vector constructs which effectively express the cfaB, cfaE and LTB proteins in Shigella spp. without affecting the ability of the Shigella strain to invade cells of the colonic epithelium following oral administration to humans
Inventors
Ranallo, Ryan T. • Venkatesan, Malabi M.
Assignees
United States Department of the Army
Publication Number
US-7759106-B2
Publication Date
2010-07-20
Expiration Date
2025-05-19
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Abstract
With the goal of creating a combination vaccine against Shigella and other diarrheal pathogens we have constructed a prototype vaccine strain of Shigella flexneri 2a (SC608) that can serve as a vector for the expression and delivery of heterologous antigens to the mucosal immune system. SC608 is an asd derivative of SC602, a well-characterized vaccine strain, which has recently undergone several phase 1 and 2 trials for safety and immunogenicity. Using non-antibiotic asd-based plasmids, we have created novel constructs for the expression of antigens from enterotoxigenic E. coli (ETEC), including CFA/I (CfaB and CfaE) and the B-subunit from heat-labile enterotoxin (LTB) in Shigella vaccine strain SC608. Heterologous protein expression levels and cellular localization are critical to immune recognition and have been verified by immunoblot analysis. Following intranasal immunization (SC608(CFAI) and SC608(CFAI/LTB) of guinea pigs, serum IgG and IgA immune responses to both the Shigella LPS and ETEC antigens can be detected by ELISA. In addition, ELISPOT analysis for ASCs from cervical lymph nodes and spleen showed similar responses. All vaccine strains conferred high levels of protection against challenge with wild-type S. flexneri 2a using the Sereny test. Furthermore, serum from guinea pigs immunized with SC608 expressing CfaB and LTB contained antibodies capable of neutralizing the cytological affects of heat-labile toxin (HLT) on Chinese Hamster Ovary (CHO) cells. These initial experiments demonstrate the validity of a multivalent invasive Shigella strain that can serve as a vector for the delivery of pathogen-derived antigens.
Core Innovation
The invention described provides vector constructs and related methodologies for preparing a combination vaccine against Shigella and enterotoxigenic Escherichia coli (ETEC), two major diarrheal pathogens. A prototype vaccine strain of Shigella flexneri 2a (SC608), an asd derivative of the well-characterized vaccine strain SC602, was constructed to serve as a vector for the expression and delivery of heterologous ETEC antigens, specifically the CfaB, CfaE, and LTB proteins. These vector constructs enable expression and export of these proteins on the bacterial surface without affecting the Shigella strain's ability to invade colonic epithelial cells after oral administration.
The problem being addressed arises from the global burden of diarrheal diseases, especially caused by Shigella spp. and ETEC, which have significant morbidity and mortality rates. Current prevention and treatment methods are inadequate. While attenuated Shigella strains have been developed and tested for safety and immunogenicity, there remains a need to create an efficacious multivalent vaccine combining protection against both Shigella and ETEC infections. This entails expressing heterologous ETEC antigens in an attenuated Shigella vaccine strain in a manner that maintains the vector's invasiveness and immunogenicity, without further attenuation or loss of tissue tropism.
The invention achieves this by designing primer pairs and plasmid vectors that allow PCR amplification and insertion of ETEC genes cfaA, cfaB, cfaE, and LTh B (LTB) into an asd-based plasmid system without antibiotic resistance markers. The expressed proteins accumulate periplasmically and assemble on the Shigella surface, preserving invasive ability. Immunization studies in guinea pigs showed measurable serum and mucosal immune responses to both Shigella LPS and ETEC antigens, as well as protection against challenge with wild-type S. flexneri 2a. The constructs demonstrate a novel strategy for expressing fimbrial antigens and support the validity of the multivalent vaccine approach.
Claims Coverage
The patent includes five independent claims covering primer pairs for PCR amplification, plasmid expression constructs, and vaccine strains, with inventive features focusing on the design and function of these constructs for expressing ETEC antigens in Shigella vectors.
Primer pairs for selective PCR amplification of ETEC genes
Primer pairs about 15-100 nucleotides in length that permit PCR amplification of the entire CfaA, CfaB, and CfaE open reading frames without the entire CFA/I operon but including the signal sequences and restriction sites. These primers enable insertion into vectors allowing expression, export, and assembly on the bacterial surface.
Expression plasmids with operably linked promoters for periplasmic antigen expression
Expression plasmids derived from pYA3098 comprising a cis-acting DNA promoter (preferably Ptrc) operably linked to open reading frames containing one or more ETEC genes (cfaA, cfaB, cfaE, LTh B). The constructs express the proteins in the periplasmic space of bacteria with export and assembly on the bacterial surface without antibiotic resistance genes.
Vectors encoding ETEC antigen gene clusters for delivery by Shigella strains
Vectors containing the cfaABCE gene cluster from ETEC H10407 designed to express combinations of cfaA, cfaB, cfaE, and LTh B genes. The open reading frames are PCR-amplified DNA fragments using the specified primers, enabling periplasmic expression and maintenance without antibiotics.
The claims cover novel primers, plasmid constructs, and corresponding vector systems that enable periplasmic expression and surface assembly of ETEC antigens in Shigella vaccine strains, contributing to multivalent vaccine development against Shigella and ETEC infections.
Stated Advantages
The vector constructs allow heterologous antigen expression without affecting Shigella's invasiveness or tissue tropism, preserving immunogenicity.
The asd-based plasmids enable stable maintenance in Shigella without the use of antibiotic resistance markers, improving safety and regulatory acceptability.
Periplasmic expression of ETEC antigens enhances immune recognition relative to cytoplasmic expression, improving vaccine efficacy.
Multivalent vaccine strains induce serum and mucosal IgG and IgA responses to both Shigella and ETEC antigens and provide high levels of protection against wild-type Shigella challenge.
Antibodies raised against expressed LTB can neutralize heat-labile toxin effects in vitro, indicating functional immune responses against ETEC enterotoxins.
Documented Applications
Use of transformed Shigella strains expressing ETEC antigens as live attenuated multivalent vaccines for oral or mucosal immunization against Shigella and ETEC-mediated Traveler's Diarrhea.
Vectors for stable expression and surface presentation of heterologous proteins in Shigella spp., enabling combination vaccines to target multiple enteric pathogens.
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