A thermostable, dry formulation inactivated Hikojima whole cell/cholera toxin B subunit oral cholera vaccine.

The feared diarrheal disease cholera remains an important global health problem. Use of oral cholera vaccine (OCV) from a global stockpile against both epidemic and endemic cholera is a cornerstone in the World Health Organisations (WHOs) global program for "Ending cholera by 2030". Three liquid inactivated whole-cell OCVs (Dukoral®, ShancholTM, and Euvichol-Plus®) are WHO prequalified and have proved to be safe and effective. However, their multicomponent composition and cold-chain requirement increase manufacturing, storage and transport costs. ShancholTM and Euvichol-Plus® OCVs used in WHOs global vaccine stockpile also lack the protective cholera toxin B-subunit (CTB) antigen present in Dukoral®, which results in suboptimal efficacy. WHOs Global Task Force on Cholera Control (GTFCC) has identified a thermostable, dry formulation vaccine as a priority for further OCV development. We describe here the development of such a vaccine, based on a lyophilized mixture of a single strain of formalin-killed Hikojima bacteria together with a low-cost, recombinantly produced CTB. The new vaccine, which is easy and inexpensive to manufacture, could be stored for at least 26 months at 25 °C and for at least 8 months at 40 °C with preservation of cell morphology and with no loss of protective Ogawa and Inaba lipopolysaccharides or CTB. It also proved to be well tolerated and to have equivalent oral immunogenicity in mice as ShancholTM and Dukoral® OCVs with regard to both serum and intestinal-mucosal antibody responses.

A phase I/II study to evaluate safety, tolerability and immunogenicity of Hillchol®, an inactivated single Hikojima strain based oral cholera vaccine, in a sequentially age descending population in Bangladesh.

BACKGROUND: The World Health Organization (WHO) recommends the use of oral cholera vaccines (OCVs) as part of an integrated control program, both in highly endemic settings and during cholera epidemics. The available and internationally recommended WHO-prequalified OCVs (Dukoral, Shanchol, Euvichol) contain multiple heat and formalin-killed V. cholerae strains of Inaba and Ogawa serotypes. MSD Wellcome Trust Hilleman Laboratories Pvt. Ltd. in technical collaboration with University of Gothenburg, Sweden has developed a new single strain OCV, Hillchol. This vaccine consists of formaldehyde-inactivated whole cell El Tor V. cholerae O1 bacteria engineered into the Hikojima serotype for stable expression of both the Ogawa (AB) and Inaba (AC) LPS antigens on the bacterial surface. We evaluated the safety and immunogenicity of this novel and potentially much less expensive OCV in comparison with Shanchol. METHODS: We conducted a randomized, non-inferiority, age-descending clinical trial of OCV (Hillchol vs. Shanchol) in the Mirpur area of Dhaka city from July 2016 to May 2017. This study was carried out in three different age cohorts (1-<5, 5-17 and ≥18 years old). Two doses of vaccine were given at 14 days intervals to 560 healthy participants. FINDINGS: No serious adverse events were reported. There were no significant differences in the rates of adverse events between the test vaccine (Hillchol) and the comparator (Shanchol) group. Serum vibriocidal antibody responses in all age groups combined were comparable for all the O1 Ogawa (59% vs. 67%; 90% CI of difference: -14.55, -0.84) and Inaba (70% vs. 71%; 90% CI of difference: -7.24, 5.77) serotypes, showing that the Hillchol vaccine was non-inferior to Shanchol. This new vaccine was also non-inferior to Shanchol in the different age strata. CONCLUSION: The safety and immunogenicity profile of the new OCV Hillchol is comparable to Shanchol in persons residing in a cholera-endemic setting. ClinicalTrials.gov number: NCT02823899.

Development of Hillchol®, a low-cost inactivated single strain Hikojima oral cholera vaccine.

Cholera remains an important global health problem with up to 4 million cases and 140,000 deaths annually. Oral cholera vaccines (OCVs) are now a cornerstone of the WHOs "Ending Cholera - A Global Roadmap to 2030" global program for the eventual elimination of cholera. There are currently three WHO prequalified OCVs available, Dukoral®, Shanchol® and Euvichol-Plus®. These vaccines are effective but due to a multiple strain composition and two different methods of inactivation, are complex and costly to manufacture. We describe here the characterization and industrial scale development of Hillchol®; a novel, likely affordable single-component OCV for low and middle-income countries. Hillchol® consists of formalin-inactivated bacteria of a stable recombinant Vibrio cholerae O1 El Tor Hikojima serotype strain expressing approximately 50% each of Ogawa and Inaba O1 LPS antigens. The novel OCV can be manufactured on an industrial scale at a low cost. Hillchol® was well tolerated in animal toxicology studies and shown to have non-inferior oral immunogenicity in mice for both intestinal-mucosal and serological immune responses when compared with a WHO-prequalified OCV. The optimized production of this single component OCV will reduce cost of OCV production and thus substantially increase vaccine availability. Based on these results, Hillchol® has been produced at a GMP facility and used successfully for clinical phase I/II studies.

Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

Preclinical immunogenicity and protective efficacy of an oral Helicobacter pylori inactivated whole cell vaccine and multiple mutant cholera toxin: A novel and non-toxic mucosal adjuvant.

Mucosal vaccines against Helicobacter pylori consisting of either whole cell bacteria or recombinant antigens can induce immune protection against challenge in mice only when co-administrated with a strong mucosal adjuvant such as cholera toxin (CT) or Escherichia coli heat labile enterotoxin (LT). The strong enterotoxicity of these adjuvants however preclude their use in human vaccines. The recently developed multiple mutant CT (mmCT) is a strong, yet practically non-toxic novel mucosal adjuvant which here was admixed with a formalin-inactivated H. pylori whole cell vaccine (WCV) as a potential vaccine candidate against H. pylori infection. We report that intragastric immunizations with H. pylori WCV together with mmCT, similar to immunization with WCV together with CT, resulted in 50-125-fold reduction in colonization of H. pylori in the stomach of mice associated with rises in both serum IgG and intestinal-mucosal IgA anti-H. pylori antibody responses and strong T cell and IFNγ and IL-17A cytokine responses. Data presented in this study also supports that the proposed vaccine can be grown in a bioreactor and would be effective against infection caused by a multitude of pathogenic H. pylori strains isolated from patients from various continents. The results warrant immunization studies in humans to evaluate the safety, immunogenicity and efficacy of the proposed H. pylori WCV and mmCT.

Publisher Correction: Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC).

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae.

Antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of the lgt gene encoding a (pro)lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in Escherichia coli and other Gram-negative organisms. The lgt gene was deleted from E. coli and complemented by the Vibrio cholerae-derived gene provided in trans on a temperature-sensitive plasmid, allowing cells to grow at 30°C but not at 37°C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed, whereby transformants were selected by growth at 39°C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting the lgt gene from V. cholerae and complementing the lesion with the corresponding gene from E. coli The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, the lgt system described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using the E. coli-derived lgt gene for complementation.IMPORTANCE Many recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues.

Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC).

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Although 22 antigenically different colonization factors (CFs) have been identified and characterized in ETEC at least 30% of clinical ETEC isolates lack known CFs. Ninety-four whole genome sequenced "CF negative" isolates were searched for novel CFs using a reverse genetics approach followed by phenotypic analyses. We identified a novel CF, CS30, encoded by a set of seven genes, csmA-G, related to the human CF operon CS18 and the porcine CF operon 987P (F6). CS30 was shown to be thermo-regulated, expressed at 37 °C, but not at 20 °C, by SDS-page and mass spectrometry analyses as well as electron microscopy imaging. Bacteria expressing CS30 were also shown to bind to differentiated human intestinal Caco-2 cells. The genes encoding CS30 were located on a plasmid (E873p3) together with the genes encoding LT and STp. PCR screening of ETEC isolates revealed that 8.6% (n = 13) of "CF negative" (n = 152) and 19.4% (n = 13) of "CF negative" LT + STp (n = 67) expressing isolates analyzed harbored CS30. Hence, we conclude that CS30 is common among "CF negative" LT + STp isolates and is associated with ETEC that cause diarrhea.