are available for meningitis, they are not effective against MenX. An efficacious, monovalent conjugate vaccine was designed against MenX and a fed-batch fermentation process was developed. The MenX polysaccharide (PS) was purified and yield estimated to be 15-fold higher than the reported elsewhere. Structure of MenX polysaccharide was confirmed by ¹H, ¹³C NMR spectroscopy analysis. Molecular weight of PS was found to be 310 kDa using HPLC-SEC coupled to refractive index (RI) detector. The MenX–Tetanus toxoid (TT) monovalent conjugate proved to be highly immunogenic in mice, and the bactericidal titers of MenX–TT conjugate were 10-fold higher than native PS. Increasing the dose of MenX–TT conjugate from 0.5 μg to 1.0 μg induced an 8-fold higher antibody titer as well as serum bactericidal titer. The current work suggests that the MenX–TT conjugate is a candidate vaccine against meningitis caused by Meningococcal group X strains.     

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10⁷ M⁻¹. The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.     

A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination

Certain pathogens recruit host complement inhibitors such as factor H (fH) to evade the immune system. Microbial complement inhibitor-binding molecules can be promising vaccine targets by eliciting Abs that neutralize this microbial defense mechanism. One such Ag, meningococcal factor H-binding protein (fHbp), was used in clinical trials before the protein was discovered to bind fH. The potential effect of fH binding on vaccine immunogenicity had not been assessed in experimental animals because fHbp binds human fH specifically. In this study, we developed a human fH transgenic mouse model. Transgenic mice immunized with fHbp vaccine had 4- to 8-fold lower serum bactericidal Ab responses than those of control mice whose native fH did not bind the vaccine. In contrast, Ab responses were unimpaired in transgenic mice immunized with a control meningococcal group C polysaccharide-protein conjugate vaccine. In transgenic mice, immunization with an fH nonbinding mutant of fHbp elicited Abs with higher bactericidal activity than that of fHbp vaccination itself. Abs elicited by the mutant fHbp more effectively blocked fH binding to wild-type fHbp than Abs elicited by fHbp that bound fH. Thus, a mutant fHbp vaccine that does not bind fH but that retains immunogenicity is predicted to be superior in humans to an fHbp vaccine that binds human fH. In the case of mutant fHbp vaccination, the resultant Ab responses may be directed more at epitopes in or near the fH binding site, which result in greater complement-mediated serum bactericidal activity; these epitopes may be obscured when human fH is bound to the wild-type fHbp vaccine.     

An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity

Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial.     

Molecular Characterization of Invasive Meningococcal Isolates from Countries in the African Meningitis Belt before Introduction of a Serogroup A Conjugate Vaccine

Background

The serogroup A conjugate meningococcal vaccine, MenAfriVac, was introduced in mass vaccination campaigns in December 2010 in Burkina Faso, Mali and Niger. In the coming years, vaccination will be extended to other African countries at risk of epidemics. To document the molecular characteristics of disease-causing meningococcal strains circulating in the meningitis belt of Africa before vaccine introduction, the World Health Organization Collaborating Centers on Meningococci in Europe and United States established a common strain collection of 773 isolates from cases of invasive meningococcal disease collected between 2004 and 2010 from 13 sub-Saharan countries.

Methodology

All isolates were characterized by multilocus sequence typing, and 487 (62%) were also analyzed for genetic variation in the surface antigens PorA and FetA. Antibiotic susceptibility was tested for part of the collection.

Principal Findings

Only 19 sequence types (STs) belonging to 6 clonal complexes were revealed. ST-5 clonal complex dominated with 578 (74.8%) isolates. All ST-5 complex isolates were remarkably homogeneous in their PorA (P1.20,9) and FetA (F3-1) and characterized the serogroup A strains which have been responsible for most epidemics during this time period. Sixty-eight (8.8%) of the 773 isolates belonged to the ST-11 clonal complex which was mainly represented by serogroup W135, while an additional 38 (4.9%) W135 isolates belonged to the ST-175 complex. Forty-eight (6.2%) serogroup X isolates from West Africa belonged to the ST-181 complex, while serogroup X cases in Kenya and Uganda were caused by an unrelated clone, ST-5403. Serogroup X, ST-181, emerged in Burkina Faso before vaccine introduction.

Conclusions

In the seven years preceding introduction of a new serogroup A conjugate vaccine, serogroup A of the ST-5 clonal complex was identified as the predominant disease-causing strain.     

Health and Economic Outcomes of Introducing the New MenB Vaccine (Bexsero) into the Italian Routine Infant Immunisation Programme

Introduction

In January 2013 a novel type of multicomponent protein-based vaccine against group B meningococcal disease was licensed by the European Medicines Agency. With the widespread use of the meningococcal serogroup C conjugate vaccines, serogroup B remains now the major cause of bacterial meningitis and septicaemia in young children in Europe. The aim of this study is to investigate the health and the economic outcomes of MenB vaccine introduction into the Italian routine mass vaccination programme.

Methods

The present work is structured in two main parts. Firstly, we assess the epidemiological burden of group B meningococcal disease using official hospitalisation and notification data from two of the most populated Italian regions (Lombardia and Piemonte) during a 6-year study period (2007-2012). Secondly, we evaluate the cost-effectiveness of the immunisation programme in Italy from the public health payer perspective under base case parameters assumptions and performing a comprehensive sensitivity analysis to assess the robustness and the uncertainty of our model results.

Results

MenB serotype is responsible for 59% of the 341 cases of Invasive Meningococcal Disease in Lombardia and Piemonte. Incidence rate for MenB infection is estimated to be 0.21/100,000/y resulting at the highest level in children ≤4 years of age. Although the new MenB vaccine can potentially prevent about one third of the disease cases in the Italian population, model results show this strategy is unlikely to be cost-effective (ICER value over €350,000/QALY) with a vaccine that prevents disease only. These results are robust under most of the sensitivity scenarios except when allowing for lower discount rates.

Discussion

The introduction of the novel vaccine into the routine immunisation schedule needs to be carefully evaluated. The new MenB vaccine has the potential to reduce the disease burden at the population level. However, from the Italian Health Service perspective, the immunisation programme is unlikely to be cost-effective at the current incidence levels and vaccine price.     

Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles

Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMV_lpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMV_dis). Antibody responses elicited in mice with nOMV_dis were compared to those elicited with nOMV_lpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMV_lpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMV_lpxl1 or OMV_dis) was versus PorA. Despite a considerable reduction of hfH binding to nOMV_dis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMV_lpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMV_dis and nOMV_lpxl1–immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model.     

Predicted Strain Coverage of a New Meningococcal Multicomponent Vaccine (4CMenB) in Spain: Analysis of the Differences with Other European Countries

Background

A novel meningococcal multicomponent vaccine, 4CMenB (Bexsero^®), has been approved in Europe, Canada, Australia and US. The potential impact of 4CMenB on strain coverage is being estimated by using Meningococcal Antigen Typing System (MATS), an ELISA assay which measures vaccine antigen expression and diversity in each strain. Here we show the genetic characterization and the 4CMenB potential coverage of Spanish invasive strains (collected during one epidemiological year) compared to other European countries and discuss the potential reasons for the lower estimate of coverage in Spain.

Material and Methods

A panel of 300 strains, a representative sample of all serogroup B Neisseria meningitidis notified cases in Spain from 2009 to 2010, was characterized by multilocus sequence typing (MLST) and FetA variable region determination. 4CMenB vaccine antigens, PorA, factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were molecularly typed by sequencing. PorA coverage was assigned to strain with VR2 = 4. The levels of expression and cross-reactivity of fHbp, NHBA and NadA were analyzed using MATS ELISA.

Findings

Global estimated strain coverage by MATS was 68.67% (95% CI: 47.77–84.59%), with 51.33%, 15.33% and 2% of strains covered by one, two and three vaccine antigens, respectively. The predicted strain coverage by individual antigens was: 42% NHBA, 36.33% fHbp, 8.33% PorA and 1.33% NadA. Coverage within the most prevalent clonal complexes (cc) was 70.37% for cc 269, 30.19% for cc 213 and 95.83% for cc 32.

Conclusions

Clonal complexes (cc) distribution accounts for variations in strain coverage, so that country-by-country investigations of strain coverage and cc prevalence are important. Because the cc distribution could also vary over time, which in turn could lead to changes in strain coverage, continuous detailed surveillance and monitoring of vaccine antigens expression is needed in those countries where the multicomponent vaccine is introduced. This is really important in countries like Spain where most of the strains are predicted to be covered by only one vaccine antigen and the chance for escape mutants to emerge with vaccine use is higher. Based on the observed data, cc213 should receive special attention as it is associated with low predicted strain coverage, and has recently emerged in Spain.     

Over‐expression of fHbp in Arabdopsis for development of meningococcal serogroup B subunit vaccine

Due to lack of commercial vaccine against the serogroup B (MenB) of Neisseria meningitides, the incidence of meningococcal disease remains high. To solve the issue, transgenic plants are used as bioreactors to produce a plant‐derived fHbp subunit vaccine. In this study, the fHbp gene was optimized according to the codon usage bias of Arabidopsis thaliana, synthesized artificially, cloned into an expression vector, driven by a seed‐specific promoter, and introduced into A. thaliana by Agrobacterium‐mediated floral‐dip transformation. Transgenic plants were identified by glufosinate selection, quickstix strips for PAT/bar tests and PCR analysis. The five plants showing higher expression of recombinant fHbp were screened through indirect ELISA. Southern blot analysis showed that the transgenic line rHF‐22 had a single‐copy integration and the highest expression of fHbp. Recombinant fHbp was purified from seeds of rHF‐22 by nitrilotriacetic acid‐mediated affinity chromatography, and the purity was 82.5%. BALB/c mice were tested for fHbp vaccine protection from lethal MenB infection, and the relative percent survival was found to be 80%. This study indicates that the recombinant fHbp produced from seeds of rHF‐22 is a potential candidate for commercial MenB vaccine. It also provides a reference for safe, cheap and large‐scale production of other plant‐made vaccines.     

FetA Antibodies Induced by an Outer Membrane Vesicle Vaccine Derived from a Serogroup B Meningococcal Isolate with Constitutive FetA Expression

Invasive meningococcal disease causes over 3500 cases each year in Europe, with particularly high incidence among young children. Among serogroup B meningococci, which cause most of the cases, high diversity in the outer membrane proteins (OMPs) is observed in endemic situations; however, comprehensive molecular epidemiological data are available for the diversity and distribution of the OMPs PorA and FetA and these can be used to rationally design a vaccine with high coverage of the case isolates. The aim of this study was to determine whether outer membrane vesicles (OMVs) derived from an isolate with constitutive FetA expression (MenPF-1 vaccine) could be used to induce antibodies against both the PorA and FetA antigens. The immunogenicity of various dose levels and number of doses was evaluated in mice and rabbits, and IgG antibody responses tested against OMVs and recombinant PorA and FetA proteins. A panel of four isogenic mutants was generated and used to evaluate the relative ability of the vaccine to induce serum bactericidal activity (SBA) against FetA and PorA. Sera from mice were tested in SBA against the four target strains. Results demonstrated that the MenPF-1 OMVs were immunogenic against PorA and FetA in both animal models. Furthermore, the murine antibodies induced were bactericidal against isogenic mutant strains, suggesting that antibodies to both PorA and FetA were functional. The data presented indicate that the MenPF-1 vaccine is a suitable formulation for presenting PorA and FetA OMPs in order to induce bactericidal antibodies, and that proceeding to a Phase I clinical trial with this vaccine candidate is justified.     

The Development of an Experimental Multiple Serogroups Vaccine for Neisseria meningitidis

A native outer membrane vesicles (NOMV) vaccine was developed from three antigenically diverse strains of Neisseria meningitidis that express the L1,8, L2, and L3,7 lipooligosaccharide (LOS) immunotypes, and whose synX, and lpxL1 genes were deleted.. Immunogenicity studies in mice showed that the vaccine induced bactericidal antibody against serogroups B, C, W, Y and X N. meningitidis strains. However, this experimental NOMV vaccine was not effective against serogroup A N. meningitidis strains. N. meningitidis capsular polysaccharide (PS) from serogroups A, C, W and Y were effective at inducing bactericidal antibody when conjugated to either tetanus toxoid or the fHbp1-fHbp2 fusion protein fHbp(1+2). The combination of the NOMV vaccine and the N. meningitidis serogroup A capsular polysaccharide (MAPS) protein conjugate was capable of inducing bactericidal antibodies against a limited number of N. meningitidis strains from serogroups A, B, C, W, Y and X tested in this study.     

A phase II, randomized study on an investigational DTPw-HBV/Hib-MenAC conjugate vaccine administered to infants in Northern Ghana

Background

Combining meningococcal vaccination with routine immunization in infancy may reduce the burden of meningococcal meningitis, especially in the meningitis belt of Africa. We have evaluated the immunogenicity, persistence of immune response, immune memory and safety of an investigational DTPw-HBV/Hib-MenAC conjugate vaccine given to infants in Northern Ghana.

Methods and Findings

In this phase II, double blind, randomized, controlled study, 280 infants were primed with DTPw-HBV/Hib-MenAC or DTPw-HBV/Hib vaccines at 6, 10 and 14 weeks of age. At 12 months of age, children in each group received a challenge dose of serogroup A+C polysaccharides. Antibody responses were assessed pre, and one month-post dose 3 of the priming schedule and pre and 1 month after administration of the challenge dose. One month post-dose 3, 87.8% and 88.2% of subjects in the study group had bactericidal meningococcal serogroup A (SBA-MenA) and meningococcal serogroup C (SBA-MenC) antibody titres ≥1∶8 respectively. Seroprotection/seropositivity rates to the 5 antigens administered in the routine EPI schedule were non-inferior in children in the study group compared to those in the control group. The percentages of subjects in the study group with persisting SBA-MenA titres ≥ 1∶8 or SBA-MenC titres ≥1∶8 at the age of 12 months prior to challenge were significantly higher than in control group (47.7% vs 25.7% and 56.4% vs 5.1% respectively). The administration of 10 μg of serogroup A polysaccharide increased the SBA-MenA GMT by 14.0-fold in the DTPW-HBV/HibMenAC-group compared to a 3.8 fold increase in the control-group. Corresponding fold-increases in SBA-MenC titres following challenge with 10 μg of group C polysaccharide were 18.8 and 1.9 respectively. Reactogenicity following primary vaccination or the administration of the challenge dose was similar in both groups, except for swelling (Grade 3) after primary vaccination which was more frequent in children in the vaccine than in the control group (23.7%; 95%CI [19.6–28.1] of doses vs 14.1%; 95% CI [10.9–17.8] of doses). Fifty-nine SAEs (including 8 deaths), none of them related to vaccination, were reported during the entire study.

Conclusions

Three dose primary vaccination with DTPw-HBV/Hib-MenAC was non-inferior to DTPw-HBV/Hib for the 5 common antigens used in the routine EPI schedule and induced bactericidal antibodies against Neisseria meningitidis of serogroups A and C in the majority of infants. Serogroup A and C bactericidal antibody levels had fallen below titres associated with protection in nearly half of the infants by the age of 12 months confirming that a booster dose is required at about that age. An enhanced memory response was shown after polysaccharide challenge. This vaccine could provide protection against 7 important childhood diseases (including meningococcal A and C) and be of particular value in countries of the African meningitis belt.

Trial Registration

Controlled-Trials.com ISRCTN35754083     

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.     

Persistence of bactericidal antibodies following early infant vaccination with a serogroup B meningococcal vaccine and immunogenicity of a preschool booster dose

Background:

The multicomponent serogroup B meningococcal (4CMenB) vaccine was recently licensed for use in Europe. There are currently no data on the persistence of bactericidal antibodies induced by use of this vaccine in infants. Our objective was to evaluate serogroup B–specific bactericidal antibodies in children aged 40–44 months previously vaccinated at 2, 4, 6 and 12 months of age.

Methods:

Participants given 4 doses of 4CMenB as infants received a fifth dose of the vaccine at 40–44 months of age. Age-matched participants who were MenB vaccine–naive received 4CMenB and formed the control group. We evaluated human complement serum bactericidal activity (hSBA) titres at baseline and 1 month after each dose of 4CMenB.

Results:

Before a booster dose at enrolment, 41%–76% of 17 participants previously vaccinated with 4CMenB in infancy had hSBA titres of 4 or greater against 4 reference strains. Before vaccination in the control group (n = 40) these proportions were similar for strains 44/76-SL (63%) and {"type":"entrez-nucleotide","attrs":{"text":"M10713","term_id":"209124","term_text":"M10713"}}M10713 (68%) but low for strains NZ98/254 (0%) and 5/99 (3%). A booster dose in the 4CMenB-primed participants generated greater increases in hSBA titres than in controls.

Interpretation:

As has been observed with other meningococcal vaccines, bactericidal antibodies waned after vaccination with 4CMenB administered according to an approved infant vaccination schedule of 2, 4, 6 and 12 months of age, but there was an anamnestic response to a booster dose at 40–44 months of age. If 4CMenB were introduced into routine vaccination schedules, assessment of the need for a booster dose would require data on the impact of these declining titres on vaccine effectiveness. ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT01027351","term_id":"NCT01027351"}}NCT01027351A vaccine against serogroup B meningococcus has recently been licensed for use in Europe¹ and is being considered for licensure in Canada. This vaccine, known as multicomponent serogroup B meningococcal (4CMenB) vaccine, consists of 3 recombinant proteins: factor H binding protein (fHbp), Neisseria adhesin A (NadA) and Neisseria heparin binding antigen (NHBA) combined with detoxified outer membrane vesicles from the strain responsible for an epidemic of serogroup B meningococcal disease in New Zealand (NZ98/254). Clinical trials of 4CMenB have shown it to be immunogenic against reference strains selected to speciScally express one of the vaccine antigens.^2–6 On the basis of these trials, the approved schedule for infants aged 2 to 5 months is 3 doses given at least 1 month apart, with a booster dose given at 12 to 23 months of age.⁷ The persistence of vaccine-induced antibodies throughout childhood following this booster dose is unknown, but it is particularly relevant because the incidence of invasive serogroup B meningococcal disease in children aged 1 to 4 years is second only to the incidence in children less than 1 year of age.⁸In this study, we assessed the persistence of these bactericidal antibodies in children aged 40–44 months who had previously received either 4CMenB or a vaccine containing the recombinant proteins alone (recombinant protein serogroup B meningococcal [rMenB] vaccine) at 2, 4, 6 and 12 months of age.³ We also assessed the immunogenicity and reactogenicity of a booster dose.     

Deconvolution of intergenic polymorphisms determining high expression of Factor H binding protein in meningococcus and their association with invasive disease

Neisseria meningitidis is a strictly human pathogen and is the major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a meningococcal surface-exposed lipoprotein that binds the human Complement factor H allowing the bacterium to evade the host innate immune response. FHbp is also a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, level of fHbp expression varies among isolates and has been correlated to differences in promoter sequences upstream of the gene. Here we elucidated the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of more than 5800 strains representative of the UK circulating isolates and we identified eleven fIR sequence alleles which represent 88% of meningococcal strains. By engineering isogenic recombinant strains where fHbp expression was under the control of each of the eleven fIR alleles, we confirmed that the fIR sequence determines a specific and distinct level of expression. Moreover, we identified the molecular basis for variation in expression through polymorphisms within key regulatory regions that are known to affect fHbp expression. We experimentally established three expression groups, high–medium–low, that correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies and the ability of the meningococcal strain to survive within human serum. By using this sequence classification and information about the variant, we predicted fHbp expression in the panel of UK strains and we observed that strains with higher expressing fIR alleles are more likely associated with invasive disease. Overall, our findings can contribute to understand and predict vaccine coverage mediated by fHbp as well as to shed light on the role of this virulence factor in determining an invasive phenotype.     

Meningococcal X polysaccharide quantification by high-performance anion-exchange chromatography using synthetic N-acetylglucosamine-4-phosphate as standard

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) is described. The polysaccharide is hydrolyzed by strong acidic treatment, and the peak of glucosamine-4-phosphate (4P-GlcN) is detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was used. This method for MenX quantification is highly selective and sensitive, and it constitutes an important analytical tool for the development of a conjugate vaccine against MenX.     

WHO/Health Canada meeting on regulatory considerations for evaluation and licensing of new meningococcal Group B vaccines,Ottawa, Canada, 3–4 October 2011

Serogroup B Neisseria meningitides (MenB) is a significant cause of endemic and epidemic outbreaks of the disease worldwide. Although polysaccharide and conjugate vaccines are available against other meningococcal serogroups, the poor immunogenicity of MenB polysaccharide has led to the development of protein-based vaccines. However, the diversity and antigenic variability of MenB strains has been a major challenge. Recently a new generation of MenB vaccines that contain conserved antigens has been developed to provide broader coverage and they are in an advanced stage of development and regulatory consideration. In October 2011, the World Health Organization and Health Canada jointly organized a consultation on regulatory considerations for the evaluation and licensing of new MenB vaccines. The aim was to seek consensus on key regulatory issues relevant to the evaluation of candidate MenB vaccines and on approaches to the standardisation of in vitro assays used in the evaluation process. Participants agreed that functional antibodies as measured in the Serum Bactericidal Activity (SBA) assay could be used to evaluate MenB vaccine efficacy and ways of improving assay standardization proposed. Approaches to bridging SBA data to large collections of strains in order to give an indication of the prospective breadth of vaccine coverage were discussed.     

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity

Background

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity.

Methods and Principal Findings

Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity.

Conclusions

The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.     

Persistence of specific bactericidal antibodies at 5 years of age after vaccination against serogroup B meningococcus in infancy and at 40 months

Background:

The multicomponent serogroup B meningococcal (4CMenB) vaccine induces antibodies against indicator strains of serogroup B meningococcus under various schedules. We investigated the persistence of antibodies in 5-year-old children 18–20 months after their last dose (at about 3.5 years of age).

Methods:

We assessed 5-year-old children who received the 4CMenB vaccine or a recombinant protein vaccine in a previous randomized trial. We also recruited 50 vaccine-naive 5-year-olds and administered 2 doses of 4CMenB to each child. We measured serum bactericidal antibody titres against 4 indicator strains of serogroup B meningococcus matched to each individual vaccine component and against 4 mismatched strains.

Results:

Of those who received the 4CMenB vaccine at 2, 4, 6, 12 and 40 months (n = 16), the percentage with protective antibody titres (≥ 1:4) at 60 months ranged from 44% to 88% against matched strains and from 13% to 81% against mismatched strains. Loss of protective titres was also observed for those who received the 4CMenB vaccine at 12, 40 and 42 months (n = 5) (80%–100% against matched strains, 60%–100% against mismatched strains) or at 40 and 42 months (n = 29) (31%–100% against matched strains, 41%–81% against mismatched strains). Administering the 4CMenB vaccine to 5-year-old children yielded protective titres against matched strains in 92%–100% and against mismatched strains in 59%–100%. The majority of these children reported injection-site pain (40/50 [80%] after dose 1, 39/46 [85%] after dose 2) and erythema (47/50 [94%] and 40/46 [87%], respectively); rates of fever were low (5/50 [10%] and 2/46 [4%], respectively).

Interpretation:

Waning of immunity by 5 years of age occurred after receipt of the 4CMenB vaccine in infancy, even with an additional booster at 40 months. The 4CMenB vaccine is immunogenic and was fairly well tolerated by 5-year-old children, although injection-site pain was noteworthy. Trial registration: ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT01027351","term_id":"NCT01027351"}}NCT01027351The multicomponent serogroup B meningococcal (4CMenB) vaccine is licensed in the European Union, Australia and Canada to prevent serogroup B meningococcal disease. It was developed using “reverse vaccinology,” in which candidate antigens were identified by interrogating the whole meningococcal genome.¹ The 4CMenB vaccine consists of 3 surface proteins (factor H binding protein [fHbp], Neisseria adhesin A [NadA] and Neisseria heparin-binding antigen [NHBA]), along with a fourth component, the outer membrane vesicle, which acts as both antigen and adjuvant.¹Group B meningococcal disease is a potentially devastating condition, with an average case fatality rate of 5.2% (data for England and Wales²), and over a third of survivors are left with measurable functional deficits.³ The incidence of laboratory-confirmed cases is about 1 per 100 000 population in England⁴ and 0.33 per 100 000 population in Canada.⁵ The recommendation of the United Kingdom Joint Committee on Vaccination and Immunisation that the 4CMenB vaccine be introduced into the routine UK immunization schedule should, if implemented, lead to a reduction in this morbidity and mortality.⁶ Data on the persistence of antibody responses following infant or toddler immunization, and after subsequent boosting, remain limited yet will be important for guiding implementation of this recommendation.We present here the results of a follow-on study investigating the persistence of antibodies 18–20 months after the last dose in 5-year-old children previously immunized under a variety of schedules with 4CMenB vaccine or another investigational vaccine (recombinant protein serogroup B meningococcal [rMenB] vaccine), which lacks the outer membrane vesicle component of the 4CMenB vaccine. Since the original infant study,⁷ 4CMenB vaccine has emerged as the preferred vaccine, because addition of the outer membrane vesicle component improves the breadth of strain coverage;⁸ however, the extension study continued follow-up for all of the original children, and all results are therefore presented here.