Development of DuoChol, a Thermostable Inactivated Whole-Cell/B-Subunit Oral Cholera Vaccine in Enteric Capsule

Abstract

Background/Objectives: Cholera remains an important global health problem. Inactivated oral cholera vaccines (OCVs) are essential in the WHO/GTFCC (World Health Organization/Global Task Force on Cholera Control) strategy to end cholera by 2030; however, global supply is insufficient, they require partial cold-chain storage, and their formulation and antigen contents leave room for improvement. We describe here the development and preclinical evaluation of DuoChol OCV, a next-generation thermostable oral vaccine designed to address these gaps.

Methods: DuoChol is a lyophilized dry-powder formulation in enteric capsules containing formalin-inactivated Vibrio cholerae O1 El Tor Ogawa and Inaba isogenic bacteria, recombinant cholera toxin B subunit (rCTB), and sucrose as stabilizer. Methods describe the construction of the novel vaccine strains, processes for the preparation and characterization of vaccine components, and the final dry formulation in enteric capsules, and in vitro and in vivo vaccine stability analyses.

Results: The newly engineered vaccine strains, together with a high-yield mixed-mode chromatography process for rCTB purification, enabled efficient and cost-effective vaccine production. Stability studies demonstrated complete preservation of O1 LPS and rCTB antigens for at least 21 months across temperatures of 4–40 °C. Moreover, regardless of storage duration or temperature, oral immunization of mice with DuoChol elicited strong serum and mucosal antibacterial and antitoxin responses that were similar to those induced by the licensed Dukoral^® OCV.

Conclusions: Its heat stability, practical enteric capsule formulation, and potential for improved efficacy compared to inactivated whole-cell only OCVs support positioning DuoChol as a promising next-generation OCV, suitable for national cholera control programs and particularly advantageous for outbreak response, where rapid deployment and early, robust protection are essential.

Introduction

Cholera remains a major global health threat, particularly in regions lacking safe water and sanitation. Infection may follow ingestion of food or water contaminated with Vibrio cholerae and cause acute watery diarrhea that can rapidly lead to dehydration and death without treatment. Nearly all cases in the past two decades have been caused by V. cholerae O1, serotypes Inaba and Ogawa, of the El Tor biotype. Despite being preventable and treatable, cholera causes an estimated 4 million cases and 140,000 deaths annually [1,2,3]. Since 2022, large epidemics across the Middle East, Sub-Saharan Africa, and Southeast Asia, driven by climate change, extreme weather, conflicts, and displacement prompted the WHO to declare cholera a Grade 3 emergency [4,5,6,7,8,9,10,11].

While cholera control traditionally relies on efforts to improve water, sanitation, and hygiene (WASH) conditions, the limitations in infrastructure in many endemic regions have made oral cholera vaccines (OCVs) an essential additional tool. Vaccine protection depends on mucosal immunity, particularly SIgA responses to lipopolysaccharide (LPS) O antigen and cholera toxin (CT) [12,13,14,15,16,17]. Early parenteral vaccines gave only modest and short-lived protection and were reactogenic, leading to their replacement by OCVs that provide multi-year protection and confer additional herd effects [18,19,20,21,22,23]. WHO currently recommends several multicomponent, inactivated whole-cell OCVs—Dukoral®, Shanchol™, and Euvichol-Plus™—for both preventive use and outbreak response. Since 2013, more than 250 million doses have been deployed through the global OCV stockpile, though demand continues to exceed supply, which was worsened by the discontinuation of Shanchol™ in 2023.

OCVs have well-documented preventive impact, with meta-analyses showing two-dose efficacy of 55% and effectiveness of 69% at 12 months, declining to 44% and 47% by 48 months [24,25,26,27,28]. Supply constraints have recently forced single-dose campaigns, raising concerns about reduced protection, especially in non-endemic settings, among young children, and high-transmission outbreaks [29,30,31,32]. New, simplified, and cheaper to produce OCVs aim to address these gaps. Euvichol-S™, a two-component formulation comprising formalin-killed classical biotype Ogawa and El Tor biotype Inaba bacteria, and Hillchol™, a single-component Hikojima-based vaccine comprising formalin-killed El Tor bacteria co-expressing the Inaba and Ogawa serotype antigens, were recently licensed [33,34].

Further improvements in formulation and antigen composition may increase both the protective impact and practicality of OCV. Current OCVs require cold-chain storage, and all except Dukoral® lack the CTB-subunit protein (CTB), which enhances short-term protection against cholera and provides cross-protection against enterotoxigenic E. coli diarrhea [35,36] but was omitted from subsequent OCVs due to cost and the requirement for co-administration with a buffer. We recently demonstrated that a lyophilized formulation containing formalin-killed V. cholerae O1 bacteria, recombinant CTB (rCTB), and sucrose was stable for over two years at 25 °C and for at least eight months at 40 °C [37].

Building on this, as described in this study, we have developed DuoChol, a dry-powder OCV in an enteric capsule containing lyophilized formalin-inactivated, newly generated isogenic V. cholerae O1 Ogawa and Inaba strains together with rCTB and sucrose as a stabilizer. Stability studies show preserved O1 LPS and rCTB antigens for at least 21 months at 4–40 °C as well as robust systemic and mucosal immunogenicity in mice, similar to that of side-by-side tested Dukoral OCV, after oral immunization.

2.7. In Vitro Stability Assays

Freshly prepared prototype DP vaccine capsules received from RISE, as well as lyophilized powder from the same lot, were analyzed (time 0, “t0”) as described below. Remaining capsules were distributed in airtight glass bottles with desiccating powder for storage at 4(±2) °C, 25(±2) °C, or 40(±2) °C; the reference powder was stored in the same way at 4(±2) °C. At 2 months (“t2”), 4 months (“t4”), 6 months (“t6”), and finally at 21 months (“t21”), three random capsules from each storage temperature were analyzed together with the reference powder stored at 4 °C.

Capsules were inspected to be intact, whereafter they were cut open, and the powder content was carefully weighed and visually inspected for any change in appearance and placed in a plastic vial. Milli-Q water was added to give a powder content concentration of 100 mg/mL. After careful mixing, the material was then analyzed side-by-side with the reference powder suspension for: (i) Optical density (OD600nm) [37]; (ii) O1 LPS antigen content by inhibition-ELISA [43]; (iii) rCTB antigen content by GM1-ELISA [37,42]; and (iv) Bacterial morphology and aggregation by light microscopy with the analytic methods described below.

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2. Materials and Methods

2.6. Lyophilization and Capsule Filling

A mixture of formalin-inactivated whole-cell components, purified rCTB, and sucrose was lyophilized for subsequent preparation of freeze-dried powder to be used for filling in capsules (see Figure 1, steps 3–6). These steps were performed at RISE (Research Institute of Sweden, Unit of Drug formulation, Södertälje). The freeze-drying (FD) procedure, earlier described [37], was used with the small modifications of using a higher concentration of sucrose (60 mg/mL) and scaling up to provide sufficient dried vaccine powder for filling 150–200 capsules. The liquid mixture used for freeze-drying contained per ml the formalin-inactivated Inaba and Ogawa components in an amount providing 0.75 mg O1 LPS antigen in each, 1 mg rCTB, 75 mg sucrose, and 5 mg buffer salts. With no loss in the lyophilization and further steps, this was calculated to provide ca 150 mg dry powder per ml liquid mixture, with the bacterial components accounting for a dry weight of 35 mg each. The inactivated bacterial suspension bulks were carefully shaken to provide an even suspension for the mixing, and the mixture was then again shaken vigorously and poured into a circular lyophilization tray with a 23 cm diameter, so that a volume of 200 mL would give a fill height of 0.48 cm. The tray was placed on the shelf of an Epsilon 2–4 LSCplus (Martin Christ GmbH, Osterode am Harz, Germany) freeze-dryer. The material was frozen at −45 °C for approximately 4 h and then subjected to a first drying cycle for 36 h at 0.0200 mbar at −30 °C, which was followed by warming the material to +20 °C and a second drying cycle for 1 h at +20 °C at 0.005 mbar.

The lyophilized “cake” was crushed and treated with a pestle and mortar to generate a free-flowing powder. The powder was transferred to a bottle that was filled with nitrogen gas to prevent moisture uptake. The whole procedure was conducted in a glove box with controlled low relative humidity. The powder was used to fill 150 size 1 enteric capsules (Capsugel® Enprotect ®, Lonza, Basel, Switzerland). Capsules were packaged in an airtight flask together with desiccating powder; the remaining powder was stored in the presence of desiccating powder at 4 °C for reference purposes.

A prototype lot of such DuoChol vaccine capsules (“Drug Product”, DP) was manufactured at RISE using the Inaba DS, Ogawa DS, and rCTB DS; the residual moisture in the final powder used for capsule filling was 0.9% (w/w). These capsules were sent to us and used for further characterization, in vitro stability, and in vivo immunogenicity testing as described below.

Terrinoni, M.; Lebens, M.R.; Nordqvist, S.L.; Nilsson, F.; Löfstrand, M.; Lynch, J.; Holmgren, J. Development of DuoChol, a Thermostable Inactivated Whole-Cell/B-Subunit Oral Cholera Vaccine in Enteric Capsule. Vaccines 2026, 14, 573. https://doi.org/10.3390/vaccines14070573

Read also our introduction article on Capsules here: