Cabana Puig, Xavier

Universitat Autònoma de Barcelona. Facultat de Biociències

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4. Expected results • Vesicles, CTB-modified and LPS-modified, were produced correctly and no problems showed up. • Results showed high levels of antibodies Anti-LPS O1 (Inaba and Ogawa), anti-LPS O139, anti-capsular O139 and anti-CTB. • All immunization groups induced a robust and long immune response with similar levels of Ig titers against both serogroups. • The protective efficacy of anti-LPS antibodies is mediated either through vibriocidal activity or inhibition of adhesion to the epithelial surface. • Only one dose was required  and the protection was always 97% in mice. • The vaccine shelf life was 5 years and storage temperature should be between 2-8°C. But the antigenicity of OMVs is not significantly compromised after prolonged heating, the viability was 60 days.7 • Price was unclear, but when large scale production takes place, prices will be cheaper than the other prequalified vaccines.   5. Benefits of the project We need cholera vaccine because cholera is endemic in developing countries. Although the provision and establishment of safe water, food, and sanitation systems are the principal way to prevent cholera, these measures cannot be implemented in the near future in most areas where there is cholera. It’s known that V. cholerae shows increased resistance to antibiotics, complicating clinical management. These concerns and the improved protection of new-generation oral cholera vaccines have an importance in order to reduce cholera cases, alongside traditional cholera prevention measures. 6. Work diffusion Regardless of whether the applicant group intends to publish the results in more impact journals in endemic areas, it's expected that the above results could be useful for professional sectors companies or users.  3. Methodology and work plan                                      The principal objective is develop a new oral cholera vaccine, which would gives more protection to prevent the disease of cholera, inducing strong immune response, than  prequalification oral vaccines.  1. Antecedents  Cholera is an acute secretory diarrhoea disease caused by intestinal infection with Vibrio cholerae. Its estimated cholera affects 3–5 million people with 100,000 dead each year, around the world.1 Infection is usually asymptomatic or mild, but sometimes can be severe. Approximately 5% of infected people develop severe cholera and without treatment, over 50% of people can die in a few hours. Cholera has probably existed on the Indian subcontinent for thousands of years. From 1817 cholera has caused seven important pandemics around the world, with thousands deads.2 The transmission happens when food and water are contaminated through the faeces of infect people. Complications of cholera are causatives by the loss of fluids and electrolytes. Cholera’s treatment consists only of quickly oral fluid replacement.  But as it happened in the past, cholera remains largely a disease of impoverishment, where sanitation water is inappropriate or absent, social unrest and displacement, and continues to be a disease of major public health concern. VACCINES FOR PREVENTING CHOLERA Xavier Cabana Puig Grau en Microbiologia, Universitat Autònoma de Barcelona    1. Harris J.B., LaRocque R.C., et al. 2012. Cholera. Lancet, 379:2466–76.  2. Plotkin S.A., Orestein W.A., Offit P.A. Vaccines. 6th Ed. Philadelphia: Saunders, 2013. Chapter 11. Pag. 141-152.  3. Madigan M.T., Martinko J.M., Dunlap P.V., Clark D.P. Brock: Biología de los microorganismes. 12th Ed. Madrid: Pearson, 2009. Chapter 9. Pag. 1152-1154.  4. WHO. 2010. The Immunological Basis for Immunization Series. Module 14: Cholera.  5. WHO. 2010. Oral cholera vaccines in mass immunization campaigns; guidance for planning and use.  6. WHO. 2009. Duncan Steele Vaccines and Immunization PATH, Seattle. Update on Cholera Vaccines.   7. Leitner D.R., Feicher S., et al. 2013. Lipopolysaccharide-modifications of a cholera vaccine candidate based on outer membrane vesicles reduce the endotoxicity and reveal the major protective antigen. Infect. Immun. doi:10.1128/IAI.01382-12. 8. Martínez E., Silvera R., et al. 2005. Cepas vivas atenuadas de Vibrio cholerae con características mejoradas de seguridad ambiental. Revista CENIC Ciencias Biológicas.36(3):147-145.  9. Kuehn M.J., Kesty N.C. 2005. Bacterial outer membrane vesicles and the host-pathogen interaction. Genes. Dev. 19:2645-2655.   V. cholerae is Gram-negative proteobacteria, comma-shaped, facultative anaerobic, with a single polar flagellum. V. cholerae have two types or serogroups where only two, are O1 and O139, can be epidemics, because they have the ability to produce choleric toxins.  Genome of V. cholerae has two pathogenic islands, where the genome has been lysogenized with a filamentous phage. It contains regulatory and virulence factors, such as ctxA and ctxB (cholera toxin genes), that help  the pathogen colonization and toxin expression (figure 1).3 Figure 1: The action of cholera enterotoxin. Cholera toxin is a heat-stable AB enterotoxin that activates a second messenger pathway, disrupting normal ion flow in the intestine.2 Currently, there are three prequalification oral vaccines for the World Health Organization (WHO), described in table 1. Duration of protection is the first problem we can see, it’s less than two years long and less than 90% of protection. In addition there are other troubles like storage, price, shelf life and presentation where is required an intervention. Table 1: Comparison of killed WC-rCTB vaccine, live CVD-HgR vaccine and reformulated WC-only vaccine.2,5,6  Figure 2: Schematic diagram showing gut defences and serum immune responses against V. cholerae infection.4  Lipopolysaccharides and cholera toxin are virulence factors, which are important for the immune response, concretely humoral immunity (figure 2). Cholera toxin induces sIgA, which neutralizes pathogens of intraluminal mucus. LPS induce IgA and IgM, which plays an important role in affinity maturation IgG, important for the development of memory B cells. A new oral recombinant vaccine was synthesized by outer membrane vesicles derived from V.  Cholerae. The idea is to use a method that can present LPS-modified with CTB-modified and it gives protection against both clinically relevant serogroups. These vesicles consist of outer membrane lipids, a subset of outer membrane proteins and soluble periplasmic components (figure 4).7 Control, checking live-cell contamination Vesicles were  resuspended in buffer,  filtered and mixed Final vaccine 5. Centrifugation and filtration of the supernatant. • pCTB  ctb-modified + β-lactomase gene + KmR • pLPS  msbB-modified + TetR • pCTB  ctb-Modified + β-lactomase gene derived + KmR • pLPS  msbB-Modified + TetR  • pCAP  otnA-Modified + CmR   Serogroup 01 Biotype Classical    Serogroup 01 Biotype El Torn   Serogroup 0139  E. coli E. coli E. coli Figure 4: Model of vesicle biogenesis. OM vesicles are proteoliposomes consisting of OM phospholipids and LPS, a subset of OM proteins, and periplasmic proteins.8 4.High Tº selection was applied to obtain Amps colonies and correct resistant deletions, by recombinase, were confirmed by PCR. 2. Objective 6. Work iff i  5. fits of the project . Expected results Characteristic WC-rCTB CVD103-HgR Reformulated WC-only Commercial name Dukoral® Orochol® or Mutachol® Vietnam: mORCVAX® India: Shanchol® Vaccine type Killed whole cells of V. cholerae O1 plus CTB recombinant  Live attenuated V. cholerae. Killed whole cells of V. cholerae O1. Vaccine composition Inaba Cairo 48 Classical and Ogawa Cairo 50 Classical (heat-killed), Inaba Phil 6973 El Tor and Ogawa Cairo 50 Classical (formalin killed) Inaba Classical strain 596B Ogawa Cairo 48/50 Classical (heater-killed). Inaba Phil6973 El Tor, Ogawa Cairo 50 Classical and 4260B O139 (formalin killed) Administration Route Oral Oral  Oral Formulation Liquid Lyophilized Liquid Regimen 2 doses  1 dose 2 doses  Age range for vaccination ≥ 2 years ≥ 2 years Vietnam: ≥ 2 years India: ≥ 1 year Duration of protection 2 years  ≥ 6 months  At least 3 years Buffer requirement  Yes  Yes  No Storage temperature 2° to 8° C 2° to 8° C 2° to 8° C Shelf life 3 years  2 years 2 years International acceptance WHO-prequalified Was WHO-prequalified until 2004  WHO-prequalified Price to the public sector per dose ~$5.25/dose for small quantity of 250.000 doses Vaccine not currently available Vietnam: ~$0.75/dose India: $1.85 per dose Protection 1st/2nd year 80%/60% 91% only six months 69%/50%  7. Bibliography 3. Conjugation and homologous recombination. 1. To construct the thermosensitive suicide plasmids pCTB/pLPS/pCAP, upstream and downstream fragments of the gene were PCR-amplified using the oligonucleotide pairs digested with the appropriate restriction enzyme, and finally ligated into the digested pCTB/pLPS/pCAP (figure3). Deletion mutants were generated using derivatives of the suicide vector pCTB/pLPS7pCAP.7 Plasmids are carrying AmpR and recombinase. 2. Obtaining our plasmids and confirmation by PCR. Figure 3: Schematic figure of one study that have shown the deletion of mshA by PCR and construction of suicide vector pCVDmshA.8 Abbreviations: pCTB: Modified ctxB gene fused to the type 2 secretion signal sequence (derived from the β-lactamase gene). pLPS: Truncated msbB (lacks a typical lipid A structure). pCAP: Mutant otnA (alteration transporter of capsule precursors).  

Vaccines for preventing cholera

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Vaccines for preventing cholera

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