Loren D. Schultz

A Novel Staphylococcus aureus Vaccine: Iron Surface Determinant B Induces Rapid Antibody Responses in Rhesus Macaques and Specific Increased Survival in a Murine S. aureus Sepsis Model

ABSTRACT Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus.

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast.

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.

Using Molecular Genetics to Improve the Production of Recombinant Proteins by the Yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has proven to be an excellent host for the production of a number of different recombinant proteins that have potential medical and commercial applications. The use of S. cerevisiae as a recombinant host has a number of advantages: (1) yeast cells are easily fermented to industrial scale using simple media; (2) yeast cells are free of endotoxin and nonpathogenic to man; (3) S. cerevisiae has well-developed genetics, which offers unparalleled possibilities for solving problems that may exist at various steps in the production of heterologous proteins through a combination of classical and molecular genetic approaches; and (4) yeast cells are capable of performing post-translational and cotranslational processing of proteins in a manner similar to higher eukaryotes. In addition, the secretion of heterologous proteins by yeast has several advantages: first, only low levels of native proteins are secreted into the culture medium, simplifying purification of a target protein; second, yeast is able in many cases to correctly fold proteins and form intramolecular disulfide bonds during secretion as demonstrated by the successful secretion of proteins containing multiple disulfide bonds in a biologically active f ~ r m . I ~ Two examples are echistatin4 and anti~tasin.~ Finally, S. cerevisiae has a number of strong promoters that are either inducible or constitutive. These promoters have been used in a variety of different yeast expression vectors that in turn can be used to readily transform yeast using several different selective markers (URA3, LEU2, T W I , etc.). As many of the above features of heterologous protein expression in yeast have been discussed in several recent they will not be discussed further here. This paper will highlight the well-defined genetics of S. cerevisiae, which enable one to engineer yeast host strains with desired genetic characteristics such that

Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae

A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the alpha-mating factor pre-pro leader sequence and galactose-inducible promoter, GAL10. Cleavage of the pre-pro leader sequence in vivo results in the secretion of a properly processed recombinant ECS with the native N-terminal glutamic acid residue. Recombinant ECS was recovered from yeast supernatants and purified by reverse phase high performance liquid chromatography. Recombinant ECS expressed and purified from yeast was identical to native ECS in its ability to inhibit platelet aggregation.

Antigenic analysis of the epstein-barr virus major membrane antigen (gp350/220) expressed in yeast and mammalian cells: Implications for the development of a subunit vaccine

The Epstein-Barr virus (EBV) major surface membrane antigen, gp350/220, was expressed in recombinant yeast cells and in several recombinant mammalian cell lines. Each of the expressed proteins was analyzed for its ability to bind to a panel of anti-gp350/220 monoclonal antibodies and to a series of anti-EBV positive human sera. The antigens also were used as immunogens for the immunization of rabbits. Each expressed protein was found to be unique both in its pattern of reactivity to the various antibodies and in the spectrum of antibody induced following animal immunization. These results suggest that cell-specific post-translational modifications critically influence the antigenic presentation of the expressed proteins. Nonetheless, all of the mammalian cell-derived versions of the membrane antigen were found capable of inducing EBV-specific neutralizing antibodies.

Mutations of the α-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast

The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture medium. To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MEL1 promoter and MEL1 sp coding sequence (MEL1sp). The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MEL1sp. S. cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1. Using site-directed mutagenesis, several mutations were introduced into the MEL1sp. Two mutations were identified which dramatically increased the secretion of both Echis and PAI-1 to levels similar to those achieved when using the yeast MF alpha 1 pre-pro secretory leader. In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase in the secretion levels of those two proteins.

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.

Regulated production of recombinant echistatin by yeast

SummaryThe α-mating factor pre-pro-leader sequence under the regulation of theGAL10 promoter was used to direct the secretion of echistatin by recombinant yeasts. Optimization of the culture medium and host strain increased the productivity of shake flask cultures twenty-fold to 8 mg/L. In fermentors, the production of echistatin was greater than 40 mg/L.

Expression and Analysis of EBV gp350 in the Yeast Saccharomyces Cerevisiae

Epstein-Barr Virus (EBV) causes infectious mononucleosis and has been implicated strongly, along with essential cofactors, as a causative agent for the development of nasopharyngeal carcinoma, Burkitt’s lymphoma, and B-cell malignancies in immune-deficient patients. The two major glycoprotein components of the virion are gp350 and gp220, which are encoded by the same gene (1). They are both capable of eliciting neutralizing antibodies in vitro (2). In addition, recent data suggest that viral gp350 can be protective in a primate model (cottontop marmoset) of EBV infection (3). Thus, gp350 may be a useful immunogen for eliciting protective immunity against EBV infections. In this report, we describe the expression in yeast (Saccharomyces cerevisiae) of the secretable form of gp350, minus the membrane anchor sequence. The yeast-derived gp375 shows structural and immunological similarities to the viral gp350.