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C K Stover

Publications and source records attributed to C K Stover.

At least 37 records · Page 2Linked to original sources
PubMed1995

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Animals↗
PubMed1995

Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria.

To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.

Amino Acid Sequence↗
PubMed1995

Bacterial vaccine vectors and bacillus Calmette-Guérin.

Recent advances in biotechnology now allow a more modern approach to the development of vaccines, particularly that of recombinant vaccines. Bacterial vaccine vectors have the advantage over viral vectors in that the former have the ability to express a greater number of antigens in different forms. Although no recombinant bacterial vaccines are currently in use, bacillus Calmette-Guérin (BCG), Salmonella species, and Escherichia coli are being developed as vaccine vectors. We review plasmid systems and mutant strains developed for the expression of foreign antigens, with particular emphasis on those developed for BCG. We describe the development of antigen expression systems as well as the immune response elicited by recombinant BCG vaccine strains to bacterial and human immunodeficiency virus (HIV) antigens. A modified recombinant BCG carrier with selection for the stable maintenance of rDNA is proposed.

Administration, Oral↗
PubMed1995

Efficacy and safety of live recombinant BCG vaccines.

BCG has a long history of safe use in humans and is one of the best adjuvants known. The use of newer production methods may further reduce the risk of adverse side-effects. Early results with experimental animals have shown BCG to be an effective live recombinant delivery vehicle for several foreign vaccine antigens. Additional refinements to the safety and efficacy of the recombinant BCG vaccine vehicle are under development.

Animals↗
PubMed1994

Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi.

The bacillus Calmette-Guerin (BCG) is a live attenuated strain of Mycobacterium bovis which offers potential advantages as a vector for mucosal delivery of antigens. Recombinant BCG elicits protective humoral immune responses to a variety of antigens. Furthermore, BCG binds specifically to microfold cells present in the epithelium overlying lymphoid follicles throughout the mucosal immune system. Here we show that a single intranasal vaccination with recombinant BCG expressing the outer-surface protein A antigen from B. burgdorferi results in a prolonged (more than one year) protective systemic IgG response and a highly sustained secretory IgA response which is disseminated throughout the mucosal immune system. Furthermore, intranasal immunization induces marked, organized lymphocyte accumulation in the proximal nasopharyngeal lymphoid tissue as well as at distal mucosal sites; the appearance and persistence of lymphoid aggregates correlates with the secretory immune responses. Thus intranasal immunization with recombinant BCG is a powerful method for inducing long-lasting secretory and systemic immune responses.

Administration, Intranasal↗
PubMed1994

Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guérin vaccines expressing pneumococcal surface protein A.

Pneumococcal surface protein A (PspA), a cell-surface protein present on all strains of pneumococci, has been shown to elicit protective antibody responses in mice in the absence of capsular polysaccharide. Whereas PspA is polymorphic, considerable cross-reactivity and cross-protection have been demonstrated among PspA proteins of pneumococci exhibiting different capsular and PspA serotypes. A gene segment encoding the nonrepetitive variable NH2-terminal portion of PspA has been cloned into three distinct recombinant Bacille Calmette-Guérin (rBCG) vectors, allowing for expression of PspA as a cytoplasmic or secreted protein, or a chimeric exported membrane-associated lipoprotein. All rBCG-PspA strains elicited comparable anti-PspA ELISA titers, ranging from 10(4) to 10(5) (reciprocal titers) in both BALB/c and C3H/HeJ mice. However, protective responses were observed only in animals immunized with the rBCG-PspA vaccines expressing PspA as a secreted protein or chimeric exported lipoprotein. In addition, anti-PspA immune sera elicited by the rBCG vaccines passively protected X-linked immunodeficient mice from lethal challenge with the highly virulent, encapsulated WU2 strain of Streptococcus pneumoniae and two additional virulent strains exhibiting heterologous PspA and capsular serotypes. These studies confirm previous PspA immunization studies showing cross-protection against heterologous serotypes of S. pneumoniae and demonstrate a potential for rBCG-based PspA vaccines to elicit protective humoral responses against pneumococcal disease in humans.

Animals↗
PubMed1994

Recombinant vaccine delivery systems and encoded vaccines.

In addition to the introductory demonstrations of genetic immunization, the past year has brought significant advances in vaccine development. Particularly encouraging are live recombinant vaccines, studies demonstrating the potential to elicit both systemic and mucosal immune responses, more studies demonstrating immune protection in animal disease models, and experimental vaccines eliciting immune responses in primates.

Animals↗
PubMed1994

Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence.

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.

Animals↗
PubMed1994

Protective immunity elicited by rBCG vaccines.

The live attenuated Mycobacterium bovis strain Bacille Calmette-Guerin (BCG) is still widely used as a vaccine against tuberculosis and therefore offers potential advantages as a safe, live vaccine vehicle for the expression and delivery of protective recombinant antigens. As an attenuated intracellular bacterium residing in macrophages, BCG would seem to be particularly suited for eliciting cellular responses and not humoral responses. Efforts to improve the potential for recombinant BCG (rBCG) vaccines to elicit protective humoral responses were undertaken by developing vectors systems which export or secrete foreign antigens. Expression of the OspA antigen of Borrelia burgdorferi, as a chimaeric membrane-associated lipoprotein, resulted in high-titred protective humoral responses when compared to cytoplasmic or secreted expression of OspA. Expression of the PspA antigen of Streptococcus pneumoniae as a secreted or membrane-associated lipoprotein by rBCG did not result in higher-titred humoral responses in comparison to PspA expressed cytoplasmically but apparently improved the quality of protective PspA specific antibodies. Based on these pre-clinical studies, rBCG vaccines for Lyme and pneumococcal diseases are being developed for clinical trials in humans. The potential for eliciting mucosal responses to antigens delivered by rBCG was also investigated. Nasal immunization was superior at eliciting substantial lasting mucosal responses at multiple mucosal sites and also resulted in systemic responses comparable to those obtained with parenteral immunization.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys.

Because the transmission of HIV is likely to occur through cell-associated virus, an effective HIV vaccine should be capable of eliciting HIV-specific CTL. We have employed the simian immunodeficiency virus (SIV)/rhesus monkey model to explore the use of the attenuated tuberculosis bacillus, Calmette Guérin bacillus (BCG), as a vaccine vehicle to elicit AIDS virus-specific CTL. BCG was engineered to express SIVmac gag under the control of hsp70 regulatory sequences. Immunization with this rBCG-SIVmac gag elicited MHC class I-restricted, CD8+ SIVmac gag-specific CTL in rhesus monkeys. In fact, SIVmac gag-specific CTL could be cloned readily from peripheral blood lymphocytes of these immunized monkeys. These findings provide further evidence for the power of BCG as a vaccine vector and its continued exploration as a vehicle for eliciting HIV-specific immunity.

Amino Acid Sequence↗
PubMed1993

Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development.

Amino Acid Sequence↗
PubMed1992

Old microbes with new faces: molecular biology and the design of new vaccines.

Rational approaches to the design of live attenuated bacterial and viral recombinant vaccine strains are leading to the manipulation of old vaccines and the generation of new ones. The two basic problems to be solved are attenuation of pathogenic strains, and the stable expression of foreign antigens.

Adenoviridae↗
PubMed1991

New use of BCG for recombinant vaccines.

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.

Animals↗
PubMed1991

Molecular cloning and sequence analysis of a Rickettsia tsutsugamushi 22 kDa antigen containing B- and T-cell epitopes.

The identification of Rickettsia tsutsugamushi T-cell epitopes is necessary for the characterization of the protective immune response (of which the T-cell response is essential) against scrub typhus rickettsiae. A T-helper cell line derived from R. tsutsugamushi (Karp strain) immune mice reacted with rickettsial protein antigens eluted from the 18-35 kDa region of polyacrylamide gels. Within this region is a 22 kDa protein which is reactive with immune serum. The gene encoding the 22 kDa scrub typhus antigen (sta22) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the sta22 gene revealed a potential open reading frame (ORF) in the sta22 sequence encoding a 22 kDa protein. A recombinant 22 kDa protein synthesized in E. coli maxicells was reactive with anti-rickettsial antibodies. The codon usage of the adenine and thymine rich sta22 sequence was similar to other previously sequenced R. tsutsugamushi genes. Computer analysis of the deduced amino acid sequence suggested that the Sta22 protein has several amphipathic regions which may be potential T-cell epitopes. The recombinant Sta22 protein eluted from polyacrylamide gels induced a strong proliferative response from the scrub typhus rickettsiae reactive T-cell line. Recognition of the R. tsutsugamushi Sta22 polypeptide by both cellular and humoral immune mechanisms implicates this antigen as one of potential importance in vaccine development.

Amino Acid Sequence↗
PubMed1991

Development of BCG as a live recombinant vector system: potential use as an HIV vaccine.

Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.

AIDS Vaccines↗