An analytical model applied to a multicenter pneumococcal enzyme-linked immunosorbent assay study.

Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.

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.

Large-scale purification and characterization of recombinant tick anticoagulant peptide.

Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.

Production of hepatitis B surface antigen (preS2 + S) by high-cell density cultivations of a recombinant yeast.

A high-cell density bioprocess has been developed for the production of hepatitis B surface protein (preS2 + S) by recombinant yeast. This fed-batch process utilizes a growth medium containing yeast extract, soy peptone and glucose which was fed at a constant rate to maintain cells in a respiratory state. Cell densities of up to 60 g l-1 dry weight were achieved, which represented a 6-fold increase over those from batch bioprocesses. This increase in cell mass was attained without compromising specific activity; therefore, volumetric productivities of six times those of batch bioprocesses were achieved.

Characterization of recombinant tick anticoagulant peptide. A highly selective inhibitor of blood coagulation factor Xa.

Tick anticoagulant peptide (TAP) is a potent, highly selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D. E., Arcuri, K. E., and Vlasuk, G. P. (1990) Science, 248, 593-596). Further detailed studies pertaining to the in vitro and in vivo evaluation of TAP require quantities of the inhibitor which cannot be isolated from ticks. To overcome this limitation we describe here the characterization of recombinant TAP (rTAP) secreted by Saccharomyces cerevisiae. Expression of rTAP was obtained using a chimeric gene containing a fusion between sequences encoding the secretory preproleader of the yeast mating pheromone alpha-factor and a synthetic sequence encoding the 60-amino acid inhibitor under the transcriptional control of a galactose-inducible promoter. Recombinant S. cerevisiae were found to secrete biologically active rTAP into the extracellular medium at levels of 0.1-0.15 g/liter. The secreted inhibitor was purified to homogeneity and found to be indistinguishable from the native inhibitor with respect to several criteria, including primary structure, amino acid composition, and electrophoretic mobility. In addition, purified rTAP and native TAP exhibited similar stoichiometric inhibition of factor Xa in vitro. The in vivo efficacy of rTAP was demonstrated using a model of low grade disseminated intravascular coagulation where the purified inhibitor was shown to significantly inhibit thromboplastin-induced fibrinopeptide A generation following an infusion into conscious rhesus monkeys. The availability of rTAP will allow a detailed evaluation of the in vitro and in vivo properties of this highly specific and potent factor Xa inhibitor.

Purification and characterization of human immunodeficiency virus (HIV) core precursor (p55) expressed in Saccharomyces cerevisiae.

The core structure of retroviruses, including the human immunodeficiency virus (HIV), consists of proteins that are initially synthesized as polyprotein precursors and then processed by a virally encoded protease yielding the mature core polypeptides. To obtain sufficient quantities of the purified HIV core precursor p55 for detailed studies, a segment of HIV DNA encoding the full length core precursor polyprotein p55 was expressed in Saccharomyces cerevisiae using a plasmid containing a constitutive galactose promoter. The expression of this DNA produced a protein with an estimated molecular size of 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this protein was immunoreactive to anti-HIV p24 antisera. Following cell lysis, freezing, and thawing, the expressed protein was an insoluble aggregate that served as the starting material for the purification process. Solubilization of the insoluble p55 with guanidine HCl followed by phenyl-Sepharose column chromatography and high performance liquid chromatography resulted in a preparation of p55 that was greater than 95% pure by SDS-PAGE, immunoreactive to anti-HIV core protein antibodies, and completely soluble in aqueous solution. The expressed p55 appeared to be myristoylated as evidenced by the incorporation of radiolabel following incubation of recombinant yeast cells with [3H]myristic acid; in addition the amino terminus of the final purified protein was blocked. Proteolytic digestion of purified p55 with synthetic HIV protease yielded the predicted amino- and carboxyl-terminal products; these were confirmed by amino acid sequence analysis. In contrast, digestion of purified p55 by the protease derived from the avian myeloblastosis virus resulted in fragments that were different in size from those produced by the HIV protease. The availability of the purified, full length water-soluble HIV core precursor will be useful in identifying agents that inhibit its processing by the HIV protease.

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.