Cold-Chain Free, Dual Vaccine for bottom billion CHILDREN

Need for a cost-effective, dual protection, cold chain free, live vaccine to protect the bottom billion children from Cholera and ETEC, the diarrheal diseases :

Diarrheal disease is the second infectious killer disease after respiratory diseases of children under the age of five years worldwide. Almost half of all diarrheal infections are predominantly caused by two major versatile enteric bacterial pathogens Vibrio cholerae and Enterotoxigenic Escherichia coli (ETEC), the causative agents of acute diarrheal disease cholera and travelers’ diarrhea respectively. Cholera’s impact is greatest in many low- and middle-income countries (LMICs). Each year 1.3 to 4.0 million cases of cholera and 21 000 to 143 000 deaths occur globally due to cholera. It is estimated 220 million cases of ETEC diarrhoea globally, with about 75 million episodes in children less than 5 years of age, resulting in between 18 700 to 42 000 deaths (WHO, 2022). ETEC is often the first bacterial illness that children experience in endemic areas during their first three years of life which adversely affects a child's physical and cognitive development. Indeed, nearly one out of every six travelers to endemic regions contract ETEC within 3 days of traveling to underdeveloped countries. Distinctly, most diarrheal patients are concurrently infected with V. cholerae and ETEC resulting in severe diarrhea. The pathogenesis of both pathogens is quite similar. The cholera toxin (CT) produced by V. cholerae O1 and the heat-labile enterotoxin (LT) of ETEC is structurally, functionally, immunologically identical and both have similar type II secretion systems for their toxins.

Cold chain free vaccine would relieve the bottlenecks and cost determinants in mass vaccination campaigns :

Vaccination in combination with water and sanitation is the most effective approach to control these diarrheal infections. Operational challenges exist for the implementation of the cholera vaccination campaign in developing countries with the existing WHO-licensed oral cholera vaccines (Dukoral®, Shanchol™, and Euvichol®), which are based on whole-cell, killed V.cholerae O1 and O139 serogroups. Killed vaccines confer short-term protection and require a booster dose as opposed to single-dose live vaccine administration. Only Dukoral with its rCTB component affords short-term cross-protection against ETEC infection. Recently, FDA approved a live cholera vaccine Vaxchora for the travelers' diarrhea market.

Although all of these vaccines are safe, they demand a cold chain supply (2-8ºC) distribution system from manufacturing to the immunization site to ensure their safety and potency. The cold-chain logistics are difficult to execute in resource-poor countries. Hence, maintaining a cold chain for two-dose schedules results in a high cost of vaccination, which poses a great challenge. Worldwide, several live attenuated vaccines against O1 and O139 are in various stages of development and evaluation. However, similar to killed cholera vaccines, live vaccine formulations are also heat-sensitive and cold chain supply-dependent. Therefore, a cold chain-free, live, attenuated cholera vaccine that can be stored at room temperature must be developed to increase its outreach to global immunization programs in LMICs.Eventually, the cold chain free formulation would reduce a) the wastage of vaccine from heat and freeze-shocks (estimated at ~ US$ 15-40 million/year across GAVI countries), b) the capital cost for cold chain equipment (estimated at ~ US$ 110 million/year) and c) decreasing the cost of stockpiling and eventually reducing the total operating costs of US$ 125-150 million / year across GAVI countries. 

The combined vaccines will be on the market near future. The existing licensed combination vaccines for children in the market are 1) Pediarix for 5 diseases (diphtheria, tetanus, pertussis, hepatitis B, and polio), 2) ProQuad for 4 diseases (measles, mumps, rubella, and varicella), 3) Kinrix for 4 diseases (Diphtheria, tetanus, pertussis, and polio) and 4) Pentacel for 5 diseases (Diphtheria, tetanus, pertussis, polio, and Hib (Haemophilus influenzae type b)). And, a strong rationale exists for dual-use /combination vaccines such as a) fewer administration and better vaccine coverage in a short time and b) higher rate of compliance with complex vaccination schedules with reduced administration cost augmented with lower storage space requirements. In this direction, dual-use/ combination of vaccines are in pipeline for the Diarrheal diseases: Cholera & ETEC, the 2nd killer disease of children below 5 years of age in Sub-Saharan African countries, Asia, and in many low- and middle-income countries (LMICs).

Development of a Prototype Cold Chain Free, Live, Dual protection vaccine against Cholera and ETEC at Technology Readiness Levels (TRL) 5 :

We at AIMST University, have developed a live, dual-protection vaccine-MyChol TM for cholera and traveler’s diarrhea. It is a live vaccine, which mimics a natural infection, is non-reactogenic, strongly immunogenic in vivo, and protects animals from a lethal wild-type challenge in extensive preclinical studies. It is a single-dose vaccine, stable and retains its potency when stored at room temperature (25°C ± 2°C) for 1 year and costs less and it would represent a great opportunity to increase its outreach for the global immunization program at a competitive cost to reinforce the Water, Sanitation and Hygiene (WASH) services supported by WHO. This prototype cold-chain free, dual-use vaccine is a first of its kind and a patent has been filed. The vaccine has already advanced to Technology Readiness Level (TRL) 5 and currently, the OECD GLP - Compliant Safety studies of the vaccine are in progress at the Malaysian Institute of Pharmaceuticals and Nutraceuticals (IPharm). The research work is being supported by the Prototype Research Grant Scheme (PRGS) and Fundamental Research Grant Scheme (FRGS), Ministry of Education (MOE), Malaysia. 

Process Validation and Technology Licensing to support the clinical development of the vaccine with a potential collaborator :

Currently, there is no exclusive ETEC vaccine; however, it can be cross-protected by a cholera vaccine, Dukoral®. Dukoral with its rCTB component affords short-term cross-protection against ETEC infection. Unfortunately, this vaccine demands a “cold chain” supply (2-8°C) in addition to multiple doses which poses a great challenge. Our cholera vaccine strain (VCUSM14P) has 2 copies of rCTB.  Hence, we have evaluated and established the immunogenicity of the prototype cholera vaccine in animal models challenged with ETEC strains. Hence it serves as a dual-use vaccine against cholera and ETEC. 

To further validate the cold chain-free, live, dual-use vaccine production process and efficacy in humans, and move towards commercialization, we would like to carry out the following. 

(a) To evaluate the protective efficacy of the vaccine in immunized animal models (mice and rabbits) challenged with ETEC strains (E. coli ST 64111, E. coli 286C2, E. coli 195, E. coli VM 75688, and E. coli E34420C) at an ICH GLP – Compliant lab. 

b) To determine the NOAEL, MRSD (maximum recommended starting dose), and HED (Human Equivalent Dose) of the dual-protection vaccine against cholera and ETEC. 

c) To compile the Preclinical Dossier to license the Process Know-How to support the clinical development. 

d) To conduct the Phase 1 Clinical trial of a dual-protective vaccine against cholera and ETEC, the diarrheal diseases.

The burden of Cholera & ETEC Diarrheal diseases:

The diarrheal disease is endemic and caused epidemics with nearly 1.7 billion cases across the globe with increasing morbidity and mortality rates every year. ETEC serves as the primary pathogen followed by V.cholerae. The diarrheal patients are concomitantly infected with V. cholerae O1 and ETEC and have severe diarrhea compared to infection with a single pathogen. ETEC is often the first bacterial illness that children experience in endemic areas during their first three years of life and resulting in stunted growth, and impaired cognitive development, leading to diminished productivity over a lifetime for millions of bottom billion people. ETEC is also the leading cause of diarrhea among travelers to the developing world. The current estimates among American and European travelers project nearly $300 million in medical costs due to travelers’ diarrhea. Indeed, nearly one out of every six travelers’ to endemic regions contract ETEC. The cholera vaccines market was valued at USD 57.39 million in 2016 and is expected to grow at a CAGR of 8.2% over the forecast period to reach USD 117.09 million by 2025. Global demand for ETEC vaccines is estimated to reach 118 million doses per year, corresponding to $600 million in revenues, 12 years post-launch. 

ETEC Vaccine landscape :

Dukoral is a killed cholera vaccine and its rCTB component affords short-term cross-protection against ETEC infection. Recently, FDA approved a live cholera vaccine Vaxchora for the travelers' diarrhea market. Similar to Dukoral, a) our live attenuated vaccine formulation contains rCTB component to induce antibodies to inactivate/neutralize LT toxin of ETEC, b) in contrast to Dukoral, our attenuated strain contains 2 copies of CTB hence it is overexpressed, c) in contrast to Dukoral, our formulation offers long-term protection due to its colonization efficiency in the intestine (serves as a constant reservoir), d) the cholera vaccine strain (VCUSM14P) is more virulent and dominant colonizer in the intestine, therefore, the formulation might prevent effective colonization of ETEC stains and e) the prototype vaccine produces high titer against cholera toxin which might induce antibodies that cross-react with and neutralize the LT toxin from ETEC, than Dukoral.

To date, no vaccines have been approved to specifically target ETEC by WHO. Therefore, there is an urgent need to develop a safe, effective, and affordable dual-use vaccine that reduces mortality and morbidity due to moderate-to-severe diarrheal diseases in infants and children less than 5 years of age in LMICs. The pipeline ETEC vaccine contains one candidate in Phase 2 studies, five candidates in early-stage Phase 1 clinical evaluation, and four candidates in preclinical development. However, to date, no vaccine was approved by WHO to specifically target the ETEC market. The combined vaccines will be in the market near future. In this context, our cold chain- free cholera and ETEC vaccine represent a better opportunity to increase the outreach of the global immunization programs. The prototype vaccine will help in a) decreasing the cost of vaccine stockpiling and b) energy cost-saving (due to avoidance of cold chain supply). An average of 5 million doses/annum are required for the next three years. The developed dual-use (cholera & ETEC) vaccine would contribute significantly to enhancing the quality of life of the bottom billion children in developing countries. In this context, we would approach the prospective vaccine formulator in Malaysia or abroad for Know-How transfer and commercialization.

Live, Genetically Attenuated, Cold-Chain-Free Cholera & ETEC Vaccine — A Scientific Journey of 20 Years :

Our cholera vaccine development journey started in 2000 at Universiti Sains Malaysia with the isolation of the hemA gene from V. cholerae, followed by the development of a gene mutant vaccine candidate VCUSM2 against V. cholerae O139 in 2006. In 2010, VCUSM2 reactogenicity was reduced by replacing its two wild-type ctxA gene copies with mutated ctxA to produce strain VCUSM14. Introducing the hemA gene into VCUSM14 created VCUSM14P, a strain with the ALA prototrophic trait and excellent colonization and immunological properties (100% protection to wild-type challenged rabbits). It was further refined at AIMST (Asian Institute of Medicine, Science and Technology) University, with the completion of single- and repeated-dose toxicity evaluations in 2019 in Sprague Dawley (SD) rats, followed by the development of a novel cold-chain-free VCUSM14P formulation in 2020. 

This 20-year-old study was funded by several federal funding agencies, including various grants from the Ministry of Science, Technology and Innovation and from the Ministry of Higher Education. Over the years, several students and staff members contributed to this project, and we acknowledge them gratefully for their contributions. Details of this journey are described below and summarized below pictures.

It is a collaborative project between USM and AIMST University for the development of a prototype cold-chain free, live oral cholera vaccine. The vaccine has already advanced to Technology Readiness Level (TRL) 5 and currently, the OECD GLP - compliant safety studies of the vaccine are in progress at the Malaysian Institute of Pharmaceuticals and Nutraceuticals (IPharm). 

We are keen to validate the production process and technology licensing to support the clinical development of the dual protective vaccine with a potential collaborator to serve the bottom billion, with a cost-effective, dual-use vaccine stored outside the cold chain to protect against cholera & ETEC diarrheal diseases. To achieve our objective, we have to evaluate the protective efficacy of the vaccine in immunized animal models (mice and rabbits) challenged with ETEC strains at an ICH GLP – Compliant lab. Mainly because, ETEC is a multivalent pathogen and has been sub-classified into different virulence types based on the production of different enterotoxins, colonization factors (CFs), coli surface antigens (CS), and the ‘O’ antigen group of the lipopolysaccharide. One challenge is that, as with the CF antigens (CFAs), LT and ST occur in varying proportions, dispersed across ETEC strains, geographic regions, and population types. Hence, researchers studying ETEC isolates and the CFAs in different geographic regions concluded that a candidate ETEC vaccine should induce anti-toxin immunity to LT and anti-CF immunity to CFA/I, CS1, CS2, CS3, CS5, and CS6 would potentially provide coverage for 80-90% of ETEC strains associated with illness in endemic areas. Eventually, the planned validation studies would determine the NOAEL, MRSD (maximum recommended starting dose), and HED (Human Equivalent Dose) of the vaccine against cholera and ETEC. After the compilation of all the Quality, Safety and Efficacy information in the Preclinical Dossier, we would license the Process Know-How to support the clinical development. Eventually, we would support the Phase 1 Clinical trial of a dual-protective vaccine against cholera and ETEC, the diarrheal diseases, the 2nd killer disease of children under 5 years of age in Sub-Saharan African countries, Asia, and in many low- and middle-income countries (LMICs).

Scientific background for the invention

Vibrio cholerae, is a facultative anaerobic bacterium that inhabits aquatic ecosystems as well as in host intestinal tract. In an aquatic environment, both toxigenic and nontoxigenic V. cholerae strains survive year-round. The toxigenic strains typically through the ingestion of contaminated water or food enter the human host, colonize the small intestine, multiply and produce the cholera toxin, which causes acute diarrheal disease. The various growth-inhibiting stresses in the environment such as deprivation of nutrients, fluctuations in temperature, salinity and oxygen level elicit stringent responses in V. cholerae involving modulation of expression of several genes to mediate their adaptation, persistence, dissemination and transmission of cholera. Therefore, we leveraged these adaptive traits of V. cholerae at an in-vitro condition that best mimics the environmental stress conditions for the extended storage period of live attenuated cholera vaccine strain at ambient temperature. The storage stability of the cold chain free vaccine formulation was evaluated for its purity, potency, and viability by phenotypic and genotypic methods over an extended storage period of 1 year at 25°C ± 2°C and 60% ± 5% humidity in the ICH compliant Stability Chamber. The vaccine has proven to be immunogenic, safe, and non-toxigenic in extensive preclinical studies with different animal models. This prototype vaccine is a first of its kind and a patent has been filed. Currently, there is no live vaccine available to protect against cholera caused by V. cholerae O139 which is more virulent than the O1 strain, invasive and widespread in eleven Asian countries and in Sub-Saharan African countries.

This cold chain vaccine formulation platform can be exploited for the development of a cost-effective, live attenuated oral vaccine against enteric pathogens such as Shigella dysenteriae causing shigellosis/ dysentery and Salmonella paratyphi causing paratyphoid fever or in combinations.

Cholera Vaccine: 

• In this current year (2022) the cold chain free, live, oral cholera vaccine will be advanced to Technology Readiness Level (TRL) 6 due to the completion of the ongoing GLP study supported by the PRGS Grant from the Ministry of Higher Education, Malaysia. 

• GMP validation of the oral liquid cholera vaccine production process will be completed by this year (2022) at a WHO-GMP facility as supported by the Strategic Research Fund, Ministry of Science, Technology & Innovation, Malaysia. 
• Completion of Phase 1 Clinical trial of cholera vaccine by 2024 supported by the Strategic Research Fund, Ministry of Science, Technology & Innovation, Malaysia. 

Dual-protective vaccine against cholera and ETEC:

• The protective efficacy of the dual vaccine in immunized animal models against the ETEC strains will be completed in an ICH GLP – Compliant lab and the NOAEL, MRSD, and HED values will be determined in the next two years through a collaborator.  
• During the next five years, the Phase 1 Clinical trial of a dual-protective vaccine against cholera and ETEC will be completed through a collaborative effort.

Sustainable Development Goals:

1. Environment

•  Live, Single-dose & Thermostable
•  No wastage of vaccine from heat- and freeze-shocks  (~US$ 15-40 million/year across GAVI).

2. Social

•  Vaccination more people/day & coverage
•  Long term protection

3. Economic

• Reduction in the capital cost for cold chain equipment (estimated at ~ US$ 110 million/year).
• Reduction in the cost of stockpiling & operating costs (US$ 125-150 million/year across GAVI).
• Competitive cost (USD 2/person) 

Theory of change in the development of prototype cold chain-free, live, oral cholera vaccine:

The theory of change in the development of a prototype cold chain-free, live, oral cholera vaccine for the bottom billion people is depicted in the following figures.

Cholera Vaccine:

Cross Protection of ETEC by the prototype Cholera vaccine:

Currently, there is no exclusive ETEC vaccine; however, it can be cross-protected by a cholera vaccine, Dukoral®. Dukoral with its rCTB component affords short-term cross-protection against ETEC infection. Unfortunately, this vaccine demands a “cold chain” supply (2-8°C) in addition to multiple doses which poses a great challenge. The vaccine strain(VCUSM14P) has 2 copies of rCTB, we have evaluated and established the immunogenicity of the prototype cholera vaccine in animal models challenged with ETEC. Hence it serves as a dual-use vaccine against cholera and ETEC. The theory of change in the development of a prototype cold chain-free, live, oral ETEC vaccine for the bottom billion people are depicted in the following figures.

Product development & Patenting: 

A thermostable live oral cholera vaccine formulation stable at room temperature for 140 days was developed. The vaccine formulation is First-of-its-kind in comparison to all existing WHO licensed oral cholera vaccines which are cold chain supply dependent. 

Patent filed: Cholera Vaccine Formulation ( PI 2018700106) in Jan'18. 

PCT Filed: MY2018/050098 in the US, EU, South Africa, and India. 

Trade Mark Registered:  "MyChol" (2576MY1)for Cholera Vaccine in July'18.   

Research Papers Published: 

• Xian, T.H., Sinniah, K., Yean, C.Y. et al. Immunogenicity and protective efficacy of a live, oral cholera vaccine formulation stored outside-the-cold-chain for 140 days. BMC Immunol 21, 29 (2020).
• Xian, T.H., Parasuraman, S., Sinniah, K., Ravichandran, M., Prabhakaran, G., 2019. Repeated dose toxicity evaluation of a cold chain-free, live, attenuated oral cholera vaccine in Sprague Dawley rats. Vaccine 37, 711–720.

We are grateful to The Ministry of Higher Education (MOHE), Malaysia, for funding this research under the Prototype Development Research Grant Scheme (PRGS) - (PRGS/2/2015/SKK10/AIMST/02/1). We are also thankful to AIMST University & Univerisiti Sains Malaysia for providing the animal house facilities to carry out the developmental research work.

The core technology powers the development of a dual-protection
vaccine against cholera and ETEC is the cold chain free, liquid oral formulation of the live attenuated vaccine strain (VCUSM14P). The vaccine is stored outside-the-cold-chain for a period of 1 year at 25°C ± 2°C and 60% ± 5% humidity. The rational attenuation made the vaccine strain a good colonizer which mimics a natural infection, non-reactogenic, strongly
immunogenic in vivo, and with 2 copies of CTB for the development of a live dual protective vaccine against cholera and ETEC.

It is a collaborative project between USM and AIMST University in Malaysia for the development of a prototype cold-chain free, live, dual-use, oral vaccine. The USM is a public university with a larger organization. The AIMST University is a private university. The research team members are staff holding various academic positions at these universities.

Our commitment to diversity, equity, and inclusion manifests in two primary ways. First, to serve the bottom billion poor children in Sub-Saharan African countries with affordable combination vaccines to protect them from diarrheal diseases that remain the second infectious killer disease after respiratory diseases of children under the age of five years. Breaking the cold chain barrier with a thermostable single-dose vaccine would relieve the significant bottlenecks and cost determinants in mass vaccination campaigns for children. Our 20 years of drive and passion have resulted in the development of a dual-use, cold chain vaccine against concomitant ETEC and V.cholera infections. A dollar a vaccine dose to serve the bottom billion to uplift their wellness. Second, we the research team at AIMST University believe that if a solution to a problem is not applicable and scalable, at least in some identifiable context, then the problem is not solved. We believe academia must strive to expand diversity with a more inclusive approach – welcoming and embracing different socioeconomic, ethnic, gender groups and creating a broader pool of thought processes and worldviews.  Our commitment to this idea resonates with my desire and responsibility to contribute as a scientist, educator, and activist to see the wellness and happiness in the last child, in the last mile.

Acquisitions, innovation and collaborations are the chief strategies adopted by vaccine manufacturers to achieve a significant position in the booming human vaccines sector. The global market value for vaccines is estimated at $85.93 billion in 2025. Vaccine manufacturing remains concentrated, with four large manufacturers (GSK, Pfizer, Merck, and Sanofi) controlling 90% of global vaccine value and five producing 60% of global volume (SII, GSK, Sanofi, BBIL and Haffkine4). Increasingly, mid-size manufacturers (mostly in Asia) are expanding their portfolios to compete in regional and new vaccine markets. However, vaccine portfolios in many pharmaceutical companies have decreased in the last decades due to the cost and time involved in vaccine development, which is much more costly and time-consuming to develop than other drugs. To circumvent this, many pharmaceutical companies are continuously investing in vaccine research in academic institutions. Furthermore, organizations including the National Institute of Health, the Wellcome Trust, and the Bill & Melinda Gates Foundation offer funding necessary for vaccine development. Therefore, we would approach the prospective vaccine formulators (Crucell, the Netherlands and Paxvax Bermuda Ltd, USA) for transfer of the Process Know-How. Alternatively, we would also seek the developmental initiatives from the Bill & Melinda Gates Foundation towards this global cause for the bottom billion who face the challenges of killer diarrheal diseases.

The research work is being supported by the Prototype Research Grant Scheme (PRGS) and Fundamental Research Grant Scheme (FRGS), Ministry of Education (MOE), Malaysia. To further validate the cold chain-free, live vaccine production process and to license the Process Know-How to support the clinical development, we would explore the avenues with the vaccine manufacturers.

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The oral cholera vaccine formulation will be evaluated in human volunteers in the first-in-human phase 0 studies to assess its safety, tolerability, and immunogenicity. The project at a cost of RM 7.9 Million is funded by the Strategic Research Fund (SRF) by the Ministry of Science, Technology and Innovation (MOSTI), Malaysia.