Jo Anne Welsch

Vaccination against Neisseria meningitidis Using Three Variants of the Lipoprotein GNA1870

Sepsis and meningitis caused by serogroup B meningococcus are devastating diseases of infants and young adults, which cannot yet be prevented by vaccination. By genome mining, we discovered GNA1870, a new surface-exposed lipoprotein of Neisseria meningitidis that induces high levels of bactericidal antibodies. The antigen is expressed by all strains of N. meningitidis tested. Sequencing of the gene in 71 strains representative of the genetic and geographic diversity of the N. meningitidis population, showed that the protein can be divided into three variants. Conservation within each variant ranges between 91.6 to 100%, while between the variants the conservation can be as low as 62.8%. The level of expression varies between strains, which can be classified as high, intermediate, and low expressors. Antibodies against a recombinant form of the protein elicit complement-mediated killing of the strains that carry the same variant and induce passive protection in the infant rat model. Bactericidal titers are highest against those strains expressing high yields of the protein; however, even the very low expressors are efficiently killed. The novel antigen is a top candidate for the development of a new vaccine against meningococcus.

The Meningococcal Vaccine Candidate GNA1870 Binds the Complement Regulatory Protein Factor H and Enhances Serum Resistance

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A ∼29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.

Protective Activity of Monoclonal Antibodies to Genome-Derived Neisserial Antigen 1870, a Neisseria meningitidis Candidate Vaccine

Genome-derived neisserial Ag (GNA) 1870 is a meningococcal vaccine candidate that can be subdivided into three variants based on amino acid sequence variability. Variant group 1 accounts for ∼60% of disease-producing group B isolates. The Ag went unrecognized until its discovery by genome mining because it is expressed in low copy number by most strains. To investigate the relationship between Ab binding to GNA1870 and complement-mediated protective functions, we prepared a panel of four murine IgG mAbs against rGNA1870 (variant 1) and evaluated their activity against nine genetically diverse encapsulated Neisseria meningitidis strains expressing subvariants of variant 1 GNA1870. Based on flow cytometry with live encapsulated bacteria, surface accessibility of the epitopes recognized by the mAbs appeared to be low in most strains. Yet mAb concentrations <1 to 5 μg/ml were sufficient to elicit bactericidal activity with human complement and/or activate C3b deposition on the bacterial surface. Certain combinations of mAbs were highly bactericidal against strains that were resistant to bactericidal activity of the respective individual mAbs. The mAbs conferred passive protection against bacteremia in infant rats challenged by strains resistant to bacteriolysis, and the protective activity paralleled the ability of the mAb to activate C3b deposition. Thus, despite low GNA1870 surface exposure, anti-GNA1870 variant 1 Abs are bactericidal and/or elicit C3b deposition and confer protection against bacteremia caused by encapsulated N. meningitidis strains expressing GNA1870 subvariant 1 proteins. The data support GNA1870 as a promising vaccine candidate for prevention of meningococcal group B disease caused by GNA1870 variant 1 strains.

Antibody to Genome-Derived Neisserial Antigen 2132, a Neisseria meningitidis Candidate Vaccine, Confers Protection against Bacteremia in the Absence of Complement-Mediated Bactericidal Activity

Genome-derived neisserial antigen 2132 (GNA2132) is a novel vaccine candidate that was identified during the Neisseria meningitidis group B strain MC58 genome-sequencing project. To assess the vaccine potential of GNA2132, we prepared antisera from mice immunized with recombinant GNA2132 (gene from strain NZ394/98). Anti-GNA2132 antibody bound to the surface of live bacteria from all 7 capsular group B or C strains tested and elicited deposition of human C3b on the bacterial surface. However, with human or infant-rat complement, anti-GNA2132 had no detectable bactericidal activity (titer, <1:4) against the nominal strain, NZ394/98, and was bactericidal against only 2 of the other 6 strains tested. These differences between strains were unrelated to GNA2132 amino acid sequence or level of protein expression. Despite lack of bactericidal activity, anti-GNA2132 antiserum passively protected infant rats against meningococcal bacteremia after challenge with all 5 resistant strains. GNA2132 is thus a promising vaccine candidate for prevention of disease caused by N. meningitidis.

Binding of complement factor H (fH) to Neisseria meningitidis is specific for human fH and inhibits complement activation by rat and rabbit sera.

ABSTRACT Complement factor H (fH), a molecule that downregulates complement activation, binds to Neisseria meningitidis and increases resistance to serum bactericidal activity. We investigated the species specificity of fH binding and the effect of human fH on downregulating rat (relevant for animal models) and rabbit (relevant for vaccine evaluation) complement activation. Binding to N. meningitidis was specific for human fH (low for chimpanzee fH and not detected with fH from lower primates). The addition of human fH decreased rat and rabbit C3 deposition on the bacterial surface and decreased group C bactericidal titers measured with rabbit complement 10- to 60-fold in heat-inactivated sera from human vaccinees. Administration of human fH to infant rats challenged with group B strain H44/76 resulted in an fH dose-dependent increase in CFU/ml of bacteria in blood 8 h later (P < 0.02). At the highest fH dose, 50 μg/rat, the geometric mean number of CFU per ml was higher than that in control animals (1,050 versus 43 [P < 0.005]). The data underscore the importance of binding of human fH for survival of N. meningitidis in vitro and in vivo. The species specificity of binding of human fH adds another mechanism toward our understanding of why N. meningitidis is strictly a human pathogen.

Complement-Dependent Synergistic Bactericidal Activity of Antibodies against Factor H–Binding Protein, a Sparsely Distributed Meningococcal Vaccine Antigen

BACKGROUND Antibodies to factor H (fH)-binding protein (fHBP), a meningococcal vaccine antigen, activate classical complement pathway serum bactericidal activity (SBA) and block binding of the complement inhibitor fH. METHODS To understand these 2 functions in protection, we investigated the interactions of human complement and 2 anti-fHBP monoclonal antibodies (MAbs) with encapsulated Neisseria meningitidis. RESULTS JAR 3 (IgG3) blocks fH binding and elicits SBA against 2 strains with naturally high fHBP expression and a low-expressing strain genetically engineered to express high fHBP levels. JAR 4 (IgG2a) does not block fH binding or elicit SBA. Neither MAb alone elicits SBA against 2 other strains with low fHBP expression, but together the MAbs increase C4b binding and elicit SBA; JAR 3 alone also is bactericidal in whole blood. In nonimmune blood, fHBP knockout mutants from high-expressing stains do not survive, but mutants of low-expressing strains do. CONCLUSIONS Expression of fHBP is a prerequisite for bacterial survival in blood only by strains with naturally high fHBP expression. In low-expressing strains, combinations of 2 nonbactericidal anti-fHBP MAbs can bind to nonoverlapping epitopes, engage C1q, activate C4, and mediate classical complement pathway SBA. In the absence of sufficient C4b binding for SBA, an individual MAb can have opsonophagocytic bactericidal activity.

Fine antigenic specificity and cooperative bactericidal activity of monoclonal antibodies directed at the meningococcal vaccine candidate factor h-binding protein.

ABSTRACT No broadly protective vaccine is available for the prevention of group B meningococcal disease. One promising candidate is factor H-binding protein (fHbp), which is present in all strains but often sparsely expressed. We prepared seven murine immunoglobulin G monoclonal antibodies (MAbs) against fHbp from antigenic variant group 2 (v.2) or v.3 (∼40% of group B strains). Although none of the MAbs individually elicited bactericidal activity with human complement, all had activity in different combinations. We used MAb reactivity with strains expressing fHbp polymorphisms and site-specific mutagenesis to identify residues that are important for epitopes recognized by six of the v.2 or v.3 MAbs and by two v.1 MAbs that were previously characterized. Residues affecting v.2 or v.3 epitopes resided between amino acids 174 and 216, which formed an eight-stranded beta-barrel in the C domain, while residues affecting the v.1 epitopes included amino acids 121 and 122 of the B domain. Pairs of MAbs were bactericidal when their respective epitopes involved residues separated by 16 to 20 Å and when at least one of the MAbs inhibited the binding of fH, a downregulatory complement protein. In contrast, there was no cooperative bactericidal activity when the distance between residues was ≥27 Å or ≤14 Å, which correlated with the inhibition of the binding of one MAb by the other MAb. Thus, a model for anti-fH MAb bactericidal activity against strains expressing low levels of fHbp requires the binding of two MAbs directed at nonoverlapping epitopes, which activates the classical complement pathway as well as inhibits fH binding. The latter increases the susceptibility of the organism to complement-mediated bacteriolysis.

Meningococcal Factor H–Binding Protein Variants Expressed by Epidemic Capsular Group A, W-135, and X Strains from Africa

BACKGROUND Meningococcal epidemics in Africa are generally caused by capsular group A strains, but W-135 or X strains also cause epidemics in this region. Factor H-binding protein (fHbp) is a novel antigen being investigated for use in group B vaccines. Little is known about fHbp in strains from other capsular groups. METHODS We investigated fHbp in 35 group A, W-135, and X strains from Africa. RESULTS The 22 group A isolates, which included each of the sequence types (STs) responsible for epidemics since 1963, and 4 group X and 3 group W-135 isolates from recent epidemics had genes encoding fHbp in antigenic variant group 1. The remaining 6 W-135 isolates had fHbp variant 2. Within each fHbp variant group, there was 92%-100% amino acid identity, and the proteins expressed conserved epitopes recognized by bactericidal monoclonal antibodies. Serum samples obtained from mice vaccinated with native outer membrane vesicle vaccines from mutants engineered to express fHbp variants had broad bactericidal activity against group A, W-135, or X strains. CONCLUSIONS Despite extensive natural exposure of the African population, fHbp is conserved among African strains. A native outer membrane vesicle vaccine that expresses fHbp variants can potentially elicit protective antibodies against strains from all capsular groups that cause epidemics in the region.

Prevalence of Factor H-Binding Protein Variants and NadA among Meningococcal Group B Isolates from the United States: Implications for the Development of a Multicomponent Group B Vaccine

BACKGROUND Two promising recombinant meningococcal protein vaccines are in development. One contains factor H-binding protein (fHBP) variants (v.) 1 and 2, whereas the other contains v.1 and 4 other antigens discovered by genome mining (5 component [5C]). Antibodies against fHBP are bactericidal against strains within a variant group. There are limited data on the prevalence of strains expressing different fHBP variants in the United States. METHODS A total of 143 group B isolates from patients hospitalized in the United States were tested for fHBP variant by quantitative polymerase chain reaction, for reactivity with 6 anti-fHBP monoclonal antibodies (MAb) by dot immunoblotting, and for susceptibility to bactericidal activity of mouse antisera. RESULTS fHBP v.1 isolates predominated in California (83%), whereas isolates expressing v.1 (53%) or v.2 (42%) were common in 9 other states. Isolates representative of 5 anti-fHBP MAb-binding phenotypes (70% of isolates) were highly susceptible to anti-fHBP v.1 or v.2 bactericidal activity, whereas 3 phenotypes were approximately 50% susceptible. Collectively, antibodies against the fHBP v.1 and v.2 vaccine and the 5C vaccine killed 76% and 83% of isolates, respectively. CONCLUSIONS Susceptibility to bactericidal activity can be predicted, in part, on the basis of fHBP phenotypes. Both vaccines have the potential to prevent most group B disease in the United States.

Protective Antibody Responses Elicited by a Meningococcal Outer Membrane Vesicle Vaccine with Overexpressed Genome-Derived Neisserial Antigen 1870

Background. Meningococcal outer membrane vesicle (OMV) vaccines are efficacious in humans but have serosubtype-specific serum bactericidal antibody responses directed at the porin protein PorA and the potential for immune selection of PorA-escape mutants.Methods. We prepared an OMV vaccine from a Neisseria meningitidis strain engineered to overexpress genome-derived neisserial antigen (GNA) 1870, a lipoprotein discovered by genome mining that is being investigated for use in a vaccine.Results. Mice immunized with the modified GNA1870-OMV vaccine developed broader serum bactericidal antibody responses than control mice immunized with a recombinant GNA1870 protein vaccine or an OMV vaccine prepared from wild-type N. meningitidis or a combination of vaccines prepared from wild-type N. meningitidis and recombinant protein. Antiserum from mice immunized with the modified GNA1870-OMV vaccine also elicited greater deposition of human C3 complement on the surface of live N. meningitidis bacteria and greater passive protective activity against meningococcal bacteremia in infant rats. A N. meningitidis mutant with decreased expression of PorA was more susceptible to bactericidal activity of anti-GNA1870 antibodies.Conclusions. The modified GNA1870-OMV vaccine elicits broader protection against meningococcal disease than recombinant GNA1870 protein or conventional OMV vaccines and also has less risk of selection of PorA-escape mutants than a conventional OMV vaccine.