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An approximately 15,000-dalton outer membrane lipoprotein of Haemophilus influenzae, the Hi-PAL (P6) protein, has been shown to elicit bactericidal and protective antibodies against both type b and nontypeable H. influenzae strains and is a vaccine candidate for these organisms. To determine whether the lipid modification of this protein is required for immunogenicity or the elicitation of biologically active antibodies, a genetic fusion was constructed that contains the sequence of mature Hi-PAL fused to the polylinker region of pUCl9. The protein expressed by this clone does not contain detectable lipid and was purified to homogeneity. This recombinant fusion protein, rPAL, elicited a strong immune response when injected into rabbits, and the antiserum reacted well with native Hi-PAL. The antiserum was bactericidal against a number of clinical nontypeable strains, duplicating the activity of anti-Hi-PAL. The anti-rPAL antiserum was also protective against type b bacteremia in the infant rat model. These results demonstrate that purified rPAL elicits antibodies with biological activities that are similar to those of anti-Hi-PAL antibodies. Thus, the lipid component of Hi-PAL is not required for either immunogenicity or elicitation of biologically active antibodies.Haemophilus influenzae strains represent a major cause of morbidity and mortality in infants and elderly adults (2,19,24). H. influenzae type b (Hib) strains are the leading causative agents of neonatal meningitis (21), and nontypeable (NT) H. influenzae strains are among the major causative agents of acute otitis media (2). The current vaccines for Hib consist of the polysaccharide capsule from Hib, polyribosyl-ribitol phosphate or oligosaccharides derived from polyribosyl-ribitol phosphate coupled to protein carriers. The protective efficacy of anti-polyribosyl-ribitol phosphate antibody is demonstrated in the infant rat meningitis model system, in which antiserum is passively transferred and the animals are challenged with virulent Hib (23). Presently, the protein-polysaccharide conjugate vaccines are licensed for use in 18-month-old children. Clinical trials of saccharide-protein vaccines are currently underway to demonstrate efficacy in infants. Although anti-polyribosyl-ribitol phosphate antibody appears to be sufficient to protect humans from Hib disease, it has no efficacy against diseases caused by NT H. influenzae. Recent attempts to identify potential vaccine components

Glycosylation analysis of recombinant glycoproteins is of importance for the biopharmaceutical industry and the production of glycoprotein pharmaceuticals. A commercially available lectin array technology was evaluated for its ability to present a reproducible fingerprint of a recombinant CTLY4-IgG fusion glycoprotein expressed in large scale CHO-cell fermentation. The glycosylation prediction from the array was compared to traditional negative mode capillary LC-MS of released oligosaccharides. It was shown that both methods provide data that allow samples to be distinguished by their glycosylation pattern. This included information about sialylation, the presence of reducing terminal galactose β1-, terminal N-acetylglucosamine β1-, and antennary distribution. With both methods it was found that a general trend of increased sialylation was associated with an increase of the antenna and reduced amount of terminal galactose β1-, while N-acetylglucosamine β1- was less affected. LC-MS, but not the lectin array, provided valuable information about the sialic acid isoforms present, including N-acetylneuraminic acid, N-glycolylneuraminic acid and their O-acetylated versions. Detected small amounts of high-mannose structures by LC-MS correlated with the detection of the same epitope by the lectin array.

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