Many of the new protein antigens being investigated as vaccine candidates for prevention of group B meningococcal disease show antigenic variability and/or variable expression by different N. meningitidis
). Achievement of broad protection by a recombinant protein-based vaccine, therefore, will likely require the use of multiple antigens (17
). However, there may be limits to the number of different recombinant proteins that can be combined into one vaccine without loss of immunogenicity. Therefore, for a multicomponent vaccine containing fHbp, it would be desirable to use a single rfHbp capable of eliciting serum bactericidal antibodies against strains expressing fHbps from the different major antigenic variant groups.
Chimeric proteins previously have been used for vaccine development in a variety of ways. One strategy employed a genetic fusion of two different antigens from the same organism to enhance cross-protection against strains with antigenic diversity (18
). An example is a multicomponent meningococcal group B vaccine that contains two fusion proteins, GNA2091 fused with fHbp v.1 and GNA2132 fused with GNA1030, along with a third antigen, rNadA (17
). In mice each of the individual fusion proteins elicited serum bactericidal antibody responses that were as high as or higher than those elicited by the respective individual recombinant protein antigens. Further, the multicomponent vaccine containing five meningococcal antigens elicited broader bactericidal responses than any of the respective individual antigens.
Another strategy has been to construct a fusion of different serologic variants (“serovars”) of an individual antigen to induce cross-protection against strains with antigenic diversity. An example is a tetravalent OspC chimeric Lyme disease vaccine that induced bactericidal antibody responses against spirochete strains expressing each of the OspC types that were incorporated into the construct (14
). In another example, four different serologic variants of domain III of the dengue fever virus envelope protein were fused together for use as a diagnostic protein (21
). These chimeric antigens consisted of repeats of an individual domain with antigenic variability. The respective variants of the domain were expressed in tandem in one protein (i.e., the same domain from different strains, A1
, etc.). In some cases, these recombinant tandem proteins can be convenient for manufacturing and quality control. However, they also can be very large and subject to improper folding or degradation.
The chimeric fHbp antigens that we investigated differed from the fusion proteins described above in that we combined different individual domains from two proteins (domain A and part of domain B from a v.1 protein with the remainder of domains B and C from a v.2 protein, i.e., A1
) to form chimeric proteins that expressed antigenically unrelated epitopes that were specific for all three fHbp v. groups. There are few examples of combining epitopes expressed by different domains into a single chimeric antigen that resulted in an effective immunogen. In one previous study, the investigators demonstrated that a truncated meningococcal rfHbp consisting only of the B and C domains elicited bactericidal titers similar to those elicited by the respective rfHbp v.1 protein containing the A domain (18
). They then constructed a hybrid of the B domain of an fHbp v.3 protein with the C domain of an fHbp v.1 protein. However, in contrast to the A1
chimeric proteins investigated in the present study, the B3
chimeric fHbp investigated previously did not elicit serum bactericidal antibody responses in mice against strains expressing fHbp from either v.1 or v.3. In retrospect, this chimeric vaccine may not have worked because the chimeric protein contained the B domain from fHbp v.3, which lacks the region of the JAR 3/5 epitope (including G121) that may be critical for eliciting high serum bactericidal antibody titers to strains expressing fHbp v.1. Also, the C domain of the v.1 protein did not contain the JAR 10, 11, 13, or 32 epitopes, which interact with bactericidal antibodies directed at fHbp v.2 or v.3. In contrast, both of the chimeric fHbps that we investigated contained the portion of the B domain of the v.1 protein with the JAR 3/5 epitope and the C domain of a v.2 protein that expressed multiple epitopes shown to interact with bactericidal MAbs against strains with fHbp from the v.2 or v.3 variant group (Fig. ).
Mice immunized with either chimeric protein vaccine I or II developed serum anti-fHbp antibodies with bactericidal activity against N. meningitidis strains that expressed fHbp v.1, v.2, or v.3, whereas the respective control wild-type rfHbp v.1 or v.2 vaccines elicited bactericidal responses predominantly against strains that expressed fHbp from the respective variant group homologous to that of the vaccine. Although the magnitudes of the serum bactericidal titers of the mice immunized with the chimeric vaccines were lower than those of control mice immunized with the corresponding wild-type rfHbp v.1 or v.2 vaccine, the data from the groups given the chimeric vaccines provide proof of concept that an individual chimeric protein can elicit serum antibodies that are bactericidal with human complement against strains expressing fHbps from all three antigenic variant groups. One limitation of our study was that we used FA, which is not suitable for use in humans. Therefore, additional studies are needed to investigate the immunogenicity of the chimeric proteins given with adjuvants that are more suitable for use in humans.
Despite engineering expression of the JAR 11 epitope in chimera I and of the JAR 32 epitope in chimera II, we found no statistically significant differences in the respective serum bactericidal antibody responses of mice immunized with either vaccine when measured against JAR 11-positive strains or JAR 32-positive strains. At the time we designed the chimeric vaccines, we did not know that binding of antibody to an epitope located near residue 174 (i.e., JAR 11 in some strains or JAR 32 in others) was not sufficient to elicit complement-mediated bactericidal activity in the absence of a second antibody that binds to an epitope associated with an ion pair at residues 180 and 192 (such as JAR 10 in some strains or JAR 33 in others) (Beernink et al., submitted). Among wild-type strains expressing fHbp v.2 or v.3, expression of JAR 32 is usually associated with expression of JAR 33, while expression of JAR 11 is usually associated with expression of JAR 10 (see, for example, our strain panel [Table ]). Therefore, in the future it may be possible to generate more effective chimeric fHbp vaccines against JAR 32-positive strains by engineering a chimeric protein that also expresses the JAR 33 epitope.