Title:  Method of delivering antigens for vaccination with a live vector

United States Patent:  6,803,231

Issued:  October 12, 2004

Inventors:  Blaser; Martin (Nashville, TN); Dworkin; Joel (Kirkwood, MO); Thompson; Stuart A. (Evans, GA)

Assignee:  Vanderbilt University (Nashville, TN)

Appl. No.:  355793

Filed:  September 21, 1999

PCT Filed:  January 30, 1998

PCT NO:  PCT/US98/01780

PCT PUB.NO.:  WO98/33386

PCT PUB. Date:  August 6, 1998

Abstract

The present invention provides a mutant C. fetus strain in which one or more sapA homologs are altered by an inserted DNA cassette so that the encoded S-layer proteins represent a chimera between the native S-layer proteins and a heterologous peptide encoded by the inserted cassette. Preferably, the heterologous peptide is an immunogen of a pathogen and the mutant strain can be used to immunize a host to develop mucosal and systemic immune responses to such immunogen.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of immunology and vaccine technology. More specifically, the present invention relates to methods of delivering antigens for vaccination with a live vector.

2. Description of the Related Art

One way in which microorganisms can alter their surface properties, allowing a fraction of the population to preadapt to environmental changes, is by varying protein expression through programmed genomic DNA rearrangements (1). Phase, antigenic, or size variation of expressed surface proteins are governed by mechanisms including transposition and DNA inversion. During transposition, a silent gene is activated by movement to an expression site where it displaces the currently expressed gene. In DNA inversion, a segment of DNA is cut, inverted, and then rejoined by a site-specific recombinase. The invertible DNA segment may contain either a promoter that directs expression of fixed structural genes or structural genes controlled by a fixed promoter. Transposition and inversion differ in both the enzymes used and in the number of genes that can be controlled (many versus two).

Campylobacter fetus, a bacterial pathogen of ungulates and humans, is covered by a paracrystalline surface (S-) layer, composed of high molecular weight S-layer proteins (SLP) that masks most of the underlying gram-negative surface features (2). More than 300 bacterial genera have been described that possess S-layers (3). The S-layer renders C. fetus cells resistant to serum killing by prohibiting the binding of C3b (4), and the S-layer proteins themselves may change, permitting antigenic variation (5,6).

These S-layer proteins are encoded by 7-9 tightly clustered and partially homologous promoterless gene cassettes (7,8). Since previous studies show that C. fetus can express alternative S-layer proteins (4-6,9), there is only a single promoter for S-layer proteins expression present on a 6.2 kb invertible element (9), and the structural genes flanking the promoter are subject to substitution (9), both the promoter and the eight structural genes (sapA and its homologs) may rearrange strictly by inversion.

Campylobacter fetus is able to colonize the mucosa of the gastrointestinal and/or genitourinary tracts of mammals, birds and reptiles. Colonization of the wild-type organism lasts for years and can cause disease. Essential for this long term colonization is the ability to produce the S-layer proteins and Campylobacter fetus has the means to change the S-layer proteins and thus the crystal struture and the particular forms of antigenicity. This antigenic variation is required for the persistence of the organism in its environmental niche.

Campylobacter fetus accomplishes this antigenic variation by posssessing 7-9 highly homologous gene cassettes, called sapA homologs (sapA, sapA1, sapA2, etc.) which encode a different S-layer protein. Each of these homologs contains a 5' region of about 600 base pairs which is completely conserved from homolog to homolog and is necessary for binding of the S-layer protein encoded by that homolog to the lipopolysaccharide molecule anchored in the bacterial outer membrane. The remainder of the open reading frame (ORF) is different for each homolog but semi-conserved regions exist. Wild type C. fetus strains are able to rearrange their chromosomal DNA so that the sapA homolog positioned downstream of a unique promoter is then expressed. This rearrangement occurs at a frequency of about 10^-4 and is recA dependent. RecA is a protein encoded by recA and which is involved in homologous recombination and in repair of breaks of DNA strands.

C. fetus S-layer proteins (SLPs) are secreted in the absence of an N-terminal signal sequence. SLP proteins contain a signal sequence located within the C-terminous of the protein and are secreted through a type I protein secretion system encoded by the sapCDEF operon of four overlapping genes. Analysis of the C-termini of four C. fetus SLPs revealed conserved structures that are potential secretion signals. A C. fetus sapD mutant neither produced nor secreted SLPs. E. coli expressing C. fetus sapA and sapCDEF secreted SapA, indicating that the sapCDEF genes were sufficient for SLP secretion.

The prior art is deficient in the lack of effective means of delivering antigens for vaccination with a live vector. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a mutant C. fetus strain in which each of the cassettes is replaced by a heterologous antigen. The sapA homologs are altered by a DNA cassette inserted so that the encoded SLP represents a chimera between the native SLP and the peptide encoded by the cassette. The inserted DNA cassettes retain 3' sapA sequences that encode the C-terminal secretion signal sequences in order to ensure secretion of the chimeric protein. Representative examples of cassettes that can be inserted in this fashion include immunogens related to Salmonella, Campylobacter jejuni, E. coli 0157:H7, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) as well as other enteric, venereal, or respiratory pathogens of humans, cattle, sheep, poultry, horses, swine, and reptiles. In this embodiment of the present invention, this strain can be used to immunize a host to develop mucosal and systemic immune responses to each of the immunogens.

In another embodiment of the present invention, there is provided a mutant C. fetus strain in which all but one of the cassettes are replaced by a heterologous antigen. In this embodiment, one of the cassettes (e.g., sapA2) remains in its native configuration and the others are mutagenized. The advantage of this construction is that it also can induce immunity to C. fetus based on the single full-length SLP produced.

In another embodiment of the present invention, there is provided a mutant C. fetus strain in which recA is mutagenized. When the RecA protein is not produced, the DNA rearrangements permitting sapA antigenic variation can not occur at any detectable frequency. Thus, the C. fetus strain can only produce one of the SLPs encoded by one sapA homolog. This strain can be used, therefore, to colonize the host briefly, i.e. until protective immunity has developed to that homolog. Subsequently, the host immune response eliminates the organism. Thus, this mutant would provide effective immunity against subsequent C. fetus infection and is useful for vaccination of ungulates (including sheep, cattle and horses) in which infectious abortion and/or infertility can occur after the wild-type infection.

In another embodiment of the present invention, there is provided a C. fetus strain in which recA is mutagenized and the expressed sapA homolog is a chimera involving a heterologous peptide. In this embodiment, a mutagenized sapA homolog expresses a chimeric protein including a heterologous antigen. The strain is then passaged in vitro so that the chimeric homolog is in the expression position and then a recA mutation is made. This strain now essentially expresses only the chimeric protein thus providing a means to immunize a host to that antigen. The duration of colonization in the host is brief and this attenuated C. fetus strain allows safe immunization for the selected antigen.

In yet another embodiment of the present invention, there is provided a mixed mutant C. fetus strains each including a sapA chimera which is also a recA mutant. In this embodiment, mutants are constructed in which a single sapA homolog is mutagenized to encode a different chimeric protein representing a different heterologous antigen. Each mutant is also RecA-deficient due to mutation in recA. A host is inoculated with a mixture of two or more of these strains to provide immunization to the requisite antigens. Each strain is short-lived in the immunized host.

In another embodiment of the present invention there is provided a strain of E. coli carrying plasmids encoding the sapCDEF proteins that permits the secretion of chimeric proteins containing the SapA C-terminal secretion signal. The secreted protein is encoded by an altered sapA homolog that has been engineered so that the 5' section of the homolog-specific region is replaced by a DNA cassette encoding a heterologous antigen. Since the chimeric protein would be secreted by E. coli into the culture medium, this system provides a method for obtaining a large amount of the chimeric protein in a relatively pure form.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations may be used herein: S-layer protein, surface layer protein; km, kanamycin-resistance gene; cm, chloramphenicol-resistance gene; NHS, normal human serum.

In the present invention, programmed gene rearrangements are employed by a variety of microorganisms, including viruses, prokaryotes, and simple eukaryotes, to control gene expression. In most instances in which organisms mediate host evasion by large families of homologous gene cassettes, the mechanism of variation is not thought to involve DNA inversion. Campylobacter fetus, a pathogenic gram-negative bacterium, reassorts a single promoter, controlling S-layer (surface) protein expression, and one or more complete open reading frames strictly by DNA inversion. Rearrangements are independent of the distance between sites of inversion. These rearrangements permit variation in protein expression from the large S-layer protein gene family and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms. Furthermore, S-layer proteins were secreted from C. fetus via a type I protein secretion system encoded by the sapCDEF operon. Secretory signal sequences for the SapA family of secreted proteins was localized to the carboxy-terminus of these proteins.

The present invention is directed to a mutant C. fetus strain useful for vaccinating an animal to Campylobacter fetus, wherein said strain is mutated to contain a DNA cassette encoding a heterologous protein. In one embodiment of the mutant C. fetus strain, a sapA homolog is altered. In one embodiment of the mutant C. fetus strain, the heterologous protein is a S-layer protein. Preferably, the encoded S-layer protein represents a chimera between the native S-layer protein and the peptide encoded by the cassette. Preferably, the cassette is selected from the group consisting of Salmonella, Shigella, Campylobacter jejuni, E. coli 0157:H7, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) and other animal pathogens. In one embodiment of the mutant C. fetus strain, the cassette contains a 5' binding region and 3' secretion signal region and the protein is inserted between said binding region and said signal region. In another embodiment of the mutant C. fetus strain, the cassette contains a 3' secretion signal but has no binding region. Preferably, the protein is an antigen or a therapeutic agent.

The present invention is also directed to a method of immunizing a host to develop mucosal and systemic immune responses to an immunogen, comprising the step of administering to said host a pharmacologically effective dose of the strain described herein.

The present invention is also directed to a mutant C. fetus strain, wherein recA is mutated so that no functional RecA protein is produced, the DNA rearrangements permitting sapA antigenic variation occur at a very low frequency and wherein said C. fetus strain can only produce one of the S-layer proteins encoded by one sapA homolog. In one embodiment, this strain contains a sapA homolog expressing a chimeric protein including a heterologous antigen.

The present invention is also directed to a mixture of mutant C. fetus strains, wherein each strain includes a sapA chimera which is also a recA mutant, wherein a single sapA homolog is mutated to encode a different chimeric protein representing a different heterologous antigen and each mutant is also RecA-deficient due to mutation in recA. The present invention is also directed to a method of immunizing a host to develop mucosal and systemic immune responses to an immunogen, comprising the step of administering to said host a pharmacologically effective dose of these strains.

The present invention is also directed to a strain of bacteria modified to express SapCDEF genes Preferably, the strain is Escherichia coli. In one embodiment of this strain, a heterologous protein is expressed as a chimeric protein composed of sequences of heterologous origin, sequences that direct the secretion of said chimeric protein to the cell surface and sequences that direct the binding of the secreted chimeric protein to the lipopolysaccharides of the bacterial cell surface via the sapCDEF directed type I secretory system. The present invention is also directed to a method of immunizing a host to generate immune responses to an immunogen, comprising the step of administering to said host a pharmacologically effective dose of this strain.

Claim 1 of 11 Claims

What is claimed is:

1. A genetically engineered mutant C. fetus strain derived from strain 23D which has an ATCC designation PTA-4754, said mutant strain contains a DNA cassette inserted into the coding sequence of a sapA homolog of said 23D strain, wherein said DNA cassette encodes a heterologous protein and expression of said DNA cassette results in surface expression of a chimeric protein comprising a 5' LPS-binding region of said sapA homolog, said heterologous protein and a 3' secretion signal region of said sapA homolog.

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