scholarly journals Development of a ΔglnA Balanced Lethal Plasmid System for Expression of Heterologous Antigens by Attenuated Vaccine Vector Strains of Vibrio cholerae

2000 ◽  
Vol 68 (1) ◽  
pp. 221-226 ◽  
Author(s):  
Edward T. Ryan ◽  
Thomas I. Crean ◽  
Sims K. Kochi ◽  
Manohar John ◽  
Angel A. Luciano ◽  
...  
Keyword(s):  
Immune Responses ◽  
Vibrio Cholerae ◽  
Antigen Expression ◽  
Single Copy ◽  
Vaccine Vector ◽  
Live Attenuated Vaccine ◽  
Primary Inoculation ◽  
Attenuated Vaccine ◽  
Growth Deficiency

ABSTRACT We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a ΔglnA derivative of V. cholerae vaccine strain Peru2. This mutant strain, Peru2ΔglnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1.8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus. Strain Peru2ΔglnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2ΔglnA, Peru2ΔglnA(pKEK71-NotI), Peru2(pTIC5), Peru2ΔglnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2ΔglnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals. However, specific anti-CtxB immune responses in serum (P ≤ 0.05) and bile (P ≤ 0.001) were significantly higher in animals that received Peru2ΔglnA(pTIC5) than in those that received ETR3, confirming the advantage of higher-level antigen expression in vivo. The development of this balanced lethal system thus permits construction and maintenance of vaccine and vector strains of V. cholerae that express high levels of immunogenic antigens from plasmid vectors without the need for antibiotic selection pressure.

scholarly journals In Vitro and In Vivo Analyses of Constitutive and In Vivo-Induced Promoters in Attenuated Vaccine and Vector Strains ofVibrio cholerae

2000 ◽  
Vol 68 (3) ◽  
pp. 1171-1175 ◽  
Author(s):  
Manohar John ◽  
Thomas I. Crean ◽  
Stephen B. Calderwood ◽  
Edward T. Ryan
Keyword(s):  
Immune Responses ◽  
Vaccine Strain ◽  
Antigen Expression ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine ◽  
In Vivo Expression ◽  
Antibody Titers ◽  
Heterologous Antigens

ABSTRACT The optimal promoter for in vivo expression of heterologous antigens by live, attenuated vaccine vector strains of Vibrio cholerae is unclear; in vitro analyses of promoter activity may not accurately predict expression of antigens in vivo. We therefore introduced plasmids expressing the B subunit of cholera toxin (CtxB) under the control of a number of promoters into V. choleraevaccine strain Peru2. We evaluated the tac promoter, which is constitutively expressed in V. cholerae, as well as the in vivo-induced V. cholerae heat shock htpGpromoter and the in vivo-induced V. cholerae iron-regulatedirgA promoter. The functionality of all promoters was confirmed in vitro. In vitro antigenic expression was highest in vaccine strains expressing CtxB under the control of thetac promoter (2 to 5 μg/ml/unit of optical density at 600 nm [OD600]) and, under low-iron conditions, in strains containing the irgA promoter (5 μg/ml/OD600). We orally inoculated mice with the various vaccine strains and used anti-CtxB immune responses as a marker for in vivo expression of CtxB. The vaccine strain expressing CtxB under the control of thetac promoter elicited the most prominent specific anti-CtxB responses in vivo (serum immunoglobulin G [IgG], P≤ 0.05; serum IgA, P ≤ 0.05; stool IgA,P ≤ 0.05; bile IgA, P ≤ 0.05), despite the finding that the tac and irgApromoters expressed equivalent amounts of CtxB in vitro. Vibriocidal antibody titers were equivalent in all groups of animals. Our results indicate that in vitro assessment of antigen expression by vaccine and vector strains of V. cholerae may correlate poorly with immune responses in vivo and that of the promoters examined, thetac promoter may be best suited for expression from plasmids of at least certain heterologous antigens in such strains.

scholarly journals Kinetics of Recombinant Adenovirus Type 5, Vaccinia Virus, Modified Vaccinia Ankara Virus, and DNA Antigen Expression In Vivo and the Induction of Memory T-Lymphocyte Responses

2008 ◽  
Vol 15 (4) ◽  
pp. 691-696 ◽  
Author(s):  
Ralf Geiben-Lynn ◽  
John R. Greenland ◽  
Kwesi Frimpong-Boateng ◽  
Norman L. Letvin
Keyword(s):  
Immune Responses ◽  
Real Time ◽  
T Lymphocyte ◽  
Recombinant Adenovirus ◽  
Antigen Expression ◽  
Vaccine Antigen ◽  
Photon Imaging ◽  
Lymphocyte Responses ◽  
Kinetics Of

ABSTRACT While a new generation of vaccine vectors has been developed for eliciting cellular immune responses, little is known about the optimal routes for their administration or about the ramifications of the kinetics of in vivo vaccine antigen expression for immunogenicity. We evaluated the kinetics of vaccine antigen expression by real-time in vivo photon imaging and showed dramatic differences in these kinetics using different vectors and different routes of administration. Further, using a gamma interferon enzyme-linked immunospot assay to measure T-lymphocyte immune responses, we observed an association between the kinetics of vaccine antigen expression in vivo and the magnitude of vaccine-elicited memory T-lymphocyte responses. These results highlight the utility of the real-time in vivo photon-imaging technology in evaluating novel immunization strategies and suggest an association between the kinetics of vaccine antigen clearance and the magnitude of vaccine-elicited T-lymphocyte memory immune responses.

scholarly journals In Vivo Assessment of NS1-Truncated Influenza Virus with a Novel SLSYSINWRH Motif as a Self-Adjuvanting Live Attenuated Vaccine

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0118934 ◽  
Author(s):  
John M. Ngunjiri ◽  
Ahmed Ali ◽  
Prosper Boyaka ◽  
Philip I. Marcus ◽  
Chang-Won Lee
Keyword(s):  
Influenza Virus ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine ◽  
In Vivo Assessment

scholarly journals A novel live attenuated RSV vaccine candidate with mutations in the L protein SAM binding site and the G protein cleavage site is protective in cotton rats and a rhesus macaque

2020 ◽  
Author(s):  
Tiffany Jenkins ◽  
Rongzhang Wang ◽  
Olivia Harder ◽  
Miaoge Xue ◽  
Phylip Chen ◽  
...  
Keyword(s):  
Binding Site ◽  
Rhesus Macaque ◽  
Vaccine Candidate ◽  
Vero Cells ◽  
Virus Yield ◽  
Vaccine Production ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine ◽  
Cotton Rats

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in children < 5 years of age worldwide, infecting the majority of infants in their first year of life. Despite the widespread impact of this virus, no vaccine is currently available. For over 50 years, live attenuated vaccines (LAV) have been shown to protect against other childhood viral infections, offering the advantage of presenting all viral proteins to the immune system for stimulation of both B and T cell responses and memory. The RSV LAV candidate described here, rgRSV-L(G1857A)-G(L208A), contains two modifications: an attenuating mutation in the S-adenosylmethionine (SAM) binding site of the viral mRNA cap methyltransferase (MTase) within the large (L) polymerase protein and a mutation in the attachment (G) glycoprotein that inhibits its cleavage during production in Vero cells, resulting in virus with a “non-cleaved G” (ncG). RSV virions containing the ncG have an increased ability to infect primary well-differentiated human bronchial epithelial (HBE) cultures which model the in vivo site of immunization, the ciliated airway epithelium. This RSV LAV candidate is produced efficiently in Vero cells, is highly attenuated in HBE cultures, efficiently induces neutralizing antibodies that are long-lasting, and provides protection against an RSV challenge in the cotton rat, without causing enhanced disease. Similar results were obtained in a rhesus macaque. Importance Globally, RSV is a major cause of death in children under one year of age, yet no vaccine is available. We have generated a novel RSV live attenuated vaccine candidate containing mutations in the L and G proteins. The L polymerase mutation does not inhibit virus yield in Vero cells, the cell type required for vaccine production, but greatly reduces virus spread in HBE cultures, a logical in vitro predictor of in vivo attenuation. The G attachment protein mutation reduces its cleavage in Vero cells, thereby increasing vaccine virus yield, making vaccine production more economical. In cotton rats, this RSV vaccine candidate is highly attenuated at a dose of 105 PFU and completely protective following immunization with 500 PFU, 200-fold less than the dose usually used in such studies. It also induced long-lasting antibodies in cotton rats and protected a rhesus macaque from RSV challenge. This mutant virus is an excellent RSV live attenuated vaccine candidate.

scholarly journals A Single-Cycle Vaccine Vector Based on Vesicular Stomatitis Virus Can Induce Immune Responses Comparable to Those Generated by a Replication-Competent Vector

2005 ◽  
Vol 79 (21) ◽  
pp. 13231-13238 ◽  
Author(s):  
Jean Publicover ◽  
Elizabeth Ramsburg ◽  
John K. Rose
Keyword(s):  
Immune Responses ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Cytotoxic T Lymphocyte ◽  
Antibody Responses ◽  
Single Cycle ◽  
Live Attenuated Vaccine ◽  
Vesicular Stomatitis ◽  
Env Protein

ABSTRACT Live attenuated vaccine vectors based on recombinant vesicular stomatitis virus (VSV) are effective in several viral disease models. In this study, we asked if a VSV vector capable of only a single cycle of replication might be an effective alternative to replication-competent VSV vectors. We compared the cellular immune responses to human immunodeficiency virus (HIV) envelope protein (Env) expressed by replication-competent and single-cycle VSV vectors and also examined the antibody response to Env. The single-cycle vector was grown by complementation with VSV G protein and then tested initially for immunogenicity when given by four different routes. When given by the intramuscular route in mice, we found that the single-cycle vector was equivalent to the replication-competent VSV vector in generating high-level primary and memory CD8 T-cell responses as well as antibody responses to Env. Cellular responses were analyzed using major histocompatibility complex class I tetramers and direct measurement of cytotoxic T-lymphocyte activity in vivo. We also found that the recall responses after boosting were equivalent in animals vaccinated with replication-competent or single-cycle vectors. Additionally, we observed recall and heightened memory responses after boosting animals with a single-cycle vector complemented with G protein from a different vesiculovirus. Because expression of HIV Env by G-deleted VSV might allow replication in human cells expressing CD4, we generated a single-cycle VSV recombinant expressing a secreted form of the HIV Env protein. This virus was just as effective as the recombinant expressing the membrane-anchored Env protein at producing CD8 T cells and antibody responses.

scholarly journals Influenza A Virus Lacking M2 Protein as a Live Attenuated Vaccine

2009 ◽  
Vol 83 (11) ◽  
pp. 5947-5950 ◽  
Author(s):  
Shinji Watanabe ◽  
Tokiko Watanabe ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Lethal Challenge ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine ◽  
Live Attenuated Influenza Vaccine

ABSTRACT Mutant influenza virus that lacks the transmembrane and cytoplasmic tail domains of M2 (M2 knockout [M2KO]) is attenuated in both cell culture and mice. Here, we examined the potency of M2KO influenza virus as a live attenuated influenza vaccine. M2KO virus grew as efficiently as the wild-type virus in cells stably expressing the wild-type M2, indicating the feasibility of efficient vaccine production. Mice intranasally vaccinated with M2KO virus developed protective immune responses and survived a lethal challenge with the wild-type virus, suggesting that the M2KO virus has potential as a live attenuated vaccine.

scholarly journals Plasmidic versus Insertional Cloning of Heterologous Genes in Mycobacterium bovis BCG: Impact on In Vivo Antigen Persistence and Immune Responses

2002 ◽  
Vol 70 (1) ◽  
pp. 303-314 ◽  
Author(s):  
I. Méderlé ◽  
I. Bourguin ◽  
D. Ensergueix ◽  
E. Badell ◽  
J. Moniz-Peireira ◽  
...  
Keyword(s):  
Immune Responses ◽  
Mycobacterium Bovis ◽  
Genetic Stability ◽  
Single Copy ◽  
Expression Cassette ◽  
Expression Levels ◽  
Antigen Persistence ◽  
Cellular Immune

ABSTRACT Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

scholarly journals Plasmid DNA Vaccine-Elicited Cellular Immune Responses Limit In Vivo Vaccine Antigen Expression through Fas-Mediated Apoptosis

2007 ◽  
Vol 178 (9) ◽  
pp. 5652-5658 ◽  
Author(s):  
John R. Greenland ◽  
Ralf Geiben ◽  
Sharmistha Ghosh ◽  
William A. Pastor ◽  
Norman L. Letvin
Keyword(s):  
Immune Responses ◽  
Dna Vaccine ◽  
Plasmid Dna ◽  
Antigen Expression ◽  
Vaccine Antigen ◽  
Cellular Immune Responses ◽  
Plasmid Dna Vaccine ◽  
Cellular Immune

scholarly journals Live attenuated SARS-CoV-2 vaccine candidate: Protective immunity without serious lung lesions in Syrian hamsters

2021 ◽  
Author(s):  
Shinya Okamura ◽  
Akiho Kashiwabara ◽  
Hidehiko Suzuki ◽  
Shiori Ueno ◽  
Paola Miyazato ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Nonstructural Protein ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Live Attenuated Vaccine ◽  
Syrian Hamsters ◽  
Attenuated Vaccine

AbstractVarious COVID-19 vaccine candidates are currently under clinical trial. However, no live attenuated vaccine has been developed yet, despite their generally high efficacy. Here, we established temperature-sensitive mutant strains of SARS-CoV-2, whose growth was significantly slower than that of the parent strain at 37°C. One of the strains, A50-18, which presented mutations in nonstructural protein 14, did not replicate at all at 37°C in vitro. In vivo experiments demonstrated that this strain replicated inefficiently in the lungs of Syrian hamsters, and intra-nasal inoculation induced sufficient anti-SARS-CoV-2-neutralizing antibodies to protect against wild type virus infection. These results suggest that the A50-18 strain could be a promising live attenuated vaccine candidate against SARS-CoV-2.One Sentence SummaryA live attenuated virus provided immunity against SARS-CoV-2 in an animal model, making it a promising vaccine candidate.

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