Haemophilus influenzae type b conjugate vaccines.

In summary, all of the Hib conjugate vaccines are highly immunogenic and efficacious in children older than 12-15 months of age, and HbOC, PRP-OMPC, and PRP-T are highly immunogenic and demonstrated to be efficacious in infants as young as 2 months old. HbOC, PRP-OMPC, and PRP-T have been licensed in numerous countries for infants and are recommended for infant immunization. However, perhaps the greatest tribute one can pay to all four Hib vaccines described in this review is to note the dramatic decrease in the incidence of Hib disease that has occurred since their introduction. In fact, according to the Morbidity and Mortality Weekly Report (March 4, 1994), the incidence of Hib disease in children less than 5 years old has declined by 95% from 41 cases per 100,000 in 1987 to 2 cases per 100,000 in 1993, timing that coincides with the availability and use of the Hib conjugate vaccines (Anderson, 1994). As universal administration is achieved and the apparent vaccine-induced reduction in carriage of Hib by the population continues, Hib vaccines may follow the lead of past vaccines (such as smallpox, measles, mumps, rubella, and polio) toward eradication of disease or at least a high degree of medical control, thereby virtually eliminating the mortality and insidious morbidity associated with invasive Hib diseases.

Characterization and evaluation of a recombinant hepatitis B vaccine expressed in yeast defective for N-linked hyperglycosylation.

The hepatitis B (HB) virus preS2 + 2 polypeptide (the M or middle envelope polypeptide) is N-glycosylated at the N4 residue of the preS2 domain when expressed in recombinant yeast. Hyperglycosylation at this amino acid residue (the addition of a large number of mannose residues to the core oligosaccharide), which occurs in common yeast strains, results in an HB vaccine with diminished immunogenicity. Hyperglycosylation can be prevented by expressing the preS2 + S polypeptide in mutant yeast strains (e.g. mnn9) which limit N-linked glycosylation to the addition of only core saccharide residues. An HB vaccine prepared from recombinant yeast expressing the non-hyperglycosylated preS2 + 2 polypeptide was of similar immunogenicity in mice to a licensed HB vaccine and was much more immunogenic in humans than the hyperglycosylated preS2 + 2 vaccine.

Immunogenicity of conjugate vaccines consisting of pneumococcal capsular polysaccharide types 6B, 14, 19F, and 23F and a meningococcal outer membrane protein complex.

In an effort to prepare pneumococcal (Pn) capsular polysaccharide (Ps) vaccines that would be immunogenic in infants, covalent conjugates were prepared for Pn types 6B, 14, 19F, and 23F. Each Ps type was covalently bound to an outer membrane protein complex from Neisseria meningitidis serogroup B and evaluated for immunogenicity in mice and infant monkeys. The conjugates induced specific anti-Ps antibody responses in mice and in infant rhesus and African green monkeys; a conjugate of 6B and outer membrane protein complex was immunogenic at Ps doses as low as 20 ng. Although low levels of the Pn group-common cell wall polysaccharide were present in all type-specific Ps preparations, anti-cell wall polysaccharide responses induced by covalent conjugates were < 1% of the total anti-Ps response after two doses of vaccine. In contrast, the anti-cell wall polysaccharide response of a noncovalent conjugate represented 41% of the anti-Ps response after two doses. Relative T-cell dependence, a requirement for the human target population of infants less than 18 months old, was demonstrated for all four Pn Ps conjugates in an athymic mouse model. Therefore, these Pn Ps-outer membrane protein complex conjugate vaccines are excellent candidates for evaluation in human infants.

Immunogenicity of a new Haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate) (PedvaxHIB).

Haemophilus influenzae type b is responsible for an estimated 15,000 to 20,000 cases of meningitis per year in the United States, mainly in children 2 months to 5 years old. The mortality rate from meningitis due to H influenzae type b infections ranges from 5% to 10%. Despite antibiotic treatment, up to 35% of survivors have permanent neurologic sequelae. In addition to meningitis, H. influenzae type b is responsible for other invasive infections, including epiglottitis, septicemia, cellulitis, septic arthritis, osteomyelitis, pneumonia, pericarditis, and otitis media; approximately 30,000 cases H influenzae diseases occur annually in the United States. The diseases peak in incidence between 6 and 12 months of age, with almost one half of the cases occurring before 1 year of age. About 75% of disease caused by H influenzae type b occurs in children younger than 24 months old. The incidence of disease is higher in children of certain groups, including blacks, Hispanics, Eskimos and Native Americans, young children attending day-care facilities, patients with asplenia or antibody-deficiency syndromes, and children of lower socioeconomic status. There is considerable evidence that antibody to the capsular polysaccharide (polyribosylribitol-phosphate [PRP] of H influenzae type b is protective.(ABSTRACT TRUNCATED AT 250 WORDS)

The application of molecular biology to the development of novel vaccines.

In summary, we have shown that yeast is the preferred host for the expression of recombinant-derived hepatitis B vaccines, and that a yeast expression system which is productive, stable and scaleable can be developed for each of the three HBV envelope proteins. The versatility of regulated and integrated yeast expression systems in the production of foreign polypeptides with biomedical utility also has been highlighted. We also have shown that careful attention to the development of recombinant clones helps to optimize the entire production process leading to highly purified products which share many biochemical properties with the plasma-derived vaccine. Furthermore, immunization with PreS2 sequences is capable of protecting chimpanzees from HBV infection. The availability of PreS2 + S and PreS1 + PreS2 + S proteins expressed in yeast now provides the opportunity for establishing the relevance of such candidate vaccines in preventing human disease, thereby highlighting the utility of molecular biology in modern vaccine development.

Cloning and expression of cDNA encoding antistasin, a leech-derived protein having anti-coagulant and anti-metastatic properties.

As a factor Xa inhibitor, antistasin is a potent anti-coagulant and anti-metastatic agent that is found in the salivary gland of the Mexican leech Haementaria officinalis. cDNA clones that encode antistasin have been isolated. Subsequent sequence analysis and comparison with the amino acid sequence of the mature protein indicates that antistasin is produced as a pre-protein containing a 17-amino acid signal peptide. Antistasin exists as at least two variants. By sequence analysis of multiple cDNA clones, we found two additional sites for amino acid substitutions, confirming variants that differ from each other by amino acid changes at a minimum of four residues. These sequence variations appear to be the result of allelic variation rather than gene duplication as deduced from DNA blot analyses. Sequence data suggest that antistasin may have evolved from a smaller ancestral gene by a duplication event giving rise to a two-fold structural homology between the N- and C-terminal halves of the molecule. Insect cells transfected with a recombinant baculovirus expressed antistasin which was biologically active and had an electrophoretic mobility identical to that of the native molecule.

Application of high-resolution n.m.r. spectroscopy to the elucidation of the structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 7F.

The specific capsular polysaccharide of Streptococcus pneumoniae type 7F (American type 51) is a high-molecular-weight neutral polymer composed of 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, L-rhamnose, and 2-O-acetyl-L-rhamnose residues. N.m.r. spectroscopy (1H and 13C), in conjunction with composition and methylation analyses, and periodate oxidation data, showed the polysaccharide to be a branched polymer with a repeating heptasaccharide unit having the following structure. (formula; see text)

Structural analysis of the specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72).

The specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72) was found to be a high molecular weight polymer composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-L-fucose, L-rhamnose, glycerol, and phosphate (2:1:1:1:1:1:1). Partial hydrolysis, dephosphorylation, methylation analysis, periodate oxidation studies, and one- and two-dimensional 1H and 13C high-field nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a 1-phosphoglycerol-substituted hexasaccharide repeating unit having the structure: (formula; see text).

A candidate vaccine for hepatitis B containing the complete viral surface protein.

The entire surface protein of hepatitis B virus serotype ayw containing the preS (preS1+preS2) and S domains has been expressed in the yeast Saccharomyces cerevisiae. Yeast containing a recombinant plasmid utilizing a constitutive promoter did not express this gene successfully due to the toxicity of the protein. A plasmid using a regulatable promoter directed expression which initiated late in the exponential phase of growth and resulted in the accumulation of high intracellular levels of the complete surface protein. The purified polypeptide aggregates into a form which, although not comprised of typical 20 nm particles, displays antigenic determinants encoded by the preS1, preS2 and S domains. Immunization of rabbits elicited the formation of antibodies directed against all three domains. This candidate vaccine will be useful for studying the contributions to viral immunity of the host response to the preS1 and preS2 domains.

Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae.

As a model system for the study of factors affecting gene expression, hepatitis B virus core antigen (HBcAg) has been expressed in the yeast Saccharomyces cerevisiae. The singularly high levels of expression achieved are approx. 40% of the soluble yeast protein. The HBcAg polypeptides are present as 28-nm particles which are morphologically indistinguishable from HBcAg particles in human plasma and are highly immunogenic in mice. The plasmid construction employed to achieve these very high levels of expression utilizes the constitutively active yeast promoter from the GAP491 gene which is fused in a way that all non-translated sequences flanking the HBcAg coding region are yeast-derived. Hybrid constructions containing 3'-nontranslated viral DNA (yeast 5') or 5'-nontranslated viral DNA (yeast 3') as well as a construction with both 5'- and 3'-nontranslated viral DNA also have been made. A comparison of these constructions for levels of HBcAg expression indicates that the strongest contributor to the high levels of protein is the presence of 5'-flanking sequences which are yeast-derived; secondarily, a significant improvement can be achieved if the 3'-flanking sequences also are yeast-derived. The high abundance of HBcAg in the highest producer is explicable in part on the basis of the very high stability in yeast cells of HBcAg polypeptides. Analysis of the HBcAg coding sequence reveals a very low index of codon bias for S. cerevisiae, largely discounting codon usage as a contributor to the high level of protein obtained.

The structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 11F (American type 11).

The specific polysaccharide of Streptococcus pneumoniae type 11F (American type 11) is composed of 2-acetamido-2-deoxy-D-glucose (one part), D-glucose (one part), D-galactose (two parts), ribitol (one part), phosphate (one part), and O-acetyl (two parts). Hydrolysis, dephosphorylation, periodate oxidation, methylation, optical rotation, and 1H and 13C nuclear magnetic resonance studies showed that the polysaccharide is an unbranched linear polymer of a ribitol-phosphate substituted repeating tetrasaccharide unit having the structure: (Formula: see text). The specific capsular polysaccharides of S. pneumoniae type 11B and 11C (American types 76 and 53) were found to have the same tetrasaccharide repeating unit as the 11F polysaccharide, but differed from it in their mode of O-acetylation and the replacement of the ribitol phosphate by glycerol phosphate in the 11C specific polysaccharide.

Structural analysis of the specific polysaccharide of Streptococcus pneumoniae type 9L (American type 49).

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 9L (American type 49) is composed of D-galactose (one part), D-glucose (one part), D-glucuronic acid (one part), 2-acetamido-2-deoxy-D-mannose (one part), and 2-acetamido-2-deoxy-D-glucose (one part). Partial acid hydrolysis, periodate oxidation, nitrous acid deamination, optical rotation, methylation, and 13C and 1H nuclear magnetic resonance studies showed that the polysaccharide is an unbranched high molecular weight linear polymer of a repeating pentasaccharide unit having the structure: (formula; see text).

The specific capsular polysaccharide of Streptococcus pneumoniae type 33F.

The specific capsular polysaccharide of Streptococcus pneumoniae type 33F (American type 70) is composed of D-galactose (5 parts), D-glucose (1 part), and O-acetyl (ca. 0.4 parts). Periodate oxidation, partial hydrolysis, and 1H and 13C nuclear magnetic resonance studies showed that the polysaccharide is a high molecular weight polymer of a repeating hexasaccharide unit having the structure: (Formula: see text)

Suppression and reversal of allergic encephalomyelitis in rhesus monkeys with basic protein and peptides.

We have extended earlier studies on the suppression of clinically evident experimental allergic encephalomyelitis (EAE) in monkeys, repeated injections of human basis protein. The results confirm that after suppressive treatment, recovered animals remain clinically normal and do not show spontaneous recurrence of symptoms. However, recovered animals are susceptible to EAE upon renewed challenge, and they develop the disease more rapidly and more severely than after the initial challenge; resuppression is also accomplished in these cases by the same methods used previously. The results indicate further that the basic protein or peptide T administered without mycobacteria is effective in suppressing the development of basic protein-induced EAE regardless of the species from which it was derived.