Immunogenicity of a 26-valent group A streptococcal vaccine

Abstract

A multivalent vaccine containing amino-terminal M protein fragments from 26 different serotypes of group A streptococci was constructed by recombinant techniques. The vaccine consisted of four different recombinant proteins that were formulated with alum to contain 400 microg of protein per dose. Rabbits were immunized via the intramuscular route at 0, 4, and 16 weeks. Immune sera were assayed for the presence of type-specific antibodies against the individual recombinant M peptides by enzyme-linked immunosorbent assay and for opsonic antibodies by in vitro opsonization tests and indirect bactericidal tests. The 26-valent vaccine was highly immunogenic and elicited fourfold or greater increases in antibody levels against 25 of the 26 serotypes represented in the vaccine. The immune sera were broadly opsonic and were bactericidal against the majority of the 26 different serotypes. Importantly, none of the immune sera cross-reacted with human tissues. Our results indicate that type-specific, protective M protein epitopes can be incorporated into complex, multivalent vaccines designed to elicit broadly protective opsonic antibodies in the absence of tissue-cross-reactive antibodies.

FIG. 1.

Schematic diagram of the four recombinant fusion proteins contained in the 26-valent M protein-based vaccine. The oligonucleotide primers used to amplify each 5′ emm gene fragment by PCR were synthesized to include the unique restriction enzyme sites indicated. The number of codons contained in each emm gene fragment is indicated below the M type. M101 was formerly stNS5, and M114 was formerly st2967. The M13 is strain M13W, which has been newly designated M94.

FIG. 2.

Summary of the 19 most common invasive emm types of group A streptococci isolated in the United States between 1998 and 2000. These data are part of ongoing studies conducted by the Active Bacterial Core Surveillance Program of the CDC. The 19 emm-types accounted for 84% of the 1,922 invasive isolates submitted during this period of time.

FIG. 3.

Type-specific M protein antibodies evoked by the 26-valent vaccine in rabbits, as determined by ELISA. Three rabbits (represented by the three bars) were each immunized with 400 μg of the 26-valent vaccine at 0, 4, and 16 weeks. ELISAs were performed with preimmune and immune sera (18 weeks) and purified recombinant dimeric peptides copying each M protein fragment (and Spa) included in the vaccine. Antibody titers of <200 were observed for all preimmune sera when tested against each of the antigens. Two of the immune sera contained significant antibody levels (serum antibody titers that increased by fourfold or greater over preimmune levels) against 24 of the 27 vaccine antigens, while the third immune serum contained significant antibody levels against 21 of the 27 antigens.

FIG. 4.

Serum opsonic antibodies evoked in rabbits by the 26-valent vaccine. Immune sera from each of three rabbits (represented by the three bars) were tested in in vitro opsonization assays using the 26 different serotypes of group A streptococci. Percent opsonization is the percentage of neutrophils that had engulfed or were associated with streptococci after rotating for 45 min in whole, nonimmune human blood containing either preimmune or immune rabbit serum. The preimmune sera resulted in ≤10% opsonization with each organism. Thirty percent or greater opsonization (a threefold increase) in the presence of immune sera was considered a positive response.

FIG. 5.

Serum bactericidal antibodies evoked in rabbits by the 26-valent vaccine. Immune sera from each of three rabbits (represented by the three bars) were tested for bactericidal activity against the 26 different serotypes of group A streptococci. Growth of each test organism during 3 h of rotation in nonimmune human blood containing preimmune rabbit serum equaled or exceeded eight generations. Percent reduction in growth in the presence of immune serum is indicated as percent killing. A positive response was considered percent killing equal to or greater than 50%.

FIG. 6.

Serum bactericidal antibodies against type 4 group A streptococci elicited in rabbits by the 26-valent vaccine. Two antisera (represented by the two bars) were tested for in vitro bactericidal activity against seven different clinical pharyngeal isolates of type 4 streptococci. One of the antisera was from a rabbit immunized at 0, 4, and 8 weeks (shaded bar), and the other was from a rabbit immunized at 0, 4, and 16 weeks (open bar). The M4 isolates were from the following five geographic locations: FL, Florida; IL, Illinois; CA, California; CT, Connecticut; SD, South Dakota. Percent killing was determined as described in the legend to Fig. 5.