International Bordetella pertussis assay standardization and harmonization meeting report. Centers for Disease Control and Prevention, Atlanta, Georgia, United States, 19-20 July 2007.

An international meeting on Bordetella pertussis assay standardization and harmonization was held at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, 19-20 July 2007. The goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the World Health Organization (WHO). The primary objectives were (1) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; (2) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; (3) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and (4) to search for a multicenter process for addressing these priority gaps. Presentations and discussions by content experts addressed these objectives. A prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. The major items included: (1) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; (2) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; (3) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; (4) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; (5) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; (6) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and (7) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines.

Enhancement of vaccine-specific cellular immunity in infants by passively acquired maternal antibody.

The known protective effects of passively acquired maternal antibody on the resistance of newborns to infections have prompted widespread interest in maternal vaccination. However, a range of animal model and human studies indicate potential inhibitory effects of maternal antibody on vaccine-specific humoral responses in infants. In the present study we have examined the relationship between maternally acquired TT-specific IgG present before DTaP vaccination and subsequent TT-specific T-cell memory responses at 12 and 18 months, in a cohort of 118 infants. We demonstrate a strong positive association between TT-specific cellular immunity as evidenced by increased IL-4, IL-5 and IL-13 responses, and maternal TT-specific IgG.

Protection against Streptococcus pneumoniae elicited by immunization with pneumolysin and CbpA.

The need for the development of cheap and effective vaccines against pneumococcal disease has necessitated the evaluation of common virulence-associated proteins of Streptococcus pneumoniae as potential vaccine antigens. In this study, we examined the capacity of active immunization with a genetic toxoid derivative of pneumolysin (PdB) and/or a fragment of choline binding protein A (CbpA; also known as PspC, Hic, and SpsA) to protect mice from intraperitoneal challenge with medium to very high doses of a highly virulent capsular type 2 pneumococcal strain, D39. The median survival times for mice immunized with the individual protein antigens in different adjuvant combinations were significantly longer than those for mice that received the respective adjuvants alone. Mice immunized with CbpA alone were significantly better protected than mice immunized with PdB alone. Correspondingly, the median survival times for mice that were immunized with a combination of PdB and CbpA were significantly longer than those for mice that received PdB alone but not significantly different from those that received CbpA alone. Mice immunized with the protein antigens in a mixture of monophospholipid A (MPL) and aluminium phosphate (AlPO4) adjuvants had higher antibody titers than mice that received the antigens in AlPO4 alone. Mice immunized with PdB in MPL plus AlPO4 were also significantly better protected than mice that received PdB in AlPO4 alone.

Heterogeneity in diphtheria-tetanus-acellular pertussis vaccine-specific cellular immunity during infancy: relationship to variations in the kinetics of postnatal maturation of systemic th1 function.

Cellular immunity to vaccines is highly variable during infancy. This study addressed the hypothesis that these responses are governed by the pace of maturational changes in adaptive immune competence, in particular, cellular functions that underlie the postnatal transition from Th2 to Th1 "bias." Tetanus-specific cytokine responses were tracked in peripheral blood mononuclear cells collected from infants at months 2, 4, 6, 12, and 18. These were compared with polyclonal responses. Results show that the Th2 component of the vaccine response develops rapidly and remains stable, unlike interferon (IFN)-gamma production, which also is initiated early but commonly declines after the final priming dose at 6 months. However, between 12 and 18 months, the IFN-gamma component of the vaccine-specific response has a spontaneous resurgence that coincides with a parallel increase in overall IFN-gamma production capacity. The Th2 component of vaccine-specific responses was more prominent in children with atopic family history.

Outer membrane protein vesicle vaccines for meningococcal disease.

Alternative strategies exist for prevention of group B Neisseria meningitidis (meningococcal) disease through vaccination (see Chapters 5 , 8 , 13 , 14 in this volume). However, the most promising approach to date has been the use of outer-membrane vesicle (OMV) vaccines for induction of bactericidal antibodies against cell-surface outer-membrane proteins (OMPs).

Animal models for meningococcal disease.

There are many in vitro systems for the study of meningococcal pathogenesis, but it is only in animal models of infection that the interactions of the bacteria with whole tissues and the humoral and cellular immune systems can be assessed. Animal-infection models are also of great importance for the assessment of the protective efficacy of existing and candidate vaccines. However, the relevance of these animal models to human disease and how well protection assessed in them corresponds to protection against human disease, must always be considered. Animal models for pathogenic Neisseria have been previously reviewed (1).

Antigen-specific responses to diphtheria-tetanus-acellular pertussis vaccine in human infants are initially Th2 polarized.

Immune responses to exogenous antigens in infant experimental animals display various degrees of Th2 polarization. Preliminary evidence from small human studies suggest a similar age-dependent response pattern to vaccines, but detailed investigations on vaccine immunity during infancy have not yet been undertaken. We report below the results of a comprehensive prospective study on responses to the tetanus component of the diphtheria, tetanus, acellular pertussis (DTaP) vaccine in a cohort of 55 healthy children, employing peripheral blood mononuclear cells (PBMC) collected at the 2-, 4-, and 6-month vaccinations and at 12 months. Antigen-specific production of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10, IL-13, and gamma interferon (IFN-gamma) was determined at each sample point, in parallel with polyclonal (phytohemagglutinin PHA-induced) cytokine responses. Our results indicate early and persistent Th2 responses to the vaccine, in contrast to a more delayed and transient pattern of IFN-gamma production. This initial disparity between the Th1 and Th2 components of the vaccine response was mirrored by patterns of polyclonally induced cytokine production, suggesting that the delayed maturation of the Th1 component of the vaccine response during infancy is secondary to developmental processes occurring within the overall Th cell system.

Developing a nontypeable Haemophilus influenzae (NTHi) vaccine.

There is a current high demand for nontypable Haemophilus influenzae (NTHi) vaccines. Various options for the composition of such vaccines are possible. Decisions about the vaccine composition have to take into account the antigenic variability of NTHi, so even complex immunogens such as whole bacteria would preferentially have a tailor-made antigenic composition. We will present a summary of NTHi vaccine development, describing research efforts from SmithKline Beecham and other laboratories. Currently, major (P1, P2, P4, P5) and minor (P6, D15, TbpA/B, ellipsis) outer membrane proteins, LPS, adhesins (HMW, Hia, pili, P5) are being studied. Preclinical results with LPD, P5 (LB1) and OMP26 from our laboratories will be described including the use of animal models of otitis and lung infection.

Cardiovascular aspects of experimental meningococcal sepsis in young and older awake piglets: age-related differences.

Severe meningococcal disease is characterized by: a high load of specific endotoxin, capillary leakage and coagulation disorders. We studied the possible age-related differences in global hemodynamic and regional blood flow responses to different dosages (1 and 10 microg/kg body weight) of rough meningococcal endotoxin in young (8 kg) and older piglets (40 kg). Animals were chronically instrumented and studied in the awake state. The response to plasma infusion (30 mL/kg in 30 min) was evaluated after placebo and endotoxin infusion. The clinical picture was similar in all groups. The mortality was 0/8, 3/8,1/8, 4/9 in young-low, young-high, old-low, and old-high dose respectively. Most important findings were that cardiac index (CI) decreased in the young animals after endotoxin infusion, while it was well preserved in the older animals; in the older animals the systemic vascular resistance dropped 20%, while in the younger ones there was no change in resistance. Conductance to the kidneys, intestines, and spleen decreased significantly more in the young animals, while the increase in conductance and flow to the liver was higher in the old animals; subsequent volume loading resulted only partly in a recovery of the hemodynamic parameters, but failed to improve oxygen delivery.

Immunogenicity of various presentation forms of PorA outer membrane protein of Neisseria meningitidis in mice.

In this study we compare different vaccine formulations containing meningococcal PorA outer membrane protein; purified PorA, outer membrane vesicles (OMV) and immune-stimulating complexes (iscom). Bactericidal antibodies could be generated by the OMV and iscom formulation but not with purified PorA using either A1PO4 or Quil-A as adjuvant. OMV and iscom formulations revealed similar immunogenicity when tested in a dose response manner, with respect to bactericidal as well as OMV-binding antibodies. The anti-OMV IgG subclass response induced by PorA in OMV formulation was found in all subclasses IgG1, IgG2a, IgG2b, IgG3. OMP-iscoms induced very high IgG1 anti-OMV antibodies but almost no IgG3 response. Also, OMP-iscoms appeared to be a potent inducer of antibodies directed against linear peptides corresponding to surface exposed loops of PorA. In addition, iscoms as well as purified PorA with Quil-A as adjuvant (but not with A1PO4) induced high levels of antibodies against purified PorA. In summary, in addition to the OMV formulation, only iscoms containing PorA are able to generate an anamnestic and bactericidal antibody response.

Immunogenicity of 2 serogroup B outer-membrane protein meningococcal vaccines: a randomized controlled trial in Chile.

CONTEXT: Meningococcal disease occurs worldwide, and serogroup B disease accounts for a large proportion of cases. Although persons younger than 4 years are at greatest risk for serogroup B meningococcal disease, vaccine efficacy has not been demonstrated in this age group. OBJECTIVE: To evaluate serum bactericidal activity (SBA) against homologous vaccine type strains and a heterologous Chilean epidemic strain of Neisseria meningitidis as a potential correlate for vaccine efficacy. DESIGN: Double-blind, randomized controlled trial conducted between March 14 and July 20, 1994. All blood samples were taken by December 1994. SETTING: Santiago, Chile, where a clonal serogroup B meningococcal disease epidemic began in 1993. PARTICIPANTS: Infants younger than 1 year (n = 187), children aged 2 to 4 years (n = 183), and adults aged 17 to 30 years (n = 173). INTERVENTION: Participants received 3 doses of outer-membrane protein (OMP) meningococcal vaccine developed in either Cuba or Norway or a control vaccine, with each dose given 2 months apart. Blood samples were obtained at baseline, prior to dose 3, and at 4 to 6 weeks after dose 3. MAIN OUTCOME MEASURE: Immune response, defined as a 4-fold or greater rise in SBA titer 4 to 6 weeks after dose 3 compared with prevaccination titer. RESULTS: Children and adult recipients of either meningococcal vaccine were more likely than controls to develop an immune response to the heterologous epidemic strain. After 3 doses of vaccine, 31% to 35% of children responded to the vaccine vs 5% to placebo; 37% to 60% of adults responded to vaccine vs 4% to placebo (P<.05 vs control for all). Infants, however, did not respond. In contrast, against homologous vaccine type strains, the response rate was 67% or higher among children and adults and 90% or higher among infants (P<.001 vs control for all). Subsequent SBA against 7 isogenic homologous target strains identified class 1 OMP as the immunodominant antigen. CONCLUSIONS: These data suggest that neither serogroup B OMP meningococcal vaccine would confer protection during a heterologous epidemic. However, epidemic strain-specific vaccines homologous for class 1 OMP are promising candidates for the control of epidemic serogroup B meningococcal disease.

Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response.

The immunogenicity of two types of Streptococcus pneumoniae capsular polysaccharide-tetanus toxoid conjugates (PS6BTT and PS14TT) was evaluated in mice. Both conjugates induced high titres of high avidity type-specific anti-PS IgG, which include all IgG isotypes except IgG2a. Repeated immunization resulted in booster responses in both cases. The antibodies induced exhibited opsonic activity, as measured in an in vitro opsonophagocytosis assay, using the mouse macrophage cell line RAW-264. Furthermore, the influence of spiking PS6BTT with free PS6B of either 1000 kDa (native) or 37 kDa was investigated. The results indicate that not only the amount but also the molecular weight of the free PS6B present in the conjugate vaccine affect the anti-PS6B immune response. Large amounts of free PS6B of both molecular weights decrease each anti-PS6B IgG isotype response. However, unlike admixture of the low molecular weight PS6B, addition of the high molecular weight PS6B leads to a rather persistent state of unresponsiveness.

Application of cystamine and N,N'-Bis(glycyl)cystamine as linkers in polysaccharide-protein conjugation.

Pneumococcal polysaccharide type 6B, 14, or 23F (35-70 kDa) was activated with cyanogen bromide and modified with cystamine. After reduction of the spacer, the thiol-containing (i.e. cysteamine-modified) polysaccharide obtained was added in a 5-10-fold molar excess to bromoacetylated tetanus toxoid to give thioether-linked polysaccharide-protein conjugates in a yield of 10-20%. This approach failed for preparing a type 19F polysaccharide-protein conjugate, possibly due to intramolecular elimination of cysteamine from the reduced 19F polysaccharide. When N,N'-bis(glycyl)cystamine was introduced as a spacer molecule, the elimination of the reduced spacer was suppressed, thus allowing preparation of a 19F polysaccharide-tetanus toxoid conjugate (15%).

Bactericidal antibody recognition of a PorA epitope of Neisseria meningitidis: crystal structure of a Fab fragment in complex with a fluorescein-conjugated peptide.

Class 1 outer membrane protein PorA of Neisseria meningitidis is a vaccine candidate against bacterial meningitis. Antibodies against PorA are able to induce complement-mediated bacterial killing and thereby play an important role in protection against meningococcal disease. Bactericidal antibodies are all directed against variable regions VR1 and VR2 of the PorA sequence, corresponding to loops 1 and 4 of a two-dimensional topology model of the porin with eight extracellular loops. We have determined the crystal structure to 2.6 A resolution of the Fab fragment of bactericidal antibody MN12H2 against meningococcal PorA in complex with a linear fluorescein-conjugated peptide TKDTNNNL derived from the VR2 sequence of sero-subtype P1.7,16 (residues 180-187) from meningococcal strain H44/76. The peptide folds deeply into the binding cavity of the Fab molecule in a type I beta-turn, with the minimal P1.16 epitope DTNNN virtually completely buried. The structure reveals H-bonds and van der Waals interactions with all minimal epitope residues and one essential salt bridge between Asp-182 of the peptide and His-31 of the MN12H2 light chain. The key components of the recognition of PorA epitope P1.16 by bactericidal antibody MN12H2 correspond well with available thermodynamic data from binding studies. Furthermore, they indicate the structural basis of an increased endemic incidence of infection by group B meningococci in England and Wales since 1981 associated with the occurrence of an Neisseria meningitidis escape mutant (strain-MC58). The observed three-dimensional conformation of the peptide provides a rationale for the development of a synthetic peptide vaccine against meningococcal disease.

Isolation and characterization of the Neisseria meningitidis lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis.

The lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis in Neisseria meningitidis has been cloned and sequenced. By complementation of a temperature-sensitive E. coli lpxD mutant, we first cloned a meningococcal chromosomal fragment that carries the lpxD homologue. Cloning and sequence analysis of chromosomal DNA downstream of lpxD revealed the presence of the fabZ and lpxA genes. This gene cluster shows high homology to the corresponding genes from several other bacterial species. The LpxA and LpxD proteins catalyze early steps in the lipid A biosynthetic pathway, adding the O- and N-linked 3-OH fatty acyl chains, respectively. In E. coli and N. meningitidis, LpxD has the same specificity, in both cases adding 3-OH myristoyl chains; in contrast to E. coli, the meningococcal LpxA protein is presumed to add 3-OH lauroyl chains instead. The established sequence points the way to further experiments to define the basis for this difference in specificity, and should allow modification of meningococcal lipid A biosynthesis through gene exchange.

On the interaction between a bactericidal antibody and a PorA epitope of Neisseria meningitidis in outer membrane vesicles: a competitive fluorescence polarization immunoassay.

This paper describes a method for determining the affinity constant (Ka) of the binding between an antibody Fab fragment and a membrane-embedded protein epitope under equilibrium conditions. Monoclonal antibody MN12H2, directed against outer membrane protein PorA of Neisseria meningitidis, is used in a competitive fluorescence polarization assay with a cyclic peptide-fluorescein conjugate as a tracer antigen. Displacement experiments with PorA-containing and PorA-deficient meningococcal outer membrane vesicles revealed highly specific binding of MN12H2 Fab to the membrane-embedded PorA P1.16 epitope with Ka of 1.5 x 10(8) M-1.

Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines.

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 micrograms or 100 micrograms protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.

Response of Neisseria meningitidis to iron limitation.

In the human body, the concentration of free iron is limiting for bacterial growth, since iron is bound to transport and storage proteins such as transferrin and lactoferrin. When grown under iron starvation, Neisseria meningitidis produces receptors for these proteins in the outer membrane. These receptors are presently being characterized at the molecular level. Here, we summarize our current knowledge of these receptors, with special emphasis on the LbpA and FrpB proteins, which are studied in our laboratories. Furthermore, the genetic and antigenic variability of these proteins and their vaccine potential are discussed.

Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide.

By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the icsA gene, which encodes the glycosyltransferase required for adding GlcNAc to Hep-II in the inner core of meningococcal LPS. This gene has homology to several LPS glycosyltransferases, notably to rfaK from Salmonella typhimurium and bplH from Bordetella pertussis, both of which encode GlcNAc transferases. Directly upstream of icsA is an ORF showing significant homology to the hypothetical protein HI0653 from the Haemophilus influenzae genome sequence, and to a lesser degree to putative glycosyltransferases from Streptococcus thermophilus and Yersinia enterocolitica. Insertional inactivation of this ORF resulted in a meningococcal strain with truncated LPS. We have named this new LPS-involved gene icsB. Differences in binding of monoclonal antibodies and in mobility on Tricine-SDS-PAGE showed that LPS from icsA and icsB mutants is similar but not identical. On the basis of these results, we postulated that the new gene encodes the glycosyltransferase required for adding Glc to Hep-I. Structural analysis of purified mutant LPS by electrospray mass spectrometry was used to verify this hypothesis. The composition determined for icsA and icsB is lipidA-(KDO)2-(Hep)2.PEA and lipidA-(KDO)2-(Hep)2.PEA-GlcNAc, respectively. The icsA and icsB genes thus form an operon encoding the glycosyltransferases required for chain elongation from the lipidA-(KDO)2-(Hep)2 basal structure, with IcsA first adding GlcNAc to Hep-II and IcsB subsequently adding Glc to Hep-I. Only then is completion of the lacto-N-neotetraose structure possible through the action of the IgtA-E genes.

Sequence variability of FrpB, a major iron-regulated outer-membrane protein in the pathogenic neisseriae.

The FrpB protein from pathogenic neisseriae is a 77 kDa iron-regulated outer-membrane protein that belongs to the family of TonB-dependent receptors and may have potential as a vaccine component. Comparison between the frpB gene from three different meningococcal strains and a published gonococcal one revealed that the region from residues 350 to 390 displays pronounced sequence variability. In a model for the topology of FrpB in the outer membrane, this region corresponds to loop 7, the longest of the predicted 13 surface-exposed loops. Binding of four out of a total of eight bactericidal monoclonal antibodies to synthetic peptides corresponding to loop 7 showed that their epitopes are located here. The frpB genes from five additional meningococcal strains were cloned and sequenced in this region. Pairwise comparisons showed different degrees of similarity.