Recombinant hyaluronate associated protein as a protective immunogen against Streptococcus equi and Streptococcus zooepidemicus challenge in mice

The capsule of Streptococcus equi, the cause of strangles, and Streptococcu s zooepidemicus, associated with equine lower airway disease, plays an impo rtant role in evasion of phagocytosis by polymorphonuclear leucocytes. It i s composed of hyaluronate, making it non-immunogenic. A hyaluronate associa ted protein (HAP) from S. equisimilis, whose gene has been sequenced [1], w as investigated (a) for its presence in S. equi and S. zooepidemicus and (b ) as an immunogen able to interfere with capsule structure and protect agai nst experimental challenge of mice. The purified capsule of S. equi contain ed a protein of similar molecular mass to the S. equisimilis protein (appro ximately 53 kDa). Polymerase chain reaction (PCR) using primers derived fro m the published sequence of S. equisimilis HAP yielded a product from S. eq ui and S. zooepidemicus of the expected size and susceptibility to restrict ion endonucleases. Subcloning of two large in frame Stul/Sspl fragments of the HAP gene from S. equi, approximately equivalent to the two halves of th e molecule, into the expression vector pGEX-3X yielded only the carboxy hal f in the correct orientation. This latter recombinant produced a GST fusion protein (HAP-GST) of the expected size that was affinity purified. Antibod ies in rabbit antiserum to the native protein in purified hyaluronate react ed strongly in immunoblots with HAP-GST Antiserum to HAP-GST, when soaked i nto filter paper strips, caused a diminution of capsule production by S. eq ui cultured on blood agar. Antiserum added into fresh rabbit blood was not opsonic for S. equi. Immunization with HAP-GST significantly reduced rhinit is in Balb/C mice challenged nasally with S. equi and significantly increas ed survival time and clearance of bacteria in CBA/CA mice challenged intrap eritoneally with S. zooepidemicus. (C) 1999 Academic Press.