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Clinical And Diagnostic Laboratory Immunology
American Society for Microbiology
Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

Sera used to identify putative hepatitis E viral proteins expressed in Pischia pastoris produced a false-positive reaction because of antibodies to a yeast protein. This report illustrates a potential problem when serological reagents are used in combination with recombinant proteins expressed in yeast.

Bacteria (e.g., Escherichia coli), yeasts (e.g., Pischia pastoris) and mammalian cells (e.g., CHO cells) are frequently used as substrates for the expression of recombinant proteins. One such use is for the development of serologic tests for the detection of antibodies to the expressed antigenic proteins.

Alternatively, expression systems may be used to identify uncharacterized proteins. However, infection with organisms related to those used for expression of the recombinant antigens can lead to the development of antibodies to the expression system, with the potential for false-positive antibody tests.

We demonstrate here how a probable P. pastoris infection, temporally related to an experimental infection with hepatitis E virus (HEV), led to an initial erroneous interpretation of serologic data.

An attempt to express open reading frame 1 (ORF1) of HEV in P. pastoris was made by standard procedures (Invitrogen, San Diego, Calif.). ORF1 has the potential to encode a polypeptide or group of polypeptides with a total size of 200 kDa. However, the products of ORF1 have not been identified, and standardized serological reagents have yet to be developed.

Therefore, crude extracts of transformed yeast cultures were evaluated for the presence of the putative antigen by comparison of Western blots (immunoblots) of preinfection and convalescent sera of a chimpanzee that had been experimentally infected with HEV previously (1).

The assumption was that antibodies unique to the convalescent serum would be specific for HEV antigens. To test the specificity of the reaction, preinfection serum from the chimpanzee that was obtained in 1986 before an experimental infection with HEV and hyperimmune convalescent serum that was obtained following a second challenge of the chimpanzee with HEV in 1989 were tested at a 1:100 dilution against the crude yeast lysate by Western blotting. A 200-kDa band was detected only with the convalescent serum, consistent with specificity for one or more HEV-encoded proteins.

However, apparent seroconversion to a 200-kDa band was also observed after extracts of P. pastoris cells which had not been transfected with the HEV ORF1 expression plasmid were used as an antigen in Western blots of the preinfection and convalescent sera. Additional sera were evaluated by enzyme-linked immunosorbent assay (ELISA) as described previously (1), with slight modifications.

Convalescent serum samples from 11 of 12 HEV-infected cynomolgus monkeys and the infected chimpanzee were positive by ELISA. However, some preinoculation sera were also positive by ELISA, suggesting prior HEV infection or reactivity with antigens unrelated to HEV infection.

A more extensive analysis was performed with additional serial serum samples obtained from the chimpanzee. For purposes of comparison, the serum samples were also tested in an ELISA based on a well-characterized recombinant antigen expressed from ORF2 (capsid protein) of HEV in insect cells (1).

Antibody to the capsid protein of HEV was detected in a pattern consistent with the challenge and rechallenge of the chimpanzee with HEV. The animal was seronegative prior to the first challenge, seroconverted postchallenge, maintained detectable antibody until the second challenge, and demonstrated an anamnestic response following the second challenge.

The demonstration of the reactivity of the chimpanzee sera to normal yeast extract, in addition to the demonstration that antibody responses to yeast and to HEV were not superimposable, led to the correct conclusion that the attempt to transform yeast with a plasmid containing ORF1 of HEV was unsuccessful.

This experience emphasizes the importance of meticulous recordkeeping and the use of complete controls. As yeast-based systems are used more frequently for the expression of foreign antigens, the problem of naturally acquired yeast infections and of a resulting serum reactivity to the yeast proteins becomes more important.

We would like to thank Doris Wong for the care of sera, Ying Huang for teaching and assistance, Gopa Raychaudhuri for helpful discussion, and Max Shapiro and the staff of BIOQUAL, Inc., for excellent animal care.

REFERENCE
Tsarev, S. A., T. S. Tsareva, S. U. Emerson, A. Z. Kapikian, J. Ticehurst, W. London, and R. H. Purcell. 1993. ELISA for antibody to hepatitis E virus (HEV) based on complete open reading frame-2 protein expressed in insect cells: identification of HEV infection in primates. J. Infect. Dis. 168:369–378. VOL. 3, 1996 NOTES 615 by on November 17, 2009 cvi.asm.org

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