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contrast, most of the tissue-crossreactive epitopes that have been identified to date occur at a distance from the amino terminus, typically in the A, B, or C repeat regions. Consequently, researchers have attempted to develop a vaccine that incorporates amino terminal fragments of several different M proteins, so that they can evoke opsonic antibodies without evoking tissue-crossreactive antibodies. This approach might be characterized as a “fragment of subunit vaccine.” By incorporating these fragments into a single construct, they hope to minimize the total amount of protein injected while representing the largest possible number of serotypes.

Development of Complex, Type-Specific M Protein Vaccines. Using specific PCR primers, researchers amplified the 5-prime regions of the M protein gene from four different serotypes—M24, M5, M6, and M19. They purified the PCR products, ligated them in a hybrid M protein gene, and inserted the gene into an expression vector, in this case a transformed E. coli. Schematically, the resulting hybrid protein contained 110 amino acids from M24, 58 from M5, and 35 each from M6 and M19. Recombinant hybrid protein was extracted from the E. coli, purified, and used as a vaccine in a series of animal tests.

Three rabbits immunized with the tetravalent protein produced significant levels of antibodies against all four serotypes of native protein. However, the level of relevant (i.e., opsonic) antibodies tended to be higher for the first two fragments (M24 and M5, nearest the amino terminus) and lower for the last two (M6 and M19, nearest the carboxy terminus). Researchers were uncertain whether this difference was a function of the size of the fragments or their position on the hybrid protein. To resolve this uncertainty, researchers added another four fragments toward the carboxy terminus—35 amino acids each from M1, M3, M18, and M2—and injected another set of rabbits with the resulting octavalent protein. Again, the immunized rabbits produced significant levels of antibodies against all of the serotypes, and again the level of opsonic antibodies tended to decline toward the carboxy terminus.

To determine whether multivalent hybrid M protein would be immunogenic when administered locally by the mucosal route, researchers constructed additional hybrid proteins consisting of the entire B subunit of E. coli labile toxin (LT-B) ligated to 15 amino acids from type 5 M protein (M5), using a proline-and glycine-rich linker. Mice that were immunized intranasally with the resulting LT-B-M5 vaccine produced opsonic serum antibodies against the M5 component, and this response afforded significant protection when the mice were challenged peritoneally with type 5 streptococci. When this experiment was repeated with a LT-B-tetravalent vaccine, however, one of the components (M19, nearest the carboxy terminus) was not immunogenic at all. These mice also developed secretory immunoglobulin-A (IGA) in their saliva, a sign of mucosal immunity, but the IgA level was not particularly high.

An alternative to this approach concentrates not on the hypervariable amino terminus but instead on epitopes in the so-called C-repeats region, epitopes that

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