Capsular polysaccharide conjugates of Haemophilus influenzae type b (Hib) are important components of several mono- or multi-valent childhood vaccines. However, their access to the most needy people is limited due to their high cost. As a step towards developing a cost effective and more immunogenic Hib conjugate vaccine, we present a method for the preparation of Hib capsular polysaccharide (PRP)-tetanus toxoid (TT) conjugates using optimized PRP chain length and conjugation conditions. Reactive aldehyde groups were introduced into the polysaccharides by controlled periodate oxidation of the native polysaccharide, which were subsequently covalently linked to hydrazide derivatized tetanus toxoid by means of reductive amination. Native polysaccharides were reduced to average 100 or 50kDa polysaccharide and 10kDa oligosaccharides in a controlled manner. Various conjugates were prepared using Hib polysaccharide and oligosaccharide yielding conjugates with polysaccharide to protein ratios in the range of 0.25-0.5 (w/w) and free saccharide levels of less than 10%. Immunization of Sprague Dawley rats with the conjugates elicited specific antibodies to PRP. The low molecular weight PRP-TT conjugates were found to be more immunogenic as compared to their high molecular weight counterparts and the PRP-TT reference vaccine.
Keywords: Conjugate vaccine; Haemophilus influenzae type b; Immunogenicity; PRP size; Reductive amination.
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