Expression Of Protective Antigen Research Articles

Protective efficacy of recombinant BCG over-expressing protective, stage-specific antigens of Mycobacterium tuberculosis

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. CysVac2, a fusion protein comprising stage-specific Mycobacterium tuberculosis antigens, provided superior protective efficacy against chronic M. tuberculosis infection in mice, compared to BCG. To determine if the delivery of CysVac2 in the context of BCG could improve BCG-induced immunity and protection, we generated a recombinant strain of BCG overexpressing CysVac2 (rBCG:CysVac2). Expression of CysVac2 in BCG was facilitated by the M. tuberculosis hspX promoter, which is highly induced inside phagocytic cells and induces strong cellular immune responses to antigens expressed under its regulation. Intradermal vaccination with rBCG:CysVac2 resulted in increased monocyte/macrophage recruitment and enhanced antigen-specific CD4+ T cell priming compared to parental BCG, indicating CysVac2 overexpression had a marked effect on rBCG induced-immunity. Further, rBCG:CysVac2 was a more potent inducer of antigen-specific multifunctional CD4+ T cells (CD4+IFN-γ+TNF+IL-2+) than BCG after vaccination of mice. This improved immunogenicity however did not influence protective efficacy, with both BCG and rBCG:CysVac2 affording comparable level of protection aerosol infection with M. tuberculosis. Boosting either BCG or rBCG:CysVac2 with the CysVac2 fusion protein resulted in a similar improvement in protective efficacy. These results demonstrate that the expression of protective antigens in BCG can augment antigen-specific immunity after vaccination but does not alter protection against infection, further highlighting the challenge of developing effective vaccines to control TB.

Recent progress in the development of anthrax vaccines

Bacillus anthracis is the etiological agent of anthrax. Although anthrax is primarily an epizootic disease; humans are at risk for contracting anthrax. The potential use of B. anthracis spores as biowarfare agent has led to immense attention. Prolonged vaccination schedule of current anthrax vaccine and variable protection conferred; often leading to failure of therapy. This highlights the need for alternative anthrax countermeasures. A number of approaches are being investigated to substitute or supplement the existing anthrax vaccines. These relied on expression of Protective antigen (PA), the key protective immunogen; in bacterial or plant systems; or utilization of attenuated strains of B. anthracis for immunization. Few studies have established potential of domain IV of PA for immunization. Other targets including the spore, capsule, S-layer and anthrax toxin components have been investigated for imparting protective immunity. It has been shown that co-immunization of PA with domain I of lethal factor that binds PA resulted in higher antibody responses. Of the epitope based vaccines, the loop neutralizing determinant, in particular; elicited robust neutralizing antibody response and conferred 97% protection upon challenge. DNA vaccination resulted in varying degree of protection and seems a promising approach. Additionally, the applicability of monoclonal and therapeutic antibodies in the treatment of anthrax has also been demonstrated. The recent progress in the direction of anthrax prophylaxis has been evaluated in this review.

An efficient fusion protein system for expression of Bacillus anthracis protective antigen as immunogenic and diagnostic antigen

Objective To produce high quantities of recombinant protective antigen (rPA) for human vaccine and diagnosis. Methods The PA gene was amplified by PCR with p XO1 plasmid as template. The PCR product was cloned into pMAL-c2X vector using the BamHI and SalI restriction enzymes. The recombinant plasmid was transformed into Escherichia coli DH5 α strain and then screened for transformation. The expression of protective antigen was analyzed by SDS-PAGE and Western blotting after isopropyl β-D-thiogalactopyranoside (IPTG) induction. Results The full-length PA gene (2.2 kb) was cloned into pMAL vector system. The recombinant vector was confirmed by restriction enzyme and PCR analysis. The expression of cytoplasmic maltose-binding protein-protective (MBP-P) antigen fusion protein was detected by SDS-PAGE and Western blotting, and obtained a 125 kDa protein band, which was similar to expected size of fusion protein. Conclusions This expression system can be used in the high production of rPA. After purification and immunization studies, the purified r PA may be used in the development of the human recombinant anthrax vaccine and also in diagnosis of anthrax disease.

Parasite Vaccines: Recent Progress in, and Problems Associated with their Development

Metazoan and protozoan parasites are major causes of human and animal disease causing extensive morbidity and mortality, particularly in tropical and sub-tropical climatic regions. WHO estimates that one person in every four is affected by parasitic worms with disease outcomes ranging from chronic symptoms, blindness, disfiguration to death. In addition, gastrointestinal parasitism is one of the greatest animal health constraints worldwide, both to commercial and subsistence farmers. While substantial progress has been made in identifying potential protective antigens, vaccine development is impaired because it is difficult, often impossible, to cultivate many of the major target parasites in vitro, most parasites present different developmental stages to the host immune system, most show considerable antigenic diversity and there is growing evidence that they directly modulate the host immune system to their advantage. While progress has been made in the last decade in the cloning and expression of protective antigens from a large number of parasites, the majority have failed to stimulate practically useful levels of protective immunity in trials. This review is intended to provide the reader with an overview of the nature of the disease problem being addressed using selected examples and the approaches being taken to develop vaccines to counter the adverse effects of infection. It is not intended to be fully comprehensive and the interested reader can access more in depth discussion from cited references and using on-line search resources

Expression Systems for Study of Mycobacterial Gene Regulation and Development of Recombinant BCG Vaccines

Successful genetic engineering of mycobacteria is crucial for developing new approaches to combat tuberculosis as well as for dissecting out the molecular basis of pathogenesis ofMycobacterium tuberculosis.We have constructed aMycobacterium–Escherichia colishuttle expression vector pSD5. It carries a modular expression cassette which provides sites for cloning of promoters, a ribosome binding site (RBS) with an appropriately placed initiation codon and multiple cloning sites for cloning the genes of interest. We also constructed pDK20, an integration proficient derivative of pSD5, by incorporating mycobacteriophage L5 integration signals in lieu of the origin of DNA replication for mycobacteria. This vector permits stable expression of genes inM.bovisBCG,M.smegmatis,andM.tuberculosisunder the transcriptional control of a mycobacterial promoter. These vectors enable the expression of a gene to be regulated by several hundred fold depending upon the strength of mycobacterial promoter. We propose that expression of protective antigens using an appropriate promoter derivative of pDK20 should help in development of recombinant BCG vaccines that can induce an optimal immune response from the host. We have further employed the integration proficient expression system for comparing the efficiency and specificity of transcriptional recognition inM.bovisBCG,M.tuberculosis,andM.smegmatis.We show that fast growingM.smegmatisand slow growingM.tuberculosisandM.bovisBCG recognize mycobacterial promoters with comparable efficiency inspite of differences in their growth rates.

Vaccines against blood-sucking arthropods

This paper provides selected personal insights on the development of vaccines against blood-sucking arthropods, with particular emphasis on vaccines against ticks. The emergence of novel or concealed antigens of haematophagous ectoparasites as candidate vaccine antigens is reviewed and the effect of feeding by the parasite on the expression of protective antigens is considered. The distribution of protective antigens through life cycle stages, the stage of the life cycle targeted by protective responses, and the nature of these responses, are commented on briefly. Concealed antigens of the gut, including the peritrophic membrane, and other internal organs, are evaluated for the role they play in induction of immunity artificially. Some of the work carried out to purify and characterise protective antigens of tick guts is described. A commentary is developed on vaccines that combine both “concealed” and “exposed” antigens. Some of the problems associated with the infestation and challenge of vaccinated hosts in the field are identified and the delivery of parasite antigens as vaccines that are both protective and “user-friendly” is emphasised as a major problem to be solved.

Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine.

The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F. de la Cruz, T.R. Fuerst, J.E. Burlein, L.A. Benson, L.T. Bennett, G.P. Bansal, J.F. Young, M.H. Lee, G.F. Hatfull et al. 1991. Nature [Lond]. 351:456; Jacobs, W.R., Jr., S.B. Snapper, L. Lugosi and B.R. Bloom. 1990. Curr. Top. Microbiol. Immunol. 155:153; Jacobs, W.R., M. Tuckman, and B.R. Bloom. 1987. Nature [Lond.]. 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses. Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens. Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100-1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein. Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth. Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response.

4883761 Pertussis toxin gene: Cloning and expression of protective antigen: Jerry M Keith, Camille Locht assigned to The United States of America as represented by the Department of Health and Human Services

4883761 Pertussis toxin gene: Cloning and expression of protective antigen: Jerry M Keith, Camille Locht assigned to The United States of America as represented by the Department of Health and Human Services

4883761 Pertussis toxin gene: Cloning and expression of protective antigen: Jerry M Keith, Camille Locht assigned to The United States of America as represented by the Department of Health and Human Services

4883761 Pertussis toxin gene: Cloning and expression of protective antigen: Jerry M Keith, Camille Locht assigned to The United States of America as represented by the Department of Health and Human Services