A process for the isolation and purification of immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b. The PRP has been purified with ethanol, Cetavlon (hexadecyltrimethyl ammonium bromide) and a phosphate containing adsorbent, hydroxylapatite. The contaminants (nucleic acid, proteins and endotoxins) are removed to the minimum level by a treatment with hydroxylapatite. Also described is a process for the preparation of a combined vaccine containing the PRP (prepared as aforementioned) and B. pertussis antigens. The vaccine elicits anti-PRP antibody and antipertussis antibody (as measured by microagglutination) formations in young animals. This sera with anti-PRP antibody exhibits a strong bactericidal activity.
A water-soluble covalent polysaccharide-diphtheria toxoid conjugate having a molecular weight between 140,000 and 4,500,000 dalton and a ribose/protein ratio between 0.25 and 0.75, capable of producing T-cell dependent antibody response to polysaccharide from H. influenzae b, is prepared by mixing a derivatized diphtheria toxoid in a substantially cyanogen halide-free solution with a cyanogen halide-activated capsular H. influenzae b polysaccharide hapten consisting of approximately equal parts of ribose, ribitol and phosphate, which polysaccharide had previously been heat sized until more than 60% attained a molecular size between 200,000 and 2,000,000 dalton.
A water-soluble covalent polysaccharide-diphtheria toxoid conjugate having a molecular weight between 140,000 and 4,500,000 dalton and a ribose/protein ratio between 0.25 and 0.75, capable of producing T-cell dependent antibody response to polysaccharide from H. influenzae b, prepared by mixing a derivatized diphtheria toxoid in a substantially cyanogen halide-free solution with a cyanogen halide-activated capsular H. influenzae b polysaccharide hapten consisting of approximately equal parts of ribose, ribitol and phosphate, which polysaccharide had previously been heat sized until more than 60% attained a molecular size between 200,000 and 2,000,000 dalton.
A combined vaccine for eliciting polyribosyl ribitol phosphate (PRP) antibody formations in warm-blooded animals has been invented. The combined vaccine comprises the capsular polysaccharide PRP isolated and purified from Haemophilus influenzae type b and antigens isolated and purified from an outer membrane component of Bordetella pertussis. A method for inducing active immunization in warm-blooded animals against systemic infection caused by the pathogen H. influenzae type b has also been invented. The method comprises administering an immunogenic amount of a combined vaccine comprising the capsular polysaccharide PRP isolated and purified from H. influenzae type b and antigens isolated and purified from an outer membrane component of B. pertussis.
A process for removing endotoxin from Gram-negative polysaccharides such as H. influenzae polyribosylribitol phosphate by adding alcohol incrementally until substantially all lipopolysaccharide precipitates.
A process for removing endotoxin from Gram-negative polysaccharides such as polyribosylribitol phosphate by solubilizing polysaccharide-containing powder derived from Gram-negative bacteria fermentation broth to provide a divalent counter ion for endotoxin and adding alcohol incrementally to induce lipopolysaccharide precipition, and mixing material resulting from the alcohol addition with a nonionic resin, a detergent and a chelating agent.
