Live Pertussis Vaccine
The Recombinant Bordetella Pertussis Bacteria - Live Vaccine and Bacterial Vector
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Gamalei Institute of Epidemiology and Microbiology, Russia, Moscow
- University of Saskatchewan / Vaccine & Infectious Disease Organization, Canada, SK, Saskatoon
Project summaryWhooping cough, or pertussis, is a human respiratory disease, caused by gram-negative bacteria Bordetella pertussis, and transmitted via respiratory droplets. Pertussis prevention is included in Russian national calendar of planned vaccination and is provided by mass inrfant and childhood immunization schedules. However, the circulation of the pathogen is not decreasing. The yearly incidence of reported pertussis is 48.5 million cases all over the world, and the annual pertussis death rate is 300 000 in infants and children (Croweroft N.S.,et al. 2003). In infants pertussis occurs more often and severe, but there were marked laboratory confirmed cases of B.pertussis infection in adolescents and adults recently (Cherry J.D., Heininger U., 2004). The study of pertussis epidemiology showed that the main reservoir of infection in susceptible to disease infants were often adolescents and adults. Also were recorded cases of B.pertussis asymptomatic carriage (Heininger U.W. et al., 2004). The mechanisms of B.pertussis infection in human organism were studied (Bassinet L., 2000).
Pertussis vaccination according to contemporary vaccination calendar requires four immunizations by DTP (whole-cell preparations) and three immunizations by acellular vaccines (Infanriks DTaP). However, DTP or acellular vaccination provide relatively short-lived immunity (for 3-5 years) and at the same time children who have withstood classic pertussis are protected from repeated infection during 10 years and more. So, it can be expected that live attenuated bacteria B.pertussis used for vaccination will provide stronger immunity lasting for a longer time than DTP or acellular vaccination.
Vaccination schedules in new century should include immunization of no less than 95% infants and it will lead to necessity of 20-40 vaccine administrations for each infant annually. As a result, the total number of injections administrated to each infant is increasing several folds. In this connection an urgency of combined vaccines development including bacterial preparations expressing heterological antigens in the human respiratory tract appeared to be topical. Among these vaccines, the live pertussis vaccines and bacterial vector constructed on the basis of attenuated microorganisms of genus Bordetella seem to be of first priority.
Main targets for attenuation are pertussis toxin (PT), dermonecrotic toxin (DNT) and tracheal cytotoxin (NCN). For elimination of DNT and TCT toxicity “knock-out” mutations disturbing appropriate genes expression were used (N.Mielcarek, 2006). РТ is the main protective component of all pertussis vaccines. Inactivation of PT activity should be achieved without modifying its immunobiological characteristics. It was sown that this problem can be solved by introducing two changes in the PT enzymatic(fermentative) center Arg9-Lys9, Glu129-Gly29 (Pizza M., et al., 1989). Bacterial strains containing “knockouted” mutaitions in dnt and tct genes and Arg9-Lys, Glu129-Glu mutations in ptx gene were constracted recently in USA and in France (Mielcarek N., et al., 2006). It was shown that nasal administration of attenuated bacteria provided protection in mice that could be compared with acellular vaccine protection.
Stevenson A. et al., 2003 demonstrated a possibility of using bacteria of genus Bordetella for expressing heterologic antigenes. In all studies, antigens were expressed as a part of protein fused with filamentous hemagglutinin (FHA), the main adhesive protein and the significant Bordetella’s protective antigen.
Use of recombinant Bordetella strains for vaccination of laboratory animals has revealed specific antibodies against Bordetella bacteria and heterological antigens (for example, Schistosoma’s mansoni glutathione S-transferases) Stevenson A. et al., 2003. The disadvantage of bacterial vectors obtained is the lack of FHA activity inactivated due to the construction of the protein fused with heterologic antigen.
We constructed B.pertussis strain containing mutant ptx and dnt genes. The bacteria of recombinant B.pertussis 35 strain produced immunologically active PT lacking the toxic activity dermonecrotic (thermolabile) toxin. Gene dnt inactivation was achieved as a result of insertion of cad gene coding for chloramphenicol resistance. The elimination of PT activity was achieved by mutations in ptx gene sequence which leaded to amino acid’s replacements Arg9-Lys9, Glu129-Gly129. Moreover, B.pertussis 35 chromosome contains genes kan and cad insertions. Integrated markers will be used for monitoring of the term of persistence and stability of recombinant strains bacterial genome structure in laboratory animals during different periods of time after vaccination. It is planned to construct bacterial strain containing more than two copies of fgaB gene. The presence of two expressing copies of fgaB gene in attenuated bacteria B.pertussis chromosome will produce augmentation of protective qualities of vaccine strain and will keep adhesive activity of bacterial vector in which FHA is used for heterologic antigens expression and expansion on the surface of recombinant B.pertussis cells.
Increasing of fgaB gene copies will be provided by constracted B.pertussis mobile genetic elements (Kirillov M.Y. et al., 1995, Necaeva E.V., 2000; Shumakov Y.L., 1993; Vorontsov V.V. et al., 2004).
Authors of this project have great experience in studding pertussis pathogenesis, bacterial mobile genetic elements, development and control of vaccine preparations for pertussis prevention and diagnostic systems for pertussis identification, and pertussis epidemiology.
The aim of studing.
Constraction of attenuated B.pertussis strain, containing no less than two copies of fgaB gene sequence coding FHA-the main pertussis adhesive protein.
Schemes of studding:
- Jmmunobiological characteristics of attenuated B.pertussis 345 strain producing genetically modified PT and lacking of DNT activity.
- fga B sequence integration in B.pertussis genetically marked mobile genetic elements structure (TnBP and RSBP) which were inserted into pKnock vector (pCVD 442) not capable of replication in B.pertussis.
- Elaboration of primers and PCR conditions selection for identification of recombination accompanied by fga B gene’s sequence amplification in attenuated B.pertussis 35 strain chromosome.
- Construction and characterization of chromosomal structure of recombinant attenuated B.pertussis 35 strain containing amplificated fga B gene.
- Comparative immunobiological characteriszation of attenuated strains B.pertussis 35 containing one or several copies of fgaB sequence in the chromosomes.
Planning investigations are high priority. For increasing of fgaB gene copy number the genetically marked variants of TnBP and RSBP, constructed and studied in our laboratory, will be used.
Development on the base of attenuated B.pertussis strain live vaccine may be used for pertussis revaccination of eldest children and adults. Mutant bacteria B.pertussis containing several fgaB gene’s copies will be vectors for construction of polyvalent live intranasal vaccines. The method of choice genes amplification in Bordetella chromosome can be used for polyvalent vaccine B.bronchiseptica strains constructing for the prevention of infection diseases in domestic animals.
Expected Results and Their Application
- The dater of immunobiological characterization of attenuated strain containing mutants in ptx and dnt genes.
- Attenuated bacterial strain, containing several fgaB gene copies in B.pertussis chromosome.
- The method for choice genes amplification in Bordetella chromosome.
- New information about B.pertussis mobile genetic elements.
- The dater of immunobiological characterization of attenuated bacterial strain containing several fgaB gene copies in B.pertussis chromosome.
The project will provide Russian researchers engaged previously in studies in the area of defense, to reorient the circle of their scientific interests and to be involved in the current project with the possibility to gain more experience in performing fundamental, as well as applied studies, within the framework of international science and technology programs for biology and medicine. The proposal also will allow the Gamaleya Institute researchers to continue to build on the collaboration already established with Canadian scientists from work on ISTC project 2927 as part of the framework for integration into the international scientific community.
The work is planed for 3 years. During first six quarters the characteristics of attenuated B.pertussis 35 strain producing genetically modified PT and devoid of DNT activity will be studied (Task 1). At the same time, the tasks 2-5, which are intended to be completed in 10 quarter will be solved. In 11-12th quarter it is intended to work through methods of antibiotic stability markers inactivation in attenuated strain chromosome. Intermediate variants of attenuated B.pertussis strain containing more than fgaB gene copy, or variants, containing more than two copies of fgaB gene, depending on results after 2-5 tasks realization will be used.
The project is complementary to interests Vaccine & Infectious Disease Organization, Canada developing the first large animal model for pertussis infection in swine. VIDO has an interest and expertise in the study of diseases of humans as well as animals and the development of vaccines using a variety of technologies.
Dr. Andrew Potter, associate Research Director of Vaccine & Infectious Disease Organization as a collaborator will provide technical advice and work with the Russian scientists in the generation, gathering and analysis of scientific data. He will monitor the project and review progress reports
In order to carry out the project, we are planning to use various combinations of standard techniques in molecular biology, biochemistry, microbiology, including culture methods for bacteria isolation, PCR analysis, nucleic acid sequencing, and computer analysis based on the data from sequencing of the PCR products
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