Structure of a protective epitope reveals the importance of acetylation of Neisseria meningitidis serogroup A capsular polysaccharide.

Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup A Neisseria meningitidis (MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to be O-acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via the O- and N-acetyl moieties through either H-bonding or CH-π interactions. In silico docking showed that 3-O-acetylation of the upstream residue is essential for antibody binding, while O-acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies.

Synergic complement-mediated bactericidal activity of monoclonal antibodies with distinct specificity.

The classical complement pathway is triggered when antigen-bound immunoglobulins bind to C1q through their Fc region. While C1q binds to a single Fc with low affinity, a higher avidity stable binding of two or more of C1q globular heads initiates the downstream reactions of the complement cascade ultimately resulting in bacteriolysis. Synergistic bactericidal activity has been demonstrated when monoclonal antibodies recognize nonoverlapping epitopes of the same antigen. The aim of the present work was to investigate the synergistic effect between antibodies directed toward different antigens. To this purpose, we investigated the bactericidal activity induced by combinations of monoclonal antibodies (mAbs) raised against factor H-binding protein (fHbp) and Neisserial Heparin-Binding Antigen (NHBA), two major antigens included in Bexsero, the vaccine against Meningococcus B, for prevention from this devastating disease in infants and adolescents. Collectively, our results show that mAbs recognizing different antigens can synergistically activate complement even when each single Mab is not bactericidal, reinforcing the evidence that cooperative immunity induced by antigen combinations can represent a remarkable added value of multicomponent vaccines. Our study also shows that the synergistic effect of antibodies is modulated by the nature of the respective epitopes, as well as by the antigen density on the bacterial cell surface.

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays.

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins.

Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines against Streptococcus pneumoniae and Salmonella enterica Serovar Typhi.

B cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/- and TdT-/- mice generated comparable antibody responses to Pneumovax23 and survived Streptococcus pneumoniae challenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/- or TdT-/- mice conferred protection. TdT+/- and TdT-/- mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity against S Typhi in vitro To test the protective immunity conferred by ViPS immunization in vivo, TdT+/- and TdT-/- mice were challenged with a chimeric Salmonella enterica serovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts for S Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/- and TdT-/- mice challenged with ViPS-expressing S Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus.

Scrub typhus, caused by Orientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targeting O. tsutsugamushi 47-kDa, groEL, and human interferon beta (IFN-ß gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.

Evaluation of immunological responses to recombinant Porin A protein (rPoA) from native strains of Neisseria meningitidis serogroups A and B using OMV as an adjuvant in BALB/c mice.

Neisseria meningitidis is one of the main causes of sepsis and meningitis, which are two serious life-threatening diseases in both children and adolescents. Porin A (porA) from both serogroup A and B were cloned into the pET28a plasmid and expressed in E. coli BL21 (DE3). The protein was expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. BALB/c mice were subcutaneously injected three times with 25 µg of the recombinant PorA. Specific total IgG antibodies and isotypes were evaluated using ELISA assay. Opsonophagocytic assay (OPA) and Serum Bactericidal assay (SBA) were performed. Results showed that vaccinated mice exhibited higher levels of anti-Porin A (p < 0.05) with a predominant IgG1 response compared to the control group. Results from in vitro experiments indicated that N. meningitidis was opsonized with immunized-mice sera, and compared to non-immunized mice, immunized mice displayed significantly increased phagocytic uptake and effective intracellular killing. In this study, serogroup B N. meningitidis OMV of strain CSBPI G-245 and complete and incomplete Freund's adjuvant were used. Results demonstrated that Porin A could be a valuable target for the development of immunotherapeutic strategies against N. meningitidis.

Dose sparing and the lack of a dose-response relationship with an influenza vaccine in adult and elderly patients - a randomized, double-blind clinical trial.

AIMS: The currently licensed seasonal trivalent influenza vaccines contain 15 µg haemagglutinin per strain for adult, and up to 60 µg for elderly patients. However, due to recent shortages, dose sparing to increase production capacity would be highly desirable. In the present study, we attempted to find a dose-response relationship for immunogenicity and, thus, the optimal dose for seasonal influenza vaccines in adult and elderly patients. METHODS: A total of 256 subjects, including adult (aged 18-60 years) and elderly (aged over 60 years) individuals, were enrolled. Subjects were randomly assigned in a 1:1:1:1 ratio to receive a whole-virion, aluminium-adjuvanted trivalent influenza vaccine containing 3.5, 6, 9 or 15 µg haemagglutinin of seasonal A/H1N1, A/H3N2 and B influenza antigens manufactured by Omninvest Ltd., Hungary. Serum antibody titres against the vaccine virus strains were measured by haemagglutination inhibition. RESULT: All vaccines were well tolerated. All four vaccines fulfilled all three immunogenicity licensing criteria, as determined by the European Committee for Proprietary Medicinal Products (CPMP)/Biotechnology Working Party (BWP)/214/96 guideline for all three virus strains and both age groups. The 3.5 µg vaccine showed 28% less seroconversion compared to the 15 µg dose in terms of influenza AH3N2 in the adult group (95% confidence interval -51, -3; P < 0.05). All other doses showed no significant difference in immunogenicity compared with the licensed vaccine containing 15 µg haemagglutinin. CONCLUSIONS: Our data suggested that significant dose sparing is possible with the use of whole-virion vaccines and aluminium adjuvants, without compromising safety. This could have significant economic and public health impacts.

Impaired Immunogenicity of Meningococcal Neisserial Surface Protein A in Human Complement Factor H Transgenic Mice.

Neisserial surface protein A (NspA) is a highly conserved outer membrane protein previously investigated as a meningococcal vaccine candidate. Despite eliciting serum bactericidal activity in mice, a recombinant NspA vaccine failed to elicit serum bactericidal antibodies in a phase 1 clinical trial in humans. The discordant results may be explained by the recent discovery that NspA is a human-specific ligand of the complement inhibitor factor H (FH). Therefore, in humans but not mice, NspA would be expected to form a complex with FH, which could impair human anti-NspA protective antibody responses. To investigate this question, we immunized human FH transgenic BALB/c mice with three doses of recombinant NspA expressed in Escherichia coli microvesicles, with each dose being separated by 3 weeks. Three of 12 (25%) transgenic mice and 13 of 14 wild-type mice responded with bactericidal titers of ≥1:10 in postimmunization sera (P = 0.0008, Fisher's exact test). In contrast, human FH transgenic and wild-type mice immunized with a control meningococcal native outer membrane vesicle vaccine had similar serum bactericidal antibody responses directed at PorA, which is not known to bind human FH, and a mutant factor H binding protein (FHbp) antigen with a >50-fold lower level of FH binding than wild-type FHbp antigen binding.Thus, human FH can impair anti-NspA serum bactericidal antibody responses, which may explain the poor immunogenicity of the NspA vaccine previously tested in humans. A mutant NspA vaccine engineered to have decreased binding to human FH may increase protective antibody responses in humans.

Fecal diagnostics in combination with serology: best test to establish STEC-HUS.

BACKGROUND: In the majority of pediatric patients, the hemolytic-uremic syndrome (HUS) is caused by an infection with Shiga toxin-producing Escherichia coli (STEC), mostly serotype O157. It is important to discriminate between HUS caused by STEC and complement-mediated HUS (atypical HUS) due to differences in treatment and outcome. As STEC and its toxins can only be detected in the patient's stool for a short period of time after disease onset, the infectious agent may go undetected using only fecal diagnostic tests. Serum antibodies to lipopolysaccharide (LPS) of STEC persist for several weeks and may therefore be of added value in the diagnosis of STEC. METHODS: All patients with clinical STEC-HUS who were treated at Radboud University Medical Center between 1990 and 2014 were included in this retrospective single-center study. Clinical and diagnostic microbiological data were collected. Immunoglobulin M (IgM) antibodies against LPS of STEC serotype O157 were detected by a serological assay (ELISA). RESULTS: Data from 65 patients weres available for analysis. Fecal diagnostic testing found evidence of an STEC infection in 34/63 patients (54 %). Serological evidence of STEC O157 was obtained in an additional 16 patients. This is an added value of 23 % (p < 0.0001) when the serological antibody assay is used in addition to standard fecal diagnostic tests to confirm the diagnosis STEC-HUS. This added value becomes especially apparent when the tests are performed more than 7 days after the initial manifestation of the gastrointestinal symptoms. CONCLUSIONS: The serological anti-O157 LPS assay clearly makes a positive contribution when used in combination with standard fecal diagnostic tests to diagnose STEC-HUS and should be incorporated in clinical practice.

Characterization of the chaperonin GroEL in Mycoplasma gallisepticum.

Mycoplasma gallisepticum (MG) is a common and widespread cause of chronic respiratory disease in poultry. In this study, antigenic proteins were identified from MG membrane using two-dimensional gel electrophoresis (2-DE) analysis followed by Western blot and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), including translation elongation factor Tu, dihydrolipoamide acetyltransferase (E2) component of pyruvate dehydrogenase complex, trigger factor, chaperone protein DnaK, heat shock protein GroEL and so on. Furthermore, recombinant MG GroEL protein was successfully expressed in E. coli BL21 (DE3) with pET-28a (+) vector and found to possess ATPase activity and contributed to the refolding of recombinant MG PrpC protein. Complement-dependent bactericidal assay indicated that the rabbit antisera against MG rGroEL had satisfactory bactericidal effect, which is similar to the chicken antisera induced by MG-inactivated vaccine, suggesting MG GroEL is a protective antigen, could be used as a novel vaccine candidate. This study is the first report of the biological characterization of chaperone GroEL protein in MG.

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. NCT01027351.

Outer membrane protein OlpA contributes to Moraxella catarrhalis serum resistance via interaction with factor H and the alternative pathway.

Factor H is an important complement regulator of the alternative pathway commonly recruited by pathogens to achieve increased rates of survival in the human host. The respiratory pathogen Moraxella catarrhalis, which resides in the mucosa, is highly resistant to the bactericidal activity of serum and causes otitis media in children and respiratory tract infections in individuals with underlying diseases. In this study, we show that M. catarrhalis binds factor H via the outer membrane protein OlpA. M. catarrhalis serum resistance was dramatically decreased in the absence of either OlpA or factor H, demonstrating that this inhibition of the alternative pathway significantly contributes to the virulence of M. catarrhalis.

Evasion of killing by human antibody and complement through multiple variations in the surface oligosaccharide of Haemophilus influenzae.

The lipopolysaccharide (LPS) of H. influenzae is highly variable. Much of the structural diversity is derived from phase variation, or high frequency on-off switching, of molecules attached during LPS biosynthesis. In this study, we examined the dynamics of LPS phase variation following exposure to human serum as a source of antibody and complement in multiple H. influenzae isolates. We show that lic2A, lgtC and lex2A switch from phase-off to phase-on following serial passage in human serum. These genes, which control attachment of a galα1-4gal di-galactoside structure (lic2A and lgtC phase-on) or an alternative glucose extension (lex2A phase-on) from the same hexose moiety, reduce binding of bactericidal antibody to conserved inner core LPS structures. The effects of the di-galactoside and alternative glucose extension were also examined in the context of the additional LPS phase variable structures phosphorylcholine (ChoP) and sialic acid. We found that di-galactoside, the alternative glucose extension, ChoP, and sialic acid each contribute independently to bacterial survival in the presence of human complement, and have an additive effect in combination. We propose that LPS phase variable extensions serve to shield conserved inner core structures from recognition by host immune components encountered during infection.

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.

ZnuD, a potential candidate for a simple and universal Neisseria meningitidis vaccine.

Neisseria meningitidis serogroup B (MenB) is a major cause of bacterial sepsis and meningitis, with the highest disease burden in young children. Available vaccines are based on outer membrane vesicles (OMVs) obtained from wild-type strains. However, particularly in toddlers and infants, they confer protection mostly against strains expressing the homologous protein PorA, a major and variable outer membrane protein. In the quest for alternative vaccine antigens able to provide broad MenB strain coverage in younger populations, but potentially also across all age groups, ZnuD, a protein expressed under zinc-limiting conditions, may be considered a promising candidate. Here, we have investigated the potential value of ZnuD and show that it is a conserved antigen expressed by all MenB strains tested except for some strains of clonal complex ST-8. In mice and guinea pigs immunized with ZnuD-expressing OMVs, antibodies were elicited that were able to trigger complement-mediated killing of all the MenB strains and serogroup A, C, and Y strains tested when grown under conditions of zinc limitation. ZnuD is also expressed during infection, since anti-ZnuD antibodies were detected in sera from patients. In conclusion, we confirm the potential of ZnuD-bearing OMVs as a component of an effective MenB vaccine.

Use of expanded Neisseria meningitidis serogroup B panels with the serum bactericidal antibody assay for the evaluation of meningococcal B vaccine effectiveness.

INTRODUCTION: Neisseria meningitidis serogroup B (NmB) antigens are inherently diverse with variable expression among strains. Prediction of meningococcal B (MenB) vaccine effectiveness therefore requires an assay suitable for use against large panels of epidemiologically representative disease-causing NmB strains. Traditional serum bactericidal antibody assay using exogenous human complement (hSBA) is limited to the quantification of MenB vaccine immunogenicity on a small number of indicator strains. AREAS COVERED: Additional and complementary methods for assessing strain coverage developed previously include the Meningococcal Antigen Typing System (MATS), Meningococcal Antigen Surface Expression (MEASURE) assay, and genotyping approaches, but these do not estimate vaccine effectiveness. We provide a narrative review of these methods, highlighting a more recent approach involving the hSBA assay in conjunction with expanded NmB strain panels: hSBA assay using endogenous complement in each vaccinated person's serum (enc-hSBA) against a 110-strain NmB panel and the traditional hSBA assay against 14 (4 + 10) NmB strains. EXPERT OPINION: The enc-hSBA is a highly standardized, robust method that can be used in clinical trials to measure the immunological effectiveness of MenB vaccines under conditions that mimic real-world settings as closely as possible, through the use of endogenous complement and a diverse, epidemiologically representative panel of NmB strains.

Meningococcal disease refers to illnesses caused by the bacterium Neisseria meningitidis (meningococcus), including infections of the brain lining and spinal cord (meningitis) and bloodstream (septicemia). It is rare but often severe and can be deadly. Invasive meningococcal disease can be prevented through vaccination. Nearly all cases are caused by six serogroups (types) of meningococci, including meningococcal serogroup B. Vaccines are available against meningococcal serogroup B but, because of the uncommonness of the disease, standard clinical trials could not be performed to prove these vaccines are effective. Instead, an indirect measure, called the 'hSBA assay' (serum bactericidal antibody assay using human complement), is used to measure the ability of vaccines to provide protection against specific N. meningitidis strains that have antigens (substances that cause the immune system to react) sharing characteristics with components of the vaccines. However, meningococcal serogroup B strains are diverse in the genetic composition and expression of vaccine antigens. Hence, a large number of N. meningitidis serogroup B strains would have to be tested to make sure that the vaccine is effective against these strains. This is not feasible using the traditional hSBA assay, which requires a human complement (a protein system, which is part of the immune system) that has not come from the vaccinated person and is difficult and time-consuming to source. Recently, an alternative hSBA assay was developed that uses the complement present in each vaccinated person's blood (endogenous complement) and which overcomes these challenges. By allowing testing against a broad panel of N. meningitidis serogroup B strains, this new assay may enable a more accurate estimation of the effectiveness of vaccines against serogroup B meningococci.

Immunogenicity and tolerability of recombinant serogroup B meningococcal vaccine administered with or without routine infant vaccinations according to different immunization schedules: a randomized controlled trial.

CONTEXT: In the absence of an effective vaccine, serogroup B Neisseria meningitidis (MenB) remains a major cause of invasive disease in early childhood in developed countries. OBJECTIVE: To determine the immunogenicity and reactogenicity of a multicomponent MenB vaccine (4CMenB) and routine infant vaccines when given either concomitantly or separately. DESIGN, SETTING, AND PARTICIPANTS: Phase 2b, multicenter, open-label, parallel-group, randomized controlled study of 1885 infants enrolled at age 2 months from August 2008 to July 2010 in Europe. INTERVENTION: Participants were randomized 2:2:1:1 to receive (1) 4CMenB at 2, 4, and 6 months with routine vaccines (7-valent pneumococcal and combined diphtheria, tetanus, acellular pertussis, inactivated polio, hepatitis B, Haemophilus influenzae type b vaccines); (2) 4CMenB at 2, 4, and 6 months and routine vaccines at 3, 5, and 7 months; (3) 4CMenB with routine vaccines at 2, 3, and 4 months; or (4) routine vaccines alone at 2, 3, and 4 months. MAIN OUTCOME MEASURES: Percentage of participants with human complement serum bactericidal activity (hSBA) titer of 1:5 or greater against 3 MenB strains specific for vaccine antigens (NZ98/254, 44/76-SL, and 5/99). RESULTS: After three 4CMenB vaccinations, 99% or more of infants developed hSBA titers of 1:5 or greater against strains 44/76-SL and 5/99. For NZ98/254, this proportion was 79% (95% CI, 75.2%-82.4%) for vaccination at 2, 4, and 6 months with routine vaccines, 86.1% (95% CI, 82.9%-89.0%) for vaccination at 2, 4, and 6 months without routine vaccines, and 81.7% (95% CI, 76.6%-86.2%) for vaccination at 2, 3, and 4 months with routine vaccines. Responses to routine vaccines given with 4CMenB were noninferior to routine vaccines alone for all antigens, except for the responses to pertactin and serotype 6B pneumococcal polysaccharide. Fever was seen following 26% (158/602) to 41% (247/607) of 4CMenB doses when administered alone, compared with 23% (69/304) to 36% (109/306) after routine vaccines given alone and 51% (306/605) to 61% (380/624) after 4CMenB and routine vaccines administered together. CONCLUSION: A 4CMenB vaccine is immunogenic against reference strains when administered with routine vaccines at 2, 4, and 6 or at 2, 3, and 4 months of age, producing minimal interference with the response to routine infant vaccinations. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00721396.

Defense from the Group A Streptococcus by active and passive vaccination with the streptococcal hemoprotein receptor.

BACKGROUND: The worldwide burden of the Group A Streptococcus (GAS) primary infection and sequelae is considerable, although immunization programs with broad coverage of the hyper variable GAS are still missing. We evaluate the streptococcal hemoprotein receptor (Shr), a conserved streptococcal protein, as a vaccine candidate against GAS infection. METHODS: Mice were immunized intraperitoneally with purified Shr or intranasally with Shr-expressing Lactococcus lactis. The resulting humoral response in serum and secretions was determined. We evaluated protection from GAS infection in mice after active or passive vaccination with Shr, and Shr antiserum was tested for bactericidal activity. RESULTS: A robust Shr-specific immunoglobulin (Ig) G response was observed in mouse serum after intraperitoneal vaccination with Shr. Intranasal immunization elicited both a strong IgG reaction in the serum and a specific IgA reaction in secretions. Shr immunization in both models allowed enhanced protection from systemic GAS challenge. Rabbit Shr antiserum was opsonizing, and mice that were administrated with Shr antiserum prior to the infection demonstrated a significantly higher survival rate than did mice treated with normal rabbit serum. CONCLUSIONS: Shr is a promising vaccine candidate that is capable of eliciting bactericidal antibody response and conferring immunity against systemic GAS infection in both passive and active vaccination models.

Validation and use of a serum bactericidal antibody assay for Neisseria meningitidis serogroup X in a seroprevalence study in Niger, West Africa.

Invasive meningococcal disease (IMD) affects approximately 1.2 million people worldwide annually. Prevention of IMD is mostly provided through vaccination; however, no licensed vaccine is currently available to protect against meningococcal serogroup X associated infection. Limited data are available on the natural immunity to Neisseria meningitidis serogroup X within the African sub-Saharan meningitis belt. The objective of the study was to provide an overview of natural immunity to serogroup X within a community in the African meningitis belt prior to the introduction of a pentavalent conjugate vaccine (NmCV-5). Prior to its introduction, a validated assay to assess vaccine efficacy was also required. This study therefore incorporated two objectives: a seroprevalence study to assess natural immunity in serum samples (n = 377) collected from Niger, West Africa in 2012, and the validation of a serogroup X serum bactericidal antibody (SBA) assay. Seroprevalence data obtained found that natural immunity to N. meningitidis serogroup X were present in 52.3% of study participants. The highest putative protective titres (≥8) to serogroup X were seen in age group 5-14 years-old (73.9%) and lowest in ages < 1 year old (0%). The SBA assay was successfully validated for selectivity/specificity, precision/reproducibility, linearity, and stability. This study demonstrated the suitability of the serogroup X SBA assay in clinical trials for future meningococcal conjugate vaccines containing serogroup X polysaccharides.

The Neisseria meningitidis macrophage infectivity potentiator protein induces cross-strain serum bactericidal activity and is a potential serogroup B vaccine candidate.

A gene encoding a 29-kDa protein from Neisseria meningitidis serogroup B strain MC58 with homology to the macrophage infectivity potentiator (MIP) protein of Legionella pneumophila was cloned and expressed in Escherichia coli, and the purified soluble recombinant protein (rMIP) was used for immunization studies. Analysis of the predicted amino acid sequences of MIP from 13 well-characterized meningococcal strains, isolated from carriers or patients and differing in serogroup, serotype, and subtype, showed that the protein was highly conserved (98 to 100%), with only three distinct sequence types (designated I, II, and III) found. Western blotting showed that the MIP protein was expressed at similar levels by all of these strains. Immunization of mice with type I MC58 rMIP in detergent micelles and liposomes containing monophosphoryl lipid A (MPLA) induced high levels of surface-reactive antibodies with serum bactericidal activity (SBA) titers of 1/1,024 against the homologous strain. Bactericidal antibodies were also induced with the protein in saline alone and liposomes alone (titers, 1/128) but not following adsorption to Al(OH)(3). Significantly, antisera raised against type I rMIP administered in saline or liposomes killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/256). Taken together, these findings suggest that rMIP can provide cross-strain protection against meningococci and should be considered a potential antigen for inclusion in new vaccines against meningococcal infection.