Purified capsular polysaccharides of bacteria have been used to prepare vaccines against the cognate bacteria. Resulting immune responses have often been less satisfactory than desirable, particularly in very young children or individuals with immature or deficient immunological systems. The Haemophilus influenzae type b capsular polysaccharide by itself, for example, fails to provide an adequate immune response in infants. Thus this polysaccharide is ineffective in providing protection against the serious pediatric medical problems caused by H. influenza type b bacterial infections.
Enhancement of the immunogenicity of polysaccharides may often be accomplished by combining them with proteins [Schneerson et al., "Haemophilus Influenzae Type b Polysaccharide-Protein Conjugates: Model for a New Generation of Capsular Polysaccharide Vaccines," New Dev. With Hum. & Vet. Vaccines., 77-94 (1980); Schneerson, et al., J. Exptl. Med., 152:361 (1980); Anderson, Infection and Immunity, 39:233 (1983)].
Care must be exercised in the selection of the protein which is to be combined with these polysaccharides, however, as certain proteins (e.g. pertussinogen) are non-specific stimulators of the immune system in infants. These proteins can, to a degree, enhance the immune response to polysaccharide antigens, but unfortunately such non-specific activation leads to unwanted biological effects such as reactogenicity. The much preferred specific enhanced immune response to these polysaccharides can be achieved in infants by "conjugating" these polysaccharides to appropriate proteins, as first reported by Goebel et al., [J. Exptl. Med. 50:521-531 (1929)].
The means of combining the polysaccharide and protein must also be carefully considered. If, as is believed, the immunological enhancement is realized as a result of the molecular proximity of the polysaccharide determinants to the protein "carrier" determinants, these moieties should not easily separate in the biological system. Non-covalent complexes, arising from the polyanionic character of the polysaccharides and the polycationic character of "carrier" proteins, may stimulate immune responses, but these complexes are chemically labile and the resultant immune responses appear to show T-cell independency, and may be poorly reproducible. By contrast, covalent conjugates of polysaccharides and protein possess much greater chemical stability and may demonstrate T-cell dependent immune responses, and good reproducibility.
Covalent polysaccharide-protein conjugates have been disclosed in the literature, but the exact nature of the covalent linkage has not been proven or quantified since the only assay for covalency has been activity in vivo and the processes disclosed in the literature have been difficult to reproduce. Haemophilus influenzae type b or Streptococcus pneumoniae type 6A polysaccharides (PnPs6A) was reacted with cyanogen bromide, then with adipic acid dihydrazide, then "coupled" with tetanus toxoid or horseshoe crab hemocyanin proteins in Schneerson et al., (J. Exptl. Med. 1.52:361 (1980); Infection and Immunity 40:245 (1983)). Pneumococcal type 19 F polysaccharide (PnPs19 F) was coupled to bovine serum albumin directly by forming imines (Schiff bases) from the reducing ends of the polysaccharides and the pendant amine groups (i.e., lysines) of the protein, then reducing these imines with sodium cyanoborohydride [Lin et al. Immunology 46:333 (1982)].
Additionally, polysaccharides linked to diazotized aromatic amines were coupled to the protein's tyrosines in K. K. Nixdorff et al., [Immunology 29:87 (1975)]and polysaccharides linked to isothiocyanates, which were then linked to the pendant amino groups of the protein's lysine in Svenson et al., [J. Immunolog. Methods 25:323 (1979)]. In each case, however, the resulting conjugate was characterized only by its gel permeation chromatographic behavior. In still another example [Nutani et al., Infection and Immunity 36:971 (1982)]the polysaccharide, pullulan, was activated with cyanuric chloride, then reacted with tetanus toxoid. In this case, the conjugates were characterized by electrophoresis and only shown to be different from the starting materials.
Marburg et al., in U.S. Pat. No. 4,695,624, describe covalently-modified bacterial polysaccharides and chemically-stable conjugates of such polysaccharides with covalently-modified immunogenic membrane proteins, viral protein subunits, synthetic polypeptides, bacterial toxoids or other suitable immunogenic proteins, which conjugates are useful components of immunogenic anti-bacterial vaccines. The polysaccharide and protein entities are coupled through bigeneric spacers containing a covalent thioether group. These spacers are atom chains linking polysaccharide and protein macromolecules. One end of the spacer originates with a covalently modified polysaccharide, while the other end originates with the functionalized protein.
The instant invention differs from the invention disclosed in the U.S. Pat. No. 4,695,624 in that a conjugate vaccine is produced that has an increased polysaccharide to protein ratio and has the ability to induce immune responses protective against infection by more than one bacterial pathogen.