Vaccines made from recombinant yeast cells

Comment Vaccines made from recombinant yeast cells Production of polypeptide and protein antigens through recombinant DNA technology in prokaryotic and certain eukaryotic cells in culture is facilitating the development of new vaccines that are safe, efficacious, and economically feasible to manufacture. A current example is that of human hepatitis B vaccine that, to the present, has been produced commercially using hepatitis B viral surface antigen (HBsAg) purified from the plasma of human carriers chronically infected with the virus. Production of plasma-derived vaccine is limited by the available supply of suitable carrier plasma and by the need to apply highly technical procedures to purify the antigen as well as to ensure inactivation of all infectious agents that might be present in human plasma. Several groups of workers have successfully transformed yeast cells (S a c c h a r o m y c e s cerevisiae or baker's yeast) with plasmids bearing the HBsAg gene circumscribed by the necessary promoter and terminator yeast sequences. Highly purified HBsAg is obtained from the disrupted yeast cells by a series of chromatographic separations. Yeast-derived HBsAg is not glycosylated (approximately 50-75% of plasma-derived HBsAg also is not glycosylated) but has the same amino acid sequence. It raises the same antibody responses qualitatively and quantitatively in vaccinees, and it affords the same protective efficacy in animals and in human beings as does the plasma-derived HBsAg. Hepatitis B vaccines prepared from yeast cells are expected to be licensed soon in several countries. To the contrary, transformed prokaryotic cells, such as Escherichia coli, have not yielded economically acceptable amounts of antigen. Transformed continuous mammalian cells have also been used to produce HBsAg which resembles HBsAg obtained from human plasma in economically acceptable amounts. A substantial body of human clinical experience has been collected to date with yeast-derived vaccine, but no similar body of data is presently available for vaccines prepared from mammalian cell-derived antigens 1. Additionally, questions have been raised concerning the safety of vaccines made from antigens produced in continuous mammalian cell lines, some of which are tumorigenic and some of which contain integrated DNA seq-

uences initially derived from oncogenic agents such as SV40 and bovine papilloma viruses. All the mammalian cell producer systems are transformed with plasmids whose expression of HBsAg is driven by powerful promoters derived from oncogenic viruses. Furthermore, some of the cell lines are known to carry endogenous retroviruses as well. Though removal of all DNA from the HBsAg preparations might ensure safety from possible iatrogenically transmitted carcinogenicity, such data are impossible to obtain because of the limitations in ability of currently available tests to detect DNA present in very small amounts. The presence of genes or fragments of DNA in final vaccine products is of some concern, since cancer is a single cell phenomenon and since a single segment of DNA of appropriate composition and site of integration might suffice to transform a single cell and initiate neoplasia. The theoretical and practical issues raised here are especially sobering since the vaccine is targeted for eventual use in hundreds of millions of normal persons in the world population including newborns. Benefit-to-risk assessments for mammalian cell-derived vaccines will need to be made and considered in the light of alternative sources of the antigen such as recombinant yeast. The potential risks associated with residual cellular and viral promoter DNA in vaccines will be considered by a multidisciplinary group of international experts of the highest calibre in a meeting to be convened by the World Health Organization this

year 1, and it is anticipated that worthwhile progress will be made towards resolving this difficult problem. The kinds of cells that are considered acceptable for preparing vaccines for use in human beings have a long history of controversy2-6. Some confusion exists with respect to the biology of (a) the R A S s c gene, that is present as a part of the normal genome of Sacc h a r o m y c e s cerevisiae 7" ~, and (b) the Ty element of yeast. The yeast R A S s c gene is not equivalent to the ras protooncogenes of mammalian cells, even though the two genes are distantly related in the evolutionary sense 9. The yeast R A S s c gene, as it occurs in yeast and unlike the mammalian ras gene, does not transform NIH3T3 cells that are used to test for neoplastic transformation 1°. In order for it to function as a transforming principle in a mammalian cell, each of three separate and very specific changes in a R A S s c gene are required 1°' 1~ These multiple modifications are (a) a precise point mutation; (b) the shortening of the R A S s c gene to delete the codons for a specific 117 amino acids; and (c) the precise linkage of the mutated and truncated R A S s c gene to a strong promoter sequence derived from a mammalian cell. Furthermore, yeast genes p e r se are incapable of being transcribed in mammalian cells. In addition, yeast cells expressing the altered coding form of R A S s c lose their ability to sporulate and have greatly reduced viability; due to this strong negative selection pressure, they have poor, if not nonexistent survival probability. Thus, the R A S s c gene is an irrelevant risk factor for safety of hepatitis B vaccines produced in yeast. The Ty elements of yeast are transposons 12 that replicate through an RNA intermediate which is capable of encoding a reverse transcriptase activity and of being packaged into subcellular particles that may appear 'virus-like' when observed by electron microscopy 13'u. They are not viruses and they have been shown to be incapable of horizontal Vaccine, Vol. 4, June 1986

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Comment

transmission. Despite the proviruslike organization of their genome, there is no genetic element in Ty resembling the envelope (env) gene of retroviruses required for proviral transmission~5'~6' i.e. entry into cells. Ty lacks any oncogenes. In addition, yeast and mammalian cells are so divergent genetically that none of the signals necessary to transcribe and translate yeast genetic material are found in mammalian cells; hence, the Ty genome cannot be expressed in mammalian cells. The reverse transcriptase encoded by Ty, which is active at 30°C, a permissive temperature for the growth of yeast, is totally inactive at 37°C. Futhermore, all Tyassociated activities become detectable only in strains of yeast genetically engineered to permit overexpression of Ty by a factor of 106; such strains grow very poorly due to negative selective pressure. By all these criteria, the Ty elements pose no risk to human health with respect to products made in yeast.

Maurice R. Hilleman,

Merck Institute for Therapeutic Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486, USA

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and

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Ronald Ellis,

Department of Virus and Cell Biology, Merck Institute for Therapeutic Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486, USA

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References 1 World Health Organization. Recombinant DNA hepatitis B vaccines. Lancet, in press 2 Hilleman, M.R. Line cell saga - An argument in favor of production of biologics in cancer cells. In: Cell Substrates (Eds Petricciani, J.C., Hopps, H.E. and Chappie, P.J.), Plenum Publishing, New York, 1979, p. 47 3 Petricciani, J.C. Old issues confront biotechnology and new products. Vaccine 1984, 2, 235 4 Petricciani, J.C. Safety issues relating to the use of mammalian cells as hosts. 18th Congress, Joint lABS/WHO Symposium on Standardization and Control of Biologicals produced by Recombinant DNA Technology, Geneva 1983, Dev. Biol. Stand. 1985, 59, t49 5 Petricciani, J.C. Workshop on the use of abnormal cells to produce new products: A summary. In: Vaccines 85. (Eds Lerner, R.A., Chanock, R.M. and Brown, F.), Cold Spring Harbor, New York, 1985, p. 383 6 Patzer, E.J. and Lasky, L.A. Use of recombinant mammalian cell lines for vaccine production. Vaccine 1984, 2, 234 7 DeFeo-Jones, D., Scolnick, E.M., Koller, R. and Dhar, R. Ras-related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature 1983, 306, 707

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Powers, S., Kataoka, T., Fasano, O., Goldfarb, M., Strathern, J., Broach, J. and Wigler, M. Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell 1984, 36, 607 Wheals, A.E. Oncogene homologues in yeast. Bioessays 1983, 3, 108 DeFeo-Jones, D., Tatchell, K., Robinson, L.C., Sigal, I.S., Vass, W.C., Lowy, D.R. and Scolnick, E.M. Mammalian and yeast ras gene products: Biological function in their heterologous systems. Science 1985, 228, 179 Gibbs, J.B., Sigal, I.S. and Scolnick, E.M. Biochemical properties of normal and oncogenic ras p21. Trends Biochem. Sci. 1985, 18, 350 8oeke, J.D., Garfinkel, D.J., Styles, C.A. and Fink, G.R. Ty elements transpose through an RNA intermediate. Cell 1985, 40, 491 Garfinkle, D.J., Boeke, J.D. and Fink, G.R Ty element transposition: Reverse transcriptase and virus-like particles. Cell 1985, 42, 507 Mellor, J., Malim, MH., Gull, K., Tuite, MF., McCready, S., Dibbayawan, T., Kingsman, S.M. and Kingsman, A.J. Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast. Nature 1985, 318, 583 Clare, J. and Farabaugh, P. Nucleotide sequence of a yeast Ty element: Evidence for an unusual mechanism of gene expression. Proc. Nail Acad. ScL USA 1985, 82, 2829 Hauber, J., Nelbock-Hochstetter, P. and Feldmann, H. Nucleotide sequence and characteristics of a Ty element from yeast. Nucleic Acids Res. 1985, 13, 2745