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

2015 ◽  
Vol 84 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Eduardo Lujan ◽  
Rolando Pajon ◽  
Dan M. Granoff
Keyword(s):  
Transgenic Mice ◽  
Outer Membrane ◽  
Protein A ◽  
Surface Protein ◽  
Factor H ◽  
Antibody Responses ◽  
Complement Factor ◽  
Wild Type ◽  
Protective Antibody ◽  
Bactericidal Antibody

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 inEscherichia colimicrovesicles, 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.

scholarly journals Does Binding of Complement Factor H to the Meningococcal Vaccine Antigen, Factor H Binding Protein, Decrease Protective Serum Antibody Responses?

2013 ◽  
Vol 20 (8) ◽  
pp. 1099-1107 ◽  
Author(s):  
Dan M. Granoff ◽  
Sanjay Ram ◽  
Peter T. Beernink
Keyword(s):  
Transgenic Mice ◽  
Meningococcal Disease ◽  
Binding Protein ◽  
Factor H ◽  
Antibody Responses ◽  
Vaccine Antigen ◽  
Single Amino Acid ◽  
Complement Factor ◽  
Meningococcal Vaccine ◽  
Bactericidal Antibody

ABSTRACTFactor H binding protein (fHbp) is a principal antigen in a multicomponent meningococcal vaccine recently licensed in Europe for prevention of serogroup B diseases. The protein recruits the complement downregulator, factor H (fH), to the bacterial surface, which enables the organism to resist complement-mediated bacteriolysis. Binding is specific for human fH. In preclinical studies, mice and rabbits immunized with fHbp vaccines developed serum bactericidal antibody responses, which in humans predict protection against developing meningococcal disease. These studies, however, were in animals whose fH did not bind to the vaccine antigen. Here we review the immunogenicity of fHbp vaccines in human fH transgenic mice. The data suggest that animals with high serum human fH concentrations have impaired protective antibody responses. Further, mutant fHbp vaccines with single amino acid substitutions that decrease fH binding are superior immunogens, possibly by unmasking epitopes in the fH binding site that are important for eliciting serum bactericidal antibody responses. Humans immunized with fHbp vaccines develop serum bactericidal antibody, but achieving broad coverage in infants required incorporation of additional antigens, including outer membrane vesicles, which increased rates of fever and local reactions at the injection site. The experimental results in transgenic mice predict that fHbp immunogenicity can be improved in humans by using mutant fHbp vaccines with decreased fH binding. These results have important public health implications for developing improved fHbp vaccines for control of serogroup B meningococcal disease and for development of vaccines against other microbes that bind host molecules.

scholarly journals A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes

2015 ◽  
Vol 112 (48) ◽  
pp. 14823-14828 ◽  
Author(s):  
Monica Konar ◽  
Rolando Pajon ◽  
Peter T. Beernink
Keyword(s):  
Thermal Stability ◽  
Electrostatic Interactions ◽  
Double Mutant ◽  
Factor H ◽  
Antibody Responses ◽  
Vaccine Antigen ◽  
Complement Factor ◽  
Wild Type ◽  
Protective Antibody ◽  
Terminal Domain

Factor H binding protein (FHbp) is part of two vaccines recently licensed for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp is classified in three phylogenic variant groups that have limited antigenic cross-reactivity, and FHbp variants in one of the groups have low thermal stability. In the present study, we replaced two amino acid residues, R130 and D133, in a stable FHbp variant with their counterparts (L and G) from a less stable variant. The single and double mutants decreased thermal stability of the amino- (N-) terminal domain compared with the wild-type protein as measured by scanning calorimetry. We introduced the converse substitutions, L130R and G133D, in a less stable wild-type FHbp variant, which increased the transition midpoint (Tm) for the N-terminal domain by 8 and 12 °C; together the substitutions increased the Tm by 21 °C. We determined the crystal structure of the double mutant FHbp to 1.6 Å resolution, which showed that R130 and D133 mediated multiple electrostatic interactions. Monoclonal antibodies specific for FHbp epitopes in the N-terminal domain had higher binding affinity for the recombinant double mutant by surface plasmon resonance and to the mutant expressed on meningococci by flow cytometry. The double mutant also had decreased binding of human complement Factor H, which in previous studies increased the protective antibody responses. The stabilized mutant FHbp thus has the potential to stabilize protective epitopes and increase the protective antibody responses to recombinant FHbp vaccines or native outer membrane vesicle vaccines with overexpressed FHbp.

scholarly journals Clumping Factor A Interaction with Complement Factor I Increases C3b Cleavage on the Bacterial Surface of Staphylococcus aureus and Decreases Complement-Mediated Phagocytosis

2010 ◽  
Vol 78 (4) ◽  
pp. 1717-1727 ◽  
Author(s):  
Pamela S. Hair ◽  
Charlene G. Echague ◽  
Amber M. Sholl ◽  
Justin A. Watkins ◽  
Joan A. Geoghegan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Protein ◽  
Factor H ◽  
Complement Factor ◽  
Wild Type ◽  
Bacterial Surface ◽  
Complement Control Protein ◽  
Factor I ◽  
Protein Factor ◽  
Fibrinogen Binding

ABSTRACT The human complement system is important in the immunological control of Staphylococcus aureus infection. We showed previously that S. aureus surface protein clumping factor A (ClfA), when expressed in recombinant form, bound complement control protein factor I and increased factor I cleavage of C3b to iC3b. In the present study, we show that, compared to the results for the wild type, when isogenic ClfA-deficient S. aureus mutants were incubated in serum, they bound less factor I, generated less iC3b on the bacterial surface, and bound fewer C3 fragments. It has been shown previously that two amino acids in ClfA (P336 and Y338) are essential for fibrinogen binding. However, S. aureus expressing ClfA(P336A Y338S) was less virulent than ClfA-deficient strains in animal models. This suggested that ClfA contributed to S. aureus virulence by a mechanism different than fibrinogen binding. In the present study, we showed that S. aureus expressing ClfA(P336A Y338S) was more susceptible to complement-mediated phagocytosis than a ClfA-null mutant or the wild type. Unlike ClfA, ClfA(P336A Y338S) did not enhance factor I cleavage of C3b to iC3b and inhibited the cofactor function of factor H. Fibrinogen enhanced factor I binding to ClfA and the S. aureus surface. Twenty clinical S. aureus strains all expressed ClfA and bound factor I. High levels of factor I binding by clinical strains correlated with poor phagocytosis. In summary, our results suggest that the interaction of ClfA with factor I contributes to S. aureus virulence by a complement-mediated mechanism.

scholarly journals Human Factor H (FH) Impairs Protective Meningococcal Anti-FHbp Antibody Responses and the Antibodies Enhance FH Binding

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Isabella Costa ◽  
Rolando Pajon ◽  
Dan M. Granoff
Keyword(s):  
Transgenic Mice ◽  
Bactericidal Activity ◽  
Binding Protein ◽  
Vaccine Safety ◽  
Factor H ◽  
Antibody Responses ◽  
Wild Type ◽  
Serum Samples ◽  
Serum Bactericidal Activity ◽  
4Cmenb Vaccine

ABSTRACT The meningococcal 4CMenB vaccine (Bexsero; Novartis) contains four antigens that can elicit serum bactericidal activity, one of which is factor H (FH)-binding protein (FHbp). FHbp specifically binds human complement FH. When humans are immunized, FHbp is expected to form a complex with FH, which could affect immunogenicity and safety. Wild-type mice (whose FH does not bind to FHbp) and human FH transgenic mice were immunized with three doses of 4CMenB, and their responses were compared. There were no significant differences between the serum bactericidal responses of transgenic and wild-type mice to strains with all of the antigens mismatched for 4CMenB except PorA or NadA. In contrast, against a strain mismatched for all of the antigens except FHbp, the transgenic mice had 15-fold weaker serum bactericidal antibody responses (P = 0.0006). Binding of FH downregulates complement. One explanation for the lower anti-FHbp serum bactericidal activity in the transgenic mice is that their postimmunization serum samples enhanced the binding of FH to FHbp, whereas the serum samples from the wild-type mice inhibited FH binding. Control antiserum from transgenic mice immunized with a low-FH-binding mutant FHbp (R41S) vaccine inhibited FH binding. Two 4CMenB-vaccinated transgenic mice developed serum IgM autoantibodies to human FH. Thus, human FH impairs protective serum anti-FHbp antibody responses, in part by skewing the antibody repertoire to FHbp epitopes outside the FH binding site. FHbp vaccines that bind FH may elicit FH autoantibodies. Mutant FHbp antigens with low FH binding could improve protection and, potentially, vaccine safety in humans. IMPORTANCE Two serogroup B meningococcal vaccines contain a novel antigen called factor H (FH)-binding protein (FHbp). FHbp specifically binds human FH, a plasma protein that downregulates complement. One vaccine (4CMenB; Novartis) is licensed in Europe, Canada, and Australia. When humans are immunized, FHbp can complex with FH. We compared the immunogenicity of 4CMenB vaccine in wild-type mice, whose own FH does not bind to FHbp, and human FH transgenic mice. Transgenic mice had respective antibody responses similar to those of wild-type mice to 4CMenB antigens that do not bind FH. However, the protective antibody responses of the transgenic mice to FHbp were impaired, largely because the antibodies did not inhibit but rather enhanced the binding of FH to FHbp. Two transgenic mice developed serum IgM autoantibodies to FH. Mutant FHbp antigens with low FH binding likely will elicit greater protection in humans than FHbp vaccines that bind FH and have a lower risk of FH autoantibodies.

scholarly journals Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine.

1993 ◽  
Vol 178 (1) ◽  
pp. 197-209 ◽  
Author(s):  
C K Stover ◽  
G P Bansal ◽  
M S Hanson ◽  
J E Burlein ◽  
S R Palaszynski ◽  
...  
Keyword(s):  
Outer Surface ◽  
Protein A ◽  
Surface Protein ◽  
Antibody Responses ◽  
Target Antigen ◽  
Bacille Calmette Guerin ◽  
Protective Antibody ◽  
Outer Surface Protein A ◽  
Outer Surface Protein ◽  
Recombinant Bcg

The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F. de la Cruz, T.R. Fuerst, J.E. Burlein, L.A. Benson, L.T. Bennett, G.P. Bansal, J.F. Young, M.H. Lee, G.F. Hatfull et al. 1991. Nature [Lond]. 351:456; Jacobs, W.R., Jr., S.B. Snapper, L. Lugosi and B.R. Bloom. 1990. Curr. Top. Microbiol. Immunol. 155:153; Jacobs, W.R., M. Tuckman, and B.R. Bloom. 1987. Nature [Lond.]. 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses. Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens. Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100-1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein. Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth. Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response.

scholarly journals Meningococcal Factor H Binding Protein Vaccine Antigens with Increased Thermal Stability and Decreased Binding of Human Factor H

2016 ◽  
Vol 84 (6) ◽  
pp. 1735-1742 ◽  
Author(s):  
Raffaella Rossi ◽  
Monica Konar ◽  
Peter T. Beernink
Keyword(s):  
Thermal Stability ◽  
Binding Protein ◽  
Alternative Pathway ◽  
Factor H ◽  
Antibody Responses ◽  
Protein Vaccine ◽  
Wild Type ◽  
Complement Alternative Pathway ◽  
Content Type ◽  
Protective Antibody

Neisseria meningitidiscauses cases of bacterial meningitis and sepsis. Factor H binding protein (FHbp) is a component of two licensed meningococcal serogroup B vaccines. FHbp recruits the complement regulator factor H (FH) to the bacterial surface, which inhibits the complement alternative pathway and promotes immune evasion. Binding of human FH impairs the protective antibody responses to FHbp, and mutation of FHbp to decrease binding of FH can increase the protective responses. In a previous study, we identified two amino acid substitutions in FHbp variant group 2 that increased its thermal stability by 21°C and stabilized epitopes recognized by protective monoclonal antibodies (MAbs). Our hypothesis was that combining substitutions to increase stability and decrease FH binding would increase protective antibody responses in the presence of human FH. In the present study, we generated four new FHbp single mutants that decreased FH binding and retained binding of anti-FHbp MAbs and immunogenicity in wild-type mice. From these mutants, we selected two, K219N and G220S, to combine with the stabilized double-mutant FHbp antigen. The two triple mutants decreased FH binding >200-fold, increased the thermal stability of the N-terminal domain by 21°C, and bound better to an anti-FHbp MAb than the wild-type FHbp. In human-FH-transgenic mice, the FHbp triple mutants elicited 8- to 15-fold-higher protective antibody responses than the wild-type FHbp antigen. Collectively, the data suggest that mutations to eliminate binding of human FH and to promote conformational stability act synergistically to optimize FHbp immunogenicity.

scholarly journals Meningococcal Outer Membrane Vesicle Vaccines Derived from Mutant Strains Engineered To Express Factor H Binding Proteins from Antigenic Variant Groups 1 and 2

2008 ◽  
Vol 16 (2) ◽  
pp. 156-162 ◽  
Author(s):  
Oliver Koeberling ◽  
Serena Giuntini ◽  
Anja Seubert ◽  
Dan M. Granoff
Keyword(s):  
Outer Membrane ◽  
Mononuclear Cells ◽  
Membrane Vesicle ◽  
Factor H ◽  
Antibody Responses ◽  
Outer Membrane Vesicle ◽  
Antigenic Variant ◽  
Endotoxin Activity ◽  
Bactericidal Antibody ◽  
Group B

ABSTRACT Meningococcal outer membrane vesicle (OMV) vaccines, which are treated with detergents to decrease endotoxin activity, are safe and effective in humans. However, the vaccines elicit serum bactericidal antibody responses largely directed against PorA, which is antigenically variable. We previously prepared a native (non-detergent-treated) OMV vaccine from a mutant of group B strain H44/76 in which the lpxL1 gene was inactivated, which resulted in penta-acylated lipid A with attenuated endotoxin activity. To enhance protection, we overexpressed factor H binding protein (fHbp) from the antigenic variant 1 group. The vaccine elicited broad serum bactericidal antibody responses in mice against strains with fHbp variant 1 (∼70% of group B isolates) but not against strains with variant 2 or 3. In the present study, we constructed a mutant of group B strain NZ98/254 with attenuated endotoxin that expressed both endogenous variant 1 and heterologous fHbp variant 2. A mixture of the two native OMV vaccines from the H44/76 and NZ98/254 mutants stimulated proinflammatory cytokine responses by human peripheral blood mononuclear cells similar to those stimulated by control, detergent-treated OMV vaccines from the wild-type strains. In mice, the mixture of the two native OMV vaccines elicited broad serum bactericidal antibody responses against strains with heterologous PorA and fHbp in the variant 1, 2, or 3 group. By adsorption studies, the principal bactericidal antibody target was determined to be fHbp. Thus, native OMV vaccines from mutants expressing fHbp variants have the potential to be safe for humans and to confer broad protection against meningococcal disease from strains expressing fHbp from each of the antigenic variant groups.

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