We describe a novel fungal expression system which utilizes the Quorn(R) myco-protein fungus Fusarium graminearum A 3/5. A transformation system was developed for F. graminearum and was used to introduce the coding and regulatory regions of trypsin gene from Fusarium oxysporum. The protein was efficiently expressed, processed and secreted by the recombinant host strain. In addition, the promoter and terminator of the F. oxysporum trypsin gene have been successfully utilized to drive the expression of a cellulase gene from Scytalidium thermophilum and a lipase gene from Thermomyces lanuginosus in F. graminearum.

The fructose-1,6-bisphosphatase [Fru(1,6)P2ase] gene of the budding yeast, Kluyveromyces lactis, was cloned and sequenced. The gene encodes one open reading frame predicting a 354-amino-acid polypeptide. The polypeptide is different from other Fru(1,6)P2ases in that it contains a short amino-acid-insert region close to a basic residue located at the binding site for the allosteric inhibitor AMP. Comparison of the biochemical properties of the K. lactis enzyme with its closest homolog, the Saccharomyces cerevisiae Fru(1,6)P2ase (74% amino acid identity), reveals that the K. lactis enzyme is significantly less sensitive to AMP (Ki = 540 microM) than the S. cerevisiae enzyme (Ki = 190 microM). However, studies with a K. lactis Fru(1,6)P2ase mutant, in which the insert region (amino acids 152-160) was deleted by site-directed mutagenesis [(des-152-160)Fru(1,6)P2ase], showed that the mutant enzyme had higher sensitivity to AMP inhibition (Ki = 280 microM) than the control K. lactis enzyme. Thus, the nine-amino-acid insert region appears to be responsible for the decreased AMP inhibition shown by the K. lactis wild-type enzyme. Catabolite-repression and catabolite-inactivation studies show that, unlike the complete repression of FBP1 mRNA and inactivation of enzyme activity by glucose seen in S. cerevisiae, mRNA levels and enzyme activity of K. lactis Fru(1,6)P2ase decreased only about 2-4-fold due to the presence of glucose in the cell-culture medium.

Background: To assess the role played by the immune response in the outcome of hepatitis C virus infection, the CD4+ T-lymphocyte response to viral antigens was studied in infected individuals with different clinical courses. Methods: Using six recombinant proteins of hepatitis C virus, the study assessed the proliferative responses of peripheral blood mononuclear cells from 41 patients with chronic hepatitis C, 11 patients whose chronic hepatitis was successfully treated with interferon alfa and 11 healthy HCV seropositive individuals. Results: (1) Sixty-five percent of hepatitis C virus-seropositive individuals had CD4+ T-cell responses to viral proteins. (2) All viral proteins were immunogenic for T cells, although NS4 was the most immunogenic. (3) There was a significant correlation between the presence of CD4+ T cell responses to Core and a benign course of infection in healthy seropositives, most of whom were viremic. Conclusions: CD4+ T-cell responses to Core, although they do not coincide with virus clearance, are associated with a benign course of infection and may be required to maintain humoral and cellular responses protective against the disease.

AbstractHepatitis C virus (HCV) infection can be detected by using immunoassay techniques that measure reactivity to viral protein antigens. In this study seven discreet proteins derived from HCV genomic coding sequences have been expressed, purified and characterized. Six proteins represent the structural regions of the core (C22‐3), the envelope (E1 and E2), and the non‐structural regions NS3 (C33C), NS3–NS4 (C100‐3) and NS5. The seventh, C25, is a chimeric fusion protein containing C33C, C100‐3 and C22‐3 regions. Using these recombinant proteins, multi‐antigen radioimmunoassays and enzyme immunoassays (EIA) were designed. The fusion protein, C25, was demonstrated to be an improved antigen for serodiagnosis of HCV antibody. Use of the C25 protein accelerated HCV antibody detection by 3–46 weeks in non‐A, non‐B hepatitis seroconversion cases and significantly increased the rate of detection in a paid donor population by 20%. The C25 assay also demonstrated excellent specificity in 2446 randomly selected low prevalence samples. The repeated reactive rate in this group of samples was 0.5%.Samples from volunteer blood donors pre‐selected for repeat reactivity with the first generation C100‐3‐based HCV antibody tests (n= 175) were tested using the C25 assay. The C25 assay detected 37.7% samples as reactive and 53.1% samples as non‐reactive. This result was in agreement with all other supplementary tests that include RIBATM, multi‐antigen assay, Abbott neutralization and peptide assay. The other 9.2% samples were classified as ‘indeterminant’ because these samples were only partially in agreement with some of the above supplementary tests.The C25 enzyme‐linked immunosorbent assay (ELISA), with its improved assay sensitivity, can identify additional HCV antibody reactive cases in both hepatocellular carcinoma and cryptogenic cirrhosis patients. The C25 and multi‐antigen EIA assays were used to investigate the vertical transmission of HCV. It was observed that these improved assays are able to detect antibodies in a vertical transmission case. The C25 ELISA was also compared with a synthetic peptide assay. The C25 assay was found to be superior to the peptide assay that performed poorly in the detection of the C33C only reactive samples.

Structural and nonstructural regions of the HCV-encoded polyprotein have been expressed in recombinant yeast, bacteria, or insect cells and used to capture and measure reactive antibodies circulating in different individuals. The putative nucleocapsid protein (C) and nonstructural proteins 3-5 (NS3-NS5) were found to contain the most immunodominant epitopes. The NS3, NS4, and C regions were expressed in yeast in the form of a fused, chimeric polyprotein (C25) and a capture assay for reactive antibody was developed. This anti-C25 assay detects all previously identified HCV-seropositive cases and provides a substantially more sensitive diagnostic for both acute and chronic HCV infections than the current anti-C100-3 (NS4) assay. Anti-C25 was detected more frequently than anti-C100-3 in chronic, transfusion-associated non-A, non-B hepatitis patients from the United States (95% vs. 71%) and Japan (98% vs. 82%), in cryptogenic cirrhosis patients from the United States (62% vs. 28%), and in hepatitis B surface antigen-negative cases of hepatocellular carcinoma from Japan (83% vs. 63%). These data indicate that HCV has a greater role in these liver diseases than was previously thought. In volunteer United States blood donors sampled following the introduction of anti-C100-3 screening, the prevalence of anti-C25 and anti-C100-3 was 0.5% and 0.08%, respectively.

The ubiquitin fusion approach to gene expression in eukaryotic systems allows the production of heterologous proteins that are cleaved precisely in vivo from the ubiquitin fusion partner by an endogenous ubiquitin-specific hydrolase. Alternatively, ubiquitin fusions can be isolated from bacterial hosts, and cleaved by the hydrolase in vitro. In each case, the system gives recombinant proteins that contain authentic amino termini. We have found the system to be of particular utility for the production of human gamma interferon (gamma-IFN) and alpha-1-proteinase inhibitor (alpha-1-Pl). We have also used the system for the production of regions of the human immunodeficiency virus type-1 (HlV1) genome that were previously expressed at only low levels in yeast. These include domains of the HlV1 env gene and also the region of the HlV1 pol gene that encodes the HlV1 integrase enzyme. Surprisingly, for one of the env proteins we were able to isolate a product in which the amino-terminal Glu residue was modified by addition of an Arg residue. This arginyl-tRNA-protein-transferase catalyzed process had previously only been observed for short-lived intermediates in the ubiquitin-dependent proteolytic degradation pathway. The HlV1 integrase was found to contain the authentic amino terminus (Phe.Leu.Asn...) previously reported for virion-associated integrase.

Maximizing efficiency for the secretion of proteins from yeast requires an understanding of the rate limiting stages in secretion that can result from high levels of gene expression. Recent progress in this area has produced a number of improvements in yeast expression systems for protein secretion.

Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immune responses in infected hosts. Macaque simian immunodeficiency virus (SIVmac) is a T-lymphocytopathic lentivirus which causes an AIDS-like disease in rhesus macaques. The envelope gene of SIVmac encodes a precursor glycoprotein (gp160) which is cleaved into an external domain (gp130) and a transmembrane domain (gp32). To investigate the functional and immunological properties of the SIV external envelope glycoprotein, we have used genetically engineered mammalian cells to produce recombinant gp130 (rgp130). The rgp130 has the appropriate molecular weight, is glycosylated, and has native conformation as determined by binding to the cell receptor for SIV, the CD4 antigen. Rhesus macaques immunized with purified rgp130 formulated in muramyl dipeptide adjuvant generated high titers of antienvelope antibodies. Antibodies from these macaques were tested for in vitro virus neutralization; very low or undetectable levels of neutralization were observed. In contrast, neutralizing antibodies were readily detected in sera from goats immunized with rgp130. With respect to cell-mediated immunity, proliferative responses to rgp130 were demonstrated in peripheral blood monocyte cells (PBMC) from macaques immunized with the recombinant glycoprotein as well as in PBMC from SIV-infected animals. These results show that rgp130 is functional and immunogenic; the potential of rgp130 for protective immunization remains to be determined.

A specific assay has been developed for a blood-borne non-A, non-B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post-transfusion NANBH (PT-NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.

We have developed the yeast Kluyveromyces lactis as a host organism for the production of the milk-clotting enzyme chymosin. In contrast to Saccharomyces cerevisiae, we found that this yeast is capable of the synthesis and secretion of fully active prochymosin. Various signal sequences could be used to efficiently direct the secretion of prochymosin in Kluyveromyces, but not in S. cerevisiae. We conclude that the efficient synthetic and secretory capacity of this heterologous protein is a property of the yeast Kluyveromyces. These results have led to the development of a large scale production process for chymosin.

A nonglycosylated denatured form of human immunodeficiency virus (HIV) 1 glycoprotein gp120 (Env 2-3), which does not bind to CD4, was used with muramyl tripeptide as adjuvant to immunize HIV-seronegative healthy volunteers. In all the volunteers, three 50-micrograms injections of Env 2-3 induced priming of CD4+ T cells specific for conserved regions of the native glycosylated gp120. Moreover, we found that several major histocompatibility complex class II (DR) alleles can function as restriction molecules for presentation of conserved epitopes of gp120 to T cells, implying that a T-cell response to these epitopes can be obtained in a large fraction of the population. The possibility to prime CD4+ T cells specific for conserved epitopes of a HIV protein is particularly important in view of the lack of such cells in HIV-infected individuals and of a possible role that CD4+ T cells may play in the development of protective immunity against AIDS.

Variants of the envelope gene of the HIV-SF2 isolate of HIV-1 with deletions of one or more of the hypervariable domains of gp120 were produced in genetically engineered yeast as nonglycosylated denatured polypeptide analogs of gp120. Purified antigens were used to immunize experimental animals to determine whether the removal of hypervariable regions from this type of gp120 immunogen had any effect on (1) the ability of the antigen to elicit virus neutralizing antibodies; and (2) the isolate specificity of the neutralizing antibodies that were elicited. The results of these studies demonstrate that, in addition to the previously identified V3 domain, at least two other hypervariable regions in gp120 are capable of eliciting neutralizing antibodies in experimental animals. However, when all five of the hypervariable regions were deleted, the resulting antigen was no longer capable of eliciting neutralizing antibodies. Finally, the neutralizing antibodies elicited by all of these nonglycosylated antigens were effective against HIV-SF2, the isolate from which the antigens were derived, but were not able to neutralize two divergent isolates, HIV-BRU or HIV-Zr6.

The production of extracellular human insulin-like growth factor I (IGF-I) in yeast is deleterious to the growth of the host organism. Mutants resistant to the toxic effects of IGF-I production were isolated. A subset of these mutants produced levels of IGF-I greater than the parent strain and were due to chromosomal recessive mutations at a single locus, hpx1. The overproduction of IGF-I was independent of the original promoter and vector expression system. The mutant strains also displayed enhanced extracellular production of other heterologous proteins.

It has been proposed in several eukaryotic systems that the regulation of gene transcription involves phosphorylation of specific transcription factors. We report here that the yeast transcriptional activator ADR1 is phosphorylated in vitro by cyclic AMP-dependent protein kinase and that mutations which enhance the ability of ADR1 to activate ADH2 expression decrease ADR1 phosphorylation. We also show that increased kinase activity in vivo inhibits ADH2 expression in an ADR1 allele-specific manner. Our data suggest that glucose repression of ADH2 is in part mediated through a cAMP-dependent phosphorylation-inactivation of the ADR1 regulatory protein.