Peter

PETER BEERNINK, PhD
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RESEARCH

Meningococci cause life-threatening cases of bacterial meningitis and bloodstream infections. These infections are caused by different strains of the bacteria, which are classified into groups based on the sugar coating (“polysaccharide capsule”) that surrounds the bacteria. The polysaccharides from groups A, C, W and/or Y are used in vaccines that are licensed in the U.S. and other countries. These vaccines are highly efficacious against strains from the same groups, however they do not protect against group B strains, which cause a significant burden of disease in the U.S. and Europe. Recently, two meningococcal group B (MenB) vaccines that contain protein antigens were developed; these vaccines are licensed in the U.S., Canada, Australia and/or European Union (1).

A key antigen in the two MenB vaccines is a meningococcal surface protein known as Factor H-binding protein (FHbp). FHbp exhibits broad sequence diversity; the three variant groups (2) or two sub-families (3) can be represented in a phylogenic tree (Figure 2). The FHbp antigen in one of the licensed protein-based vaccines, MenB-4C (Bexsero®, GSK) (5, 6) is divergent from the antigen in the same variant group in the second vaccine MenB-FHbp (Trumenba®, Pfizer) (7, 8).

Figure 2

Figure 2. Phylogenic tree depicting 72 of 964 known FHbp amino acid sequence variants using MEGA 7.0 (4). The variants included in the licensed vaccines are labeled in bold and variants expressed by strains that caused recent outbreaks are labeled with smaller numbers. The scale bar indicates five percent sequence divergence.

Factor H-binding Proteins Engineered to Eliminate Binding of Factor H
The discovery that FHbp binds the human complement regulatory protein Factor H (FH) (9) raised the question of whether binding of FH to the vaccine antigen masks important epitopes (10). Together with our collaborators at the Univ. of Massachusetts, we showed that the protective antibody responses of transgenic mice that produce the human FH protein, which were vaccinated with FHbp, were lower than those of wild-type mice (11). The lower responses were specific to FHbp since the transgenic and wild-type mice had similar responses to control antigens that were administered concurrently. The detrimental effect of FH on FHbp immunogenicity was confirmed in a second study using the licensed MenB-4C vaccine (12).

The crystal structure of FHbp in a complex with a fragment of human FH made it possible to engineer an FHbp antigen that did not bind FH (13). We predicted that, by unmasking epitopes in the FH-binding site, the protective antibody responses of humans would be enhanced. We tested this hypothesis by vaccinating transgenic mice that produced human FH, and the protective antibody responses to the mutant FHbp were higher than those to the native FHbp vaccine (11). A second study with native outer membrane vesicle (NOMV) vaccines with over-expressed FHbp (wild-type or mutant) confirmed the superior protective antibody responses to the FH non-binding mutant FHbp vaccine (14). In a recent study, we used a random mutant FHbp library approach to identify new mutants with lower FH binding that elicited higher bactericidal antibody titers (Figure 3) (15).

Figure 3

Figure 3. Serum bactericidal antibody responses of human FH transgenic mice to recombinant mutant FHbp vaccines. The mutant FHbp vaccines elicited 13- to 20-fold higher bactericidal titers compared with the control wild-type (WT) FHbp vaccine (15).

Responses of Rhesus Macaques to Vaccines Containing FHbp
We recently found that rhesus macaques have two sequence polymorphisms in the complement Factor H (FH) protein; some of the macaques have an FH protein that binds strongly to FHbp whereas other animals have FH that binds weakly (16). Based on our previous studies in human FH transgenic mice, we predicted that the animals with strong FH binding (similar to humans) would have lower antibody responses to a vaccine containing FHbp than animals with weak FH binding. When we immunized macaques with strong or weak binding of FH to FHbp, we found higher protective antibody responses of the animals with weak binding of FH to FHbp (17). The differences were larger as the FHbp sequence variant in the bacterial strain diverged from the ID 1 variant contained in the vaccine (Figure 4). The differences in the antibody responses were specific to FHbp since other antigens in the vaccine elicited similar responses in the two groups (17). In a larger recent study, we tested a new mutant FHbp, which has very low binding to human FH, in infant rhesus macaques with high binding of FH to wild-type FHbp (18). Collectively, the data support further development of vaccines containing mutant FHbp molecules that do not bind FH.

Figure 4

Figure 4. Serum bactericidal antibody responses of infant rhesus macaques to three doses of licensed MenB-4C vaccine (Bexsero®, GSK). Five isogenic mutant strains were tested in which the FHbp variant had decreasing sequence identity to the ID 1 variant present in the vaccine (17). Geometric mean titers (GMT) and the 95% confidence intervals are shown.

An FHbp Antigen Engineered to Increase Thermal Stability
By changing one or two amino acids in a salt-bridge network in FHbp, we showed that this network was important for thermal stability (19). Certain FHbp variants have low thermal stability and differ in sequence in the region of this network. Therefore, by replacing two amino acid residues in a less stable variant with the corresponding residues in a more stable variant, we increased the stability of the amino-terminal domain by 21 °C (Figure 5A). The crystal structure of the stabilized FHbp mutant showed that the mutant had atomic interactions that were very similar to the naturally stable variant. Further, monoclonal antibodies bound with higher affinity to the stabilized mutant FHbp than to the wild-type FHbp by SPR and ELISA (Figure 5B). The mutants to increase thermal stability were subsequently combined with an additional mutant to decrease FH binding, which represent optimized FHbp antigens that can be used in next-generation meningococcal vaccines (20).

Figure 5
Figure 5. A. Thermal stability of FHbp wild-type (WT) and mutant. The unfolding transition for the amino-terminal domain differs between WT and mutant (39 and 60 °C, respectively). The transition for the carboxyl-terminal domain is similar (82 °C). B. Anti-FHbp monoclonal antibody (MAb) JAR 4 binds better to the mutant than to the WT as seen from a lower concentration of MAb is needed to reach a certain optical density (OD). The results were confirmed by several other techniques (19).

Bibliography

  1. Granoff DM (2013) Commentary: European Medicines Agency recommends approval of a broadly protective vaccine against serogroup B meningococcal disease. Pediatr Infect Dis J 32: 372-373. PubMed ID: 23263177
  2. Masignani V, Comanducci M, Giuliani MM, Bambini S, Adu-Bobie J et al. (2003) Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J Exp Med 197: 789-799. PubMed ID: 12642606
  3. Fletcher LD, Bernfield L, Barniak V, Farley JE, Howell A et al. (2004) Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun. 72: 2088-2100. PubMed ID: 15039331
  4. Kumar S, Stecher G, and Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets Molecular Biology and Evolution 33:1870-1874
  5. Giuliani MM, Adu-Bobie J, Comanducci M, Aricò B, Savino S et al. (2006) A universal vaccine for serogroup B meningococcus. Proc Natl Acad Sci USA 103: 10834-10839. PubMed ID: 16825336
  6. Snape MD, Dawson T, Oster P, Evans A, John TM et al. (2010) Immunogenicity of two investigational serogroup B meningococcal vaccines in the first year of life: a randomized comparative trial. Pediatr Infect Dis J 29: e71-9. PubMed ID: 20844462
  7. Jiang HQ, Hoiseth SK, Harris SL, McNeil LK, Zhu D et al. (2010) Broad vaccine coverage predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup B meningococcal disease. Vaccine 28: 6086-6093. PubMed ID: 20619376
  8. Marshall HS, Richmond PC, Nissen MD, Jiang Q, Anderson AS et al. (2012) Safety and immunogenicity of a meningococcal B bivalent rLP2086 vaccine in healthy toddlers aged 18-36 months: a phase 1 randomized-controlled clinical trial. Pediatr Infect Dis J 31: 1061-1068. PubMed ID: 22718089
  9. Madico G, Welsch JA, Lewis LA, McNaughton A, Perlman DH et al. (2006) The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance. J Immunol 177: 501-510. PMID: 16785547
  10. Meri SM, Jordens M and Jarva H (2008) Microbial complement inhibitors as vaccines. Vaccine 26 Suppl 8: I113-117. PubMed ID: 19388175
  11. Beernink PT, Shaughnessy J, Braga EM, Liu Q, Rice PA et al. (2011) A meningococcal factor H binding protein mutant that eliminates Factor H binding enhances protective antibody responses to vaccination. J Immunol 186: 3606-3614. PubMed ID: 21325619
  12. Costa I, Pajon R and Granoff DM (2014) Human factor H (FH) impairs protective meningococcal anti-FHbp antibody responses and the antibodies enhance FH binding. MBio 5: e01625-14. PubMed ID: 25161192
  13. Schneider MC, Prosser BE, Caesar JJ, Kugelberg E, Li S et al. (2009) Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates. Nature 458: 890-893. PubMed ID: 19225461
  14. Beernink, P. T., J. Shaughnessy, R. Pajon, E. M. Braga, S. Ram, and D. M. Granoff (2012) The effect of human factor H on immunogenicity of meningococcal native outer membrane vesicle vaccines with over-expressed factor H binding protein. PLoS Pathogens 8: e1002688. PubMed ID: 22589720
  15. Konar M, Rossi R, Walter H, Pajon R, Beernink PT (2015) A mutant library approach to identify improved meningococcal Factor H binding protein vaccine antigens. PLoS One. 2015 Jun 9;10(6):e0128185. PubMed ID: 26057742
  16. Konar M, Beernink PT and Granoff DM (2015) A newly-identified polymorphism in rhesus macaque complement Factor H modulates binding affinity for meningococcal FHbp. PLoS One 10: e0135996. PubMed ID: 26285122
  17. Granoff DM, Costa I, Konar M, Giuntini S, Van Rompay KK and Beernink PT (2015) Binding of complement Factor H (FH) decreases protective anti-FH binding protein antibody responses of infant rhesus macaques immunized with a meningococcal serogroup B vaccine. J Infect Dis 212: 784-792. PubMed ID: 25676468
  18. Granoff DM, Giuntini S, Gowans A, Lujan E, Sharkey K and Beernink PT (2016) Enhanced protective antibody to a mutant meningococcal Factor H-binding protein with low Factor H binding. JCI Insight 1(14): e88907. doi:10.1172/jci.insight.88907
  19. Konar M, Pajon R, Beernink PT (2015) A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes. Proc Natl Acad Sci USA 112: 14823-14828. PubMed ID: 26627237
  20. Rossi R, Konar M, Beernink PT (2016) Meningococcal Factor H binding protein vaccine antigens with increased thermal stability and decreased binding of human Factor H. Infect Immun 84: 1735-1742. PubMed ID: 27021245

Revised: Thursday, September 22, 2016 2:12 PM

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