Potential of recombinant opa proteins as vaccine candidates against hyperinvasive meningococci

Abstract

Neisseria meningitidis causes half a million cases of septicemia and meningitis globally each year. The opacity (Opa) integral outer membrane proteins from N. meningitidis are polymorphic and highly immunogenic. Particular combinations of Opa proteins are associated with the hyperinvasive meningococcal lineages that have caused the majority of serogroup B and C meningococcal disease in industrialized countries over the last 60 years. For the first time, this genetic structuring of a diverse outer membrane protein family has been used to select a novel combination of representative antigens for immunogenicity testing. Fourteen recombinant Opa variants were produced and used in murine immunizations inducing an increase in specific antimeningococcal total IgG levels. All 14 Opa proteins elicited bactericidal antibodies against at least one hyperinvasive meningococcal isolate, and most isolates from each hyperinvasive lineage were killed by at least one Opa antiserum at a titer of 1:16 or greater. Cross-reactive bactericidal antibody responses were observed among clonal complexes. A theoretical coverage of 90% can be achieved by using a particular combination of 6 Opa proteins against an isolate collection of 227 recent United Kingdom disease cases. This study indicates the potential of Opa proteins to provide broad coverage against multiple meningococcal hyperinvasive lineages.

Fig. 1.

Reverse cumulative frequency graph of ELISA data for 29 isolates showing the percentage of fold rises in the ELISA antibody level (optical density units) with Opa antisera versus negative-control serum (Freund's adjuvant only). Antisera specific to individual Opa proteins were tested against meningococci from the same clonal complex expressing both homologous and heterologous Opa proteins, and the highest fold rise for each isolate was used in this figure. The median fold rise was 5.60.

Fig. 2.

(A) Proportion of isolates from each clonal complex which expressed at least one targeted Opa protein killed in SBA experiments using at least one serum sample (at an SBA titer of ≥1 in 16) raised against Opa proteins from the same clonal complex. (B) Reverse cumulative frequency graph of SBA titer in all 24 isolates which expressed at least one targeted Opa protein and were killed in the assay. Antisera specific to individual Opa proteins were tested against meningococci from the same clonal complex expressing both homologous and heterologous Opa proteins, and the highest SBA titer for each isolate was used in this figure. The median SBA titer was 1,659.

Fig. 3.

Immunogold electron micrographs showing binding of pooled murine sera raised against the OpaB protein (50 μg) from the ST-32 complex (OpaB185) against a meningococcal isolate (BZ83) with Opa protein expressed (A) and an isolate (H44/76) which expressed no Opa proteins (B).

Fig. 4.

Summary of dot blotting and SBA analyses to assess cross-reactivity of bactericidal antibodies against isolates from clonal complexes other than that of the Opa protein used for immunization. The central box provides details of the Opa protein used for immunization and the target isolate against which bactericidal antibodies were demonstrated, with the associated SBA titer. *, positive dot blots were those rated “high-intensity” reactions by two independent observers; **, negative when tested by dot blotting; CC, clonal complex of Opa protein or target strain.

Fig. 5.

Increasing potential vaccine coverage (hypothetical and adjusted for SBA results) of 227 isolates from invasive disease in the United Kingdom between 1996 and 2001 (at least one Opa protein in their repertoire was targeted) with addition of each Opa variant by ranked frequency, showing the number of hits (i.e., HV1 or HV2) per organism. One hit occurs when an HV1 or HV2 region of an isolate that is identical to the HV1 or HV2 region from one of the Opa proteins. In this example, Opa proteins would be added in this order: 1, Opa132; 2, Opa213; 3, Opa96; 4, Opa246; 5, Opa317; 6, Opa201; 7, Opa265; 8, Opa245; 9, Opa161; 10, Opa34; 11, Opa185; 12, Opa147; 13, Opa244; 14, Opa218.