Genetic, Functional, and Immunogenic Analyses of the O-Linked Protein Glycosylation System in Neisseria meningitidis Serogroup A ST-7 Isolates.

Neisseria meningitidis exhibits a general O-linked protein glycosylation system in which pili and other extracytoplasmic proteins are glycosylated. To investigate glycan antigenicity in humans and the significance of high glycan diversity on immune escape mechanisms, we exploited serogroup A meningococcal strains and serum samples obtained from laboratory-confirmed Ethiopian patients with meningococcal disease. The 37 meningococcal isolates were sequenced, and their protein glycosylation (pgl) genotypes and protein glycosylation phenotypes were investigated in detail. An insertion sequence (IS1655) element in pglH reduced glycan variability in the majority of isolates, while phase variation strengthened glycan variability and microheterogeneity. Homologous recombination events within the pgl genes were identified in eight of the 37 isolates, and the phenotypic consequences ranged from none detected to altered glycoforms in two of the isolates in which the whole pgl locus was exchanged. Immunoblotting of sera against a complete panel of glycan-expressing mutant strains demonstrated that most of these patient sera had IgG antibodies against various neisserial protein glycan antigens. Furthermore, using a bactericidal assay comparing a wild-type meningococcal A strain and a glycosylation-null variant strain, we showed that these protein glycan antigens interfere with bactericidal killing by antibodies in patient sera. Altogether, we were largely able to link pgl genotype with glycosylation phenotype. Our study reveals that protein glycans seem to contribute to the ability of N. meningitidis to resist the bactericidal activity of human serum, possibly by masking protein epitopes important for bactericidal killing and thus protection against meningococcal disease. IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis. Extensive variability in protein glycan structure and antigenicity is due to phase variation of protein glycosylation genes and polymorphic gene content and function. The exact role(s) of glycosylation in Neisseria remains to be determined, but increasing evidence, supported by this study, suggests that glycan variability can be a strategy to escape the human immune system. The complexity of the O-linked protein glycosylation system requires further studies to fully comprehend how these bacteria utilize variation in pgl genes to produce such high glycoform diversity and to evade the human immune response.

Immunogenicity, Safety, and Efficacy of a Standalone Universal Influenza Vaccine, FLU-v, in Healthy Adults: A Randomized Clinical Trial.

Background: FLU-v is a broad-spectrum influenza vaccine that induces antibodies and cell-mediated immunity. Objective: To compare the safety, immunogenicity, and exploratory efficacy of different formulations and dosing regimens of FLU-v versus placebo. Design: Randomized, double-blind, placebo-controlled, single-center phase 2b clinical trial. (ClinicalTrials.gov: NCT02962908; EudraCT: 2015-001932-38). Setting: The Netherlands. Participants: 175 healthy adults aged 18 to 60 years. Intervention: 0.5-mL subcutaneous injection of 500 µg of adjuvanted (1 dose) or nonadjuvanted (2 doses) FLU-v (A-FLU-v or NA-FLU-v) or adjuvanted or nonadjuvanted placebo (A-placebo or NA-placebo) (2:2:1:1 ratio). Measurements: Vaccine-specific cellular responses at days 0, 42, and 180 were assessed via flow cytometry and enzyme-linked immunosorbent assay. Solicited information on adverse events (AEs) was collected for 21 days after vaccination. Unsolicited information on AEs was collected throughout the study. Results: The AEs with the highest incidence were mild to moderate injection site reactions. The difference between A-FLU-v and A-placebo in the median fold increase in secreted interferon-γ (IFN-γ) was 38.2-fold (95% CI, 4.7- to 69.7-fold; P = 0.001) at day 42 and 25.0-fold (CI, 5.7- to 50.9-fold; P < 0.001) at day 180. The differences between A-FLU-v and A-placebo in median fold increase at day 42 were 4.5-fold (CI, 2.3- to 9.8-fold; P < 0.001) for IFN-γ-producing CD4+ T cells, 4.9-fold (CI, 1.3- to 40.0-fold; P < 0.001) for tumor necrosis factor-α (TNF-α), 7.0-fold (CI, 3.5- to 18.0-fold; P < 0.001) for interleukin-2 (IL-2), and 1.7-fold (CI, 0.1- to 4.0-fold; P = 0.004) for CD107a. At day 180, differences were 2.1-fold (CI, 0.0- to 6.0-fold; P = 0.030) for IFN-γ and 5.7-fold (CI, 2.0- to 15.0-fold; P < 0.001) for IL-2, with no difference for TNF-α or CD107a. No differences were seen between NA-FLU-v and NA-placebo. Limitation: The study was not powered to evaluate vaccine efficacy against influenza infection. Conclusion: Adjuvanted FLU-v is immunogenic and merits phase 3 development to explore efficacy. Primary Funding Source: SEEK and the European Commission Directorate-General for Research and Innovation, European Member States within the UNISEC (Universal Influenza Vaccines Secured) project.

Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa.

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

Antibodies against Neisseria meningitidis serogroups A, C, W and Y in serum and saliva of Norwegian adolescents.

INTRODUCTION: The incidence of invasive meningococcal disease (IMD) among Norwegian 16-19-year-olds was 1-7/100,000 in the decade before the COVID-19 pandemic, with serogroup Y (MenY) dominance. In contrast to many other European countries, meningococcal vaccines are not part of the national immunisation program (NIP) in Norway. This cross-sectional study aimed to measure the degree of natural immunity against Neisseria meningitidis among adolescents in Norway to evaluate the need for introducing tetravalent meningococcal conjugate vaccine (MCV4) in the NIP. MATERIALS AND METHODS: Serum and saliva samples were collected from students in upper and lower secondary schools in Norway in 2018. Samples were analysed for meningococcal capsular polysaccharide (PS)-specific antibodies using a bead-based multiplex immunoassay. PS-specific antibody levels were linked to data on meningococcal carriage, vaccination status and risk factors for carriage (assessed with questionnaire) and analysed by linear regression of log transformed concentrations. A subset of samples from unvaccinated individuals was analysed for serum bactericidal antibodies (SBA). RESULTS: A total of 1344 participants, median age 16 years (range 12-24), were included in the study. Overall, 60.9% of the participants were female and 1137 (84.6%) were not vaccinated with MCV4. PS-specific antibody concentrations in serum and saliva were low among unvaccinated individuals for all serogroups and only 6.7-20.0% of the subpopulation with high PS-specific antibodies assessed with SBA had protective levels. Unvaccinated MenY carriers had higher levels of MenY anti-PS IgG in serum and IgA in saliva than those not carrying MenY. Use of Swedish snus was associated with lower anti-PS IgG levels in serum and waterpipe use with lower anti-PS IgG levels in saliva. CONCLUSION: Unvaccinated adolescents in Norway have a low degree of natural immunity against the serogroups of N. meningitidis predominating among cases of IMD in this age group. Therefore, introduction of MCV4 for adolescents in the NIP is recommended.

FLU-v, a Broad-Spectrum Influenza Vaccine, Induces Cross-Reactive Cellular Immune Responses in Humans Measured by Dual IFN-γ and Granzyme B ELISpot Assay.

Previous reports demonstrated that FLU-v, a peptide-based broad-spectrum influenza vaccine candidate, induced antibody and cellular immune responses in humans. Here, we evaluate cellular effector functions and cross-reactivity. PBMC sampled pre- (day 0) and post-vaccination (days 42 and 180) from vaccine (n = 58) and placebo (n = 27) recipients were tested in vitro for responses to FLU-v and inactivated influenza strains (A/H3N2, A/H1N1, A/H5N1, A/H7N9, B/Yamagata) using IFN-γ and granzyme B ELISpot. FLU-v induced a significant increase in the number of IFN-γ- and granzyme-B-secreting cells responding to the vaccine antigens from pre-vaccination (medians: 5 SFU/106 cells for both markers) to day 42 (125 and 40 SFU/106 cells, p < 0.0001 for both) and day 180 (75 and 20 SFU/106 cells, p < 0.0001 and p = 0.0047). The fold increase from pre-vaccination to day 42 for IFN-γ-, granzyme-B-, and double-positive-secreting cells responding to FLU-v was significantly elevated compared to placebo (medians: 16.3-fold vs. 1.0-fold, p < 0.0001; 3.5-fold vs. 1.0-fold, p < 0.0001; 3.0-fold vs. 1.0-fold, p = 0.0012, respectively). Stimulation of PBMC with inactivated influenza strains showed significantly higher fold increases from pre-vaccination to day 42 in the vaccine group compared to placebo for IFN-γ-secreting cells reacting to H1N1 (medians: 2.3-fold vs. 0.8-fold, p = 0.0083), H3N2 (1.7-fold vs. 0.8-fold, p = 0.0178), and H5N1 (1.7-fold vs. 1.0-fold, p = 0.0441); for granzyme B secreting cells reacting to H1N1 (3.5-fold vs. 1.0-fold, p = 0.0075); and for double positive cells reacting to H1N1 (2.9-fold vs. 1.0-fold, p = 0.0219), H3N2 (1.7-fold vs. 0.9-fold, p = 0.0136), and the B strain (2.0-fold vs. 0.8-fold, p = 0.0227). The correlation observed between number of cells secreting IFN-γ or granzyme B in response to FLU-v and to the influenza strains supported vaccine-induced cross-reactivity. In conclusion, adjuvanted FLU-v vaccination induced cross-reactive cellular responses with cytotoxic capacity, further supporting the development of FLU-v as a broad-spectrum influenza vaccine.

Immune responses in Omicron SARS-CoV-2 breakthrough infection in vaccinated adults.

The SARS-CoV-2 Omicron variant has more than 15 mutations in the receptor binding domain of the Spike protein enabling increased transmissibility and viral escape from antibodies in vaccinated individuals. It is unclear how vaccine immunity protects against Omicron infection. Here we show that vaccinated participants at a super-spreader event have robust recall response of humoral and pre-existing cellular immunity induced by the vaccines, and an emergent de novo T cell response to non-Spike antigens. Individuals with Omicron SARS-CoV-2 breakthrough infections have significantly increased activated SARS-CoV-2 wild type Spike-specific cytotoxic T cells, activated follicular helper (TFH) cells, functional T cell responses, boosted humoral responses, and rapid release of Spike and RBD-specific IgG+ B cell plasmablasts and memory B cells into circulation. Omicron breakthrough infection affords significantly increased de novo memory T cell responses to non-Spike viral antigens. Concerted T and B cell responses may provide durable and broad immunity.

Cost-effectiveness of meningococcal vaccination of Norwegian teenagers with a quadrivalent ACWY conjugate vaccine.

In Norway, the incidence of invasive meningococcal disease (IMD) is higher among 16-19-year-olds than in the general population. Most IMD cases among teenagers are caused by serogroup Y. Since 2011, one dose of meningococcal ACWY conjugate vaccine (MCV4) has been recommended for teenagers with out-of-pocket payment. The teenagers are usually vaccinated through the school health service at age 18. This study aimed to estimate costs and health gains of introducing MCV4 to Norwegian teenagers through the national immunization program (NIP). A Markov model was used to analyze the cost-effectiveness of universal MCV4 vaccination of either 15-year-olds or 18-years-olds. Occurrences of IMD were simulated from 15 until 23 years of age. Costs were estimated from a healthcare perspective. Sensitivity analyses evaluated the impact of vaccine price, vaccination uptake, IMD incidence and discount rate. Compared to today's practice of vaccinating 18-year-olds with out-of-pocket payment, introducing MCV4 to 15-year-olds in a NIP-setting, with 90% vaccine uptake and 50% rebate on vaccine price, prevented 3.2 hospitalizations, 0.20 sequelae and 0.47 deaths among 15-23-year-olds, annually. Total costs were reduced by €30,000 and 9.7 quality-adjusted life-years (QALYs) were gained per birth cohort. The probability of cost-effectiveness was 99.0%, assuming a willingness-to-pay threshold of €86,000/QALY for severe diseases in Norway. Cost-effectiveness was highly dependent on vaccine price. Vaccination of 18-year-olds in a NIP-setting was also cost-effective, but less than NIP-vaccination of 15-year-olds. Introduction of MCV4 to the 15-year-olds in the Norwegian NIP is likely to be cost-effective given a rebate on the vaccine price.

Household Transmission of SARS-CoV-2: A Prospective Longitudinal Study Showing Higher Viral Load and Increased Transmissibility of the Alpha Variant Compared to Previous Strains.

We studied the secondary attack rate (SAR), risk factors, and precautionary practices of household transmission in a prospective, longitudinal study. We further compared transmission between the Alpha (B.1.1.7) variant and non-Variant of Concern (non-VOC) viruses. From May 2020 throughout April 2021, we recruited 70 confirmed COVID-19 cases with 146 household contacts. Participants donated biological samples eight times over 6 weeks and answered questionnaires. SARS-CoV-2 infection was detected by real-time RT-PCR. Whole genome sequencing and droplet digital PCR were used to establish virus variant and viral load. SARS-CoV-2 transmission occurred in 60% of the households, and the overall SAR for household contacts was 50%. The SAR was significantly higher for the Alpha variant (78%) compared with non-VOC viruses (43%) and was associated with a higher viral load. SAR was higher in household contacts aged ≥40 years (69%) than in younger contacts (40-47%), and for contacts of primary cases with loss of taste/smell. Children had lower viral loads and were more often asymptomatic than adults. Sleeping separately from the primary case reduced the risk of transmission. In conclusion, we found substantial household transmission, particularly for the Alpha variant. Precautionary practices seem to reduce SAR, but preventing household transmission may become difficult with more contagious variants, depending on vaccine use and effectiveness.

Critical roles of complement and antibodies in host defense mechanisms against Neisseria meningitidis as revealed by human complement genetic deficiencies.

Certain complement defects are associated with an increased propensity to contract Neisseria meningitidis infections. We performed detailed analyses of complement-mediated defense mechanisms against N. meningitidis 44/76 with whole blood and serum from two adult patients who were completely C2 or C5 deficient. The C5-deficient patient and the matched control were also deficient in mannose-binding lectin (MBL). The proliferation of meningococci incubated in freshly drawn whole blood was estimated by CFU and quantitative DNA real-time PCR. The serum bactericidal activity and opsonophagocytic activity by granulocytes were investigated, including heat-inactivated postvaccination sera, to examine the influence of antimeningococcal antibodies. The meningococci proliferated equally in C2- and C5-deficient blood, with a 2 log(10) increase of CFU and 4- to 5-log(10) increase in DNA copies. Proliferation was modestly decreased in reconstituted C2-deficient and control blood. After reconstitution of C5-deficient blood, all meningococci were killed, which is consistent with high antibody titers being present. The opsonophagocytic activity was strictly C2 dependent, appeared with normal serum, and increased with postvaccination serum. Serum bactericidal activity was strictly dependent on C2, C5, and high antibody titers. MBL did not influence any of the parameters observed. Complement-mediated defense against meningococci was thus dependent on the classical pathway. Some opsonophagocytic activity occurred despite low levels of antimeningococcal antibodies but was more efficient with immune sera. Serum bactericidal activity was dependent on C2, C5, and immune sera. MBL did not influence any of the parameters observed.

Antibody levels in a cohort of pregnant women after the 2009 influenza A(H1N1) pandemic: Waning and association with self-reported severity and duration of illness.

BACKGROUND: A population-based pregnancy cohort was established in Norway to study potential effects of exposure to the 2009 influenza pandemic or pandemic vaccination during pregnancy. OBJECTIVES: We studied maternal A(H1N1)pdm09-specific hemagglutination inhibition (HI)-titer levels and waning in women with influenza-like illness (ILI) in pregnancy compared to vaccinated women. Moreover, we studied the association between HI-titers and self-reported severity and duration of ILI. METHODS: HI-titers against the pandemic virus were measured in maternal blood samples obtained at birth, 3-9 months after exposure, and linked with information about pregnancy, influenza and vaccination from national registries and a cohort questionnaire. RESULTS: Among 1821 pregnant women included, 43.7% were unvaccinated and 19.3% of these had ILI. HI-titers were low (geometric mean titer (GMT) 11.3) in the unvaccinated women with ILI. Higher HI-titers (GMT 37.8) were measured in the vaccinated women. Estimated HI-titer waning was similar for vaccinated women and women with ILI. Most ILI episodes were moderate and lasted 3-5 days. Women with ILI reporting specific influenza symptoms such as fever or cough had higher HI-titers than women without these symptoms. Women who reported being "very ill" or illness duration of >5 days had higher HI-titers than women reporting less severe illness or illness of shorter duration, respectively. CONCLUSIONS: Antibody waning was similar in vaccinated women and women with ILI. More severe ILI or longer duration of illness was associated with higher HI-titers. Most unvaccinated pregnant women with ILI had low HI-titers, probably due to moderate illness and HI-titer waning between exposure and sampling.

Natural immunity against capsular group X N. meningitidis following an outbreak in Togo, 2007.

BACKGROUND: Capsular group X N. meningitidis (MenX) has emerged as a cause of localized disease outbreaks in sub-Saharan Africa, but the human immune response following exposure to MenX antigens is poorly described. We therefore assessed the natural immunity against MenX in individuals who were living in an area affected by a MenX outbreak during 2007 in Togo, West Africa. During 2009, 300 healthy individuals (100 aged 3-5 years, 100 aged 13-19 years and 100 aged 20-25 years) were included in the study, and serum responses were compared with sera from age-matched controls from the U.K. and Burkina Faso. METHODS: MenX serum bactericidal antibody (SBA) was measured using rabbit complement, and antibodies against MenX polysaccharide (XPS) and outer membrane vesicles (XOMVs) were quantified by ELISA. RESULTS: The proportion of Togolese individuals with an SBA titer of ≥8 against the MenX strain was 29% (95% confidence interval (CI) 18-41) among those aged 3-5 years, 34% (95% CI 9-60) among those aged 13-19 years and 32% (95% CI 24-40) among those aged 20-25 years. These were significantly higher than observed in the control populations from the U.K (range 13-16%) and Burkina Faso (range 2-6%). CONCLUSION: In Togolese individuals, the concentration of serum IgG against XPS was higher among the two older age groups as compared to the youngest age group. Antibody concentrations against MenX PS correlated significantly with SBA titers. This supports further development of a MenX PS based conjugate vaccine. Further studies are needed to verify the ability of MenX PS to induce SBA in humans.

Cellular Immune Responses in Humans Induced by Two Serogroup B Meningococcal Outer Membrane Vesicle Vaccines Given Separately and in Combination.

MenBvac and MeNZB are safe and efficacious outer membrane vesicle (OMV) vaccines against serogroup B meningococcal disease. Antibody responses have previously been investigated in a clinical trial with these two OMV vaccines given separately (25 µg/dose) or in combination (12.5 and 12.5 µg/dose) in three doses administered at 6-week intervals. Here, we report the results from analyzing cellular immune responses against MenBvac and MeNZB OMVs in terms of antigen-specific CD4(+)T cell proliferation and secretion of cytokines. The proliferative CD4(+)T cell responses to the combined vaccine were of the same magnitude as the homologous responses observed for each individual vaccine. The results also showed cross-reactivity in the sense that both vaccine groups receiving separate vaccines responded to both homologous and heterologous OMV antigen when assayed for antigen-specific cellular proliferation. In addition, a multiplex bead array assay was used to analyze the presence of Th1 and Th2 cytokines in cell culture supernatants. The results showed that gamma interferon, interleukin-4 (IL-4), and IL-10 responses could be detected as a result of vaccination with both the MenBvac and the MeNZB vaccines given separately, as well as when given in combination. With respect to cross-reactivity, the cytokine results paralleled the observations made for proliferation. In conclusion, the results demonstrate that cross-reactive cellular immune responses involving both Th1 and Th2 cytokines can be induced to the same extent by different tailor-made OMV vaccines given either separately or in combination with half the dose of each vaccine.

Immune responses of a meningococcal A + W outer membrane vesicle (OMV) vaccine with and without aluminium hydroxide adjuvant in two different mouse strains.

Meningococci (Neisseria meningiditis) of serogroups A and W have caused large epidemics of meningitis in sub-Saharan Africa for decades, and affordable and multivalent vaccines, effective in all age groups, are needed. A bivalent serogroup A and W (A + W) meningococcal vaccine candidate consisting of deoxycholate-extracted outer membrane vesicles (OMV) from representative African disease isolates was previously found to be highly immunogenic in outbred mice when formulated with the adjuvant aluminium hydroxide (AH). OMV has been shown to have inherent adjuvant properties. In order to study the importance of AH and genetical differences between mice strains on immune responses, we compared the immunogenicity of the A + W OMV vaccine when formulated with or without AH in inbred C57BL/6J and BALB/cJ mice (Th1 and Th2 dominant strains, respectively). The immunogenicity of the vaccine was found to be comparable in the two mice strains despite their different immune profiles. Adsorption to AH increased anti-OMV IgG levels and serum bactericidal activity (SBA). The immune responses were increased by each dose for the adsorbed vaccine, but the third dose did not significantly improve the immunogenicity further. Thus, a vaccine formulation with the A and W OMV will likely benefit from including AH as adjuvant.

Heat shock protein complex vaccines induce antibodies against Neisseria meningitidis via a MyD88-independent mechanism.

BACKGROUND: Neisseria meningitidis are common colonizers of the human nasopharynx. In some circumstances, N. meningitidis becomes an opportunistic pathogen that invades tissues and causes meningitis. While a vaccine against a number of serogroups has been in effective use for many years, a vaccine against N. meningitidis group B has not yet been universally adopted. Bacterial heat shock protein complex (HSPC) vaccines comprise bacterial HSPs, purified with their chaperoned protein cargo. HSPC vaccines use the intrinsic adjuvant activity of their HSP, thought to act via Toll-like receptors (TLR), to induce an immune response against their cargo antigens. This study evaluated HSPC vaccines from N. meningitidis and the closely related commensal N. lactamica. RESULTS: The protein composition of N. lactamica and N. meningitidis HSPCs were similar. Using human HEK293 cells we found that both HSPCs can induce an innate immune response via activation of TLR2. However, stimulation of TLR2 or TLR4 deficient murine splenocytes revealed that HSPCs can activate an innate immune response via multiple receptors. Vaccination of wildtype mice with the Neisseria HSPC induced a strong antibody response and a Th1-restricted T helper response. However, vaccination of mice deficient in the major TLR adaptor protein, MyD88, revealed that while the Th1 response to Neisseria HSPC requires MyD88, these vaccines unexpectedly induced an antigen-specific antibody response via a MyD88-independent mechanism. CONCLUSIONS: N. lactamica and N. meningitidis HSPC vaccines both have potential utility for immunising against neisserial meningitis without the requirement for an exogenous adjuvant. The mode of action of these vaccines is highly complex, with HSPCs inducing immune responses via both MyD88-dependent and -independent mechanisms. In particular, these HSPC vaccines induced an antibody response without detectable T cell help.

International validation of novel pyrogen tests based on human monocytoid cells.

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.

Development and Evaluation of a Multiplex Microsphere Assay for Quantitation of IgG and IgA Antibodies against Neisseria meningitidis Serogroup A, C, W, and Y Polysaccharides.

We developed and evaluated a rapid and simple multiplex microsphere assay for the quantification of specific IgG and IgA antibodies against meningococcal serogroup A, C, W, and Y capsular polysaccharides in serum and saliva. Meningococcal polysaccharides were conjugated to distinct magnetic carboxylated microspheres, and the performance of the assay was assessed using the CDC1992 standard meningococcal reference serum and a panel of serum and saliva samples. The standard curve was linear over an eight 3-fold dilution range in the IgG assay and a seven 3-fold dilution range in the IgA assay. No cross-reactivity was discovered, and the assay showed high specificity with ≥91% homologous inhibition and ≤11% heterologous inhibition for all serogroups and immunoglobulin classes. Lower limits of detections were ≤280 pg/ml for IgG and ≤920 pg/ml for IgA antibodies. The assay was reproducible, with a mean coefficient of variation of ≤5% for intra-assay duplicates, a mean coefficient of variation of ≤20% for interassay repeated analysis with different conjugations of microspheres, and a mean coefficient of variation within 25.8% for interoperator variation. The assay showed good correlation to the standard meningococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies. This multiplex assay is robust and reliable and requires less sample volume, and less time and workload are needed than for ELISA, making this method highly relevant for serological and salivary investigations on the effect of meningococcal vaccines and for immunosurveillance studies.

Human antibody responses to pneumococcal surface protein A and capsular polysaccharides during acute and convalescent stages of invasive disease in adult patients.

The IgG antibody responses to pneumococcal surface protein A (PspA) and capsular polysaccharides in acute and convalescent-phase sera from 10 adult patients with invasive pneumococcal disease were analysed. The relatedness between the strains were characterized by capsular serotyping (1, 4, 7F, 9V, 12F and 19F), multilocus sequence typing (MLST) and sequencing of the gene coding for PspA. Immunoblotting with the patient's own infecting strain used as whole cell antigen revealed strong antibody responses to PspA in 4 of 10 patients. Two of these patients showed cross reactivity of PspA antibodies within PspA families 1 and 2 by ELISA measurements with recombinant PspA proteins. Using ELISA, we found increased levels of capsular-specific antibodies during convalescent phase in 9 of 10 patients. All patients, except one, revealed low antibody levels in their acute phase sera. The binding of serum antibodies to live pneumococci using the patient's own infective strain was measured by flow cytometry. The antibodies binding to the live pneumococci corresponded to the serotype-specific polysaccharides by ELISA. Low antibody-binding activities to their infective strain in the acute serum may explain why they were not protected.

Fast vaccine design and development based on correlates of protection (COPs).

New and reemerging infectious diseases call for innovative and efficient control strategies of which fast vaccine design and development represent an important element. In emergency situations, when time is limited, identification and use of correlates of protection (COPs) may play a key role as a strategic tool for accelerated vaccine design, testing, and licensure. We propose that general rules for COP-based vaccine design can be extracted from the existing knowledge of protective immune responses against a large spectrum of relevant viral and bacterial pathogens. Herein, we focus on the applicability of this approach by reviewing the established and up-coming COPs for influenza in the context of traditional and a wide array of new vaccine concepts. The lessons learnt from this field may be applied more generally to COP-based accelerated vaccine design for emerging infections.

Vaccines against meningococcal serogroup B disease containing outer membrane vesicles (OMV): lessons from past programs and implications for the future.

The utility of wild-type outer membrane vesicle (wtOMV) vaccines against serogroup B (MenB) meningococcal disease has been explored since the 1970s. Public health interventions in Cuba, Norway and New Zealand have demonstrated that these protein-based vaccines can prevent MenB disease. Data from large clinical studies and retrospective statistical analyses in New Zealand give effectiveness estimates of at least 70%. A consistent pattern of moderately reactogenic and safe vaccines has been seen with the use of approximately 60 million doses of three different wtOMV vaccine formulations. The key limitation of conventional wtOMV vaccines is their lack of broad protective activity against the large diversity of MenB strains circulating globally. The public health intervention in New Zealand (between 2004-2008) when MeNZB was used to control a clonal MenB epidemic, provided a number of new insights regarding international and public-private collaboration, vaccine safety surveillance, vaccine effectiveness estimates and communication to the public. The experience with wtOMV vaccines also provide important information for the next generation of MenB vaccines designed to give more comprehensive protection against multiple strains.

Mucosal immunity in healthy adults after parenteral vaccination with outer-membrane vesicles from Neisseria meningitidis serogroup B.

BACKGROUND: Nasopharyngeal carriage of meningococcus or related species leads to protective immunity in adolescence or early adulthood. This natural immunity is associated with mucosal and systemic T cell memory. Whether parenteral Neisseria meningitidis serogroup B (MenB) vaccination influences natural mucosal immunity is unknown. OBJECTIVES: To determine whether parenteral MenB vaccination affects mucosal immunity in young adults and whether this immunity differs from that induced in the blood. METHODS: Otherwise healthy volunteers were immunized with MenB outer membrane vesicle vaccine before and after routine tonsillectomy. Mucosal and systemic immunity were assessed in 9 vaccinees and 12 unvaccinated control subjects by measuring mononuclear cell proliferation, cytokine production, Th1/Th2 surface marker expression, and antibody to MenB antigens. RESULTS: Parenteral vaccination induced a marked increase in systemic T cell immunity against MenB and a Th1 bias. In contrast, although mucosal T cell proliferation in response to MenB neither increased nor decreased following vaccination, mononuclear cell interferon gamma, interleukin (IL)-5, and IL-10 production increased, and the Th1/Th2 profile lost its Th1 bias. CONCLUSIONS: Parenteral MenB vaccination selectively reprograms preexisting naturally acquired mucosal immunity. As new-generation protein-based MenB vaccine candidates undergo evaluation, the impact of these vaccines on mucosal immunity in both adults and children will need to be addressed.