Saving Children Under Five: Cold-Chain Free, 3-in-1 Vaccine for Co-infectious Diarrheal Diseases

1) Diarrheal Diseases, the Silent Killer in Children under Five:

• Global burden:

Diarrheal diseases are the second leading cause of death in children under five, with 525,000 annual deaths in underdeveloped countries (UNICEF, 2022) and with an economic burden of US$ 4 Billion/year, globally.

• Co-infections of the Diarrheal Diseases in Children:

Over 65% of diarrheal diseases globally are caused by three major pathogens: Vibrio cholerae (Cholera), Enterotoxigenic E. coli (ETEC), and Group A Rotavirus (RVA). These concurrently co-infect children in resource-limited settings, worsening health outcomes. The pathogenesis of V. cholerae and ETEC is quite similar.

• Diagnostic Challenges of Watery Diarrheal Diseases:

Diagnosing specific diarrheal diseases caused by Rotavirus/ETEC/Cholera is challenging because they all cause similar watery diarrhea symptoms. Hence, it is challenging to differentiate them solely based on clinical presentation, patient characteristics, or disease severity, which hinders effective intervention and treatment.

• Bottom 2 Billion Children at Risk:

RVA causes 40% of severe diarrhea globally, with 90% of cases in Africa and Asia. 

Cholera impacts 2 billion, causing 200,000 deaths in 69 countries; WHO targets a 90% reduction by 2030. 

ETEC affects 220 million annually, leading to 75 million cases impacting physical and cognitive development and 42,000 deaths in children. 

ETEC also causes traveler’s diarrhea in 40% of visitors to underdeveloped countries. 

Despite WHO's initiatives, including rotavirus vaccinations in 114 nations, rotavirus causes over 2 million hospitalizations and 185,000 deaths annually.

2) Safety and Co-infection Protection: Gaps in Diarrheal Vaccines:

• Limitations in Licensed Vaccines:

Existing Rota vaccines (Rotarix®, RotaTeq®, Rotavac®, and RotaSIIL) and Cholera vaccines (Dukoral®, Shanchol® and Euvichol®) focus on individual pathogens, resulting in gaps in addressing co-infections and safety concerns. Additionally, the lack of an exclusive ETEC vaccine, despite its prioritization by WHO, represents a significant research gap. However, a cholera toxin B subunit (CT-B) based cholera vaccine offers cross-protection against ETEC infection.

 • The Cold Chain Challenge: 

All existing diarrheal vaccines demand cold-chain supply at 2-8°C (32% of the vaccine cost), and 68% for the multiple doses hinders access to diarrheal vaccines in resource-limited areas. 

• Challenges with Existing Live Rotavirus Vaccines: 

Post-licensure studies of rotavirus vaccination highlight risks like intussusception (intestinal blockage), environmental enteric dysfunction, virus shedding in stool, virulence reversion and cold-chain requirements, necessitating the need for non-replicating oral DNA rotavirus vaccines. 

3) Urgent Need for a Safer and Oral DNA Rota Vaccine:

• Safety of DNA Rota Vaccine: No live viruses, no risk of intussusception, no viral shedding, and transmission. DNA vaccine provides both humoral and cell-mediated immunity.

• Current Delivery Challenges: Injections/nebulizers require multiple doses of DNA vaccine, cumbersome compared to oral delivery. Therefore, developing an oral Multi-Epitope DNA Vaccine for Rotavirus will maximize its potential benefits. 

4) Next-generation Combined Diarrheal Vaccines:

Currently, no combined vaccine on the market (valued at USD 3.1 billion) protects against Rotavirus, ETEC, Cholera, or co-infections. To address this gap, this project aims to fast-track the development of a single-dose, cold-chain-free, 3-in-1 oral vaccine for co-infectious diarrheal pathogens in children under five, prioritizing safety, accessibility, and effectiveness.

Saving Children under Five: A Combined 3-in-1 Vaccine Solution Against Co-infectious Diarrheal Diseases

A) Building on Success: Leveraging MyChol^TM Technology:

2-in-1 Vaccine Innovation

• Joint Effort: AIMST University and USM (University Sains Malaysia) have developed MyChol^TM, a pioneering Prototype 2-in-1, live, cold-chain free, oral vaccine for Cholera and ETEC - travelers' diarrhea. This dual-protection vaccine is the culmination of 20 years of research.

• MyChol^TM mimics a natural infection, stimulating a long-lasting immune response. It targets both cholera toxin (CT) and heat-labile toxin (LT), preventing infections from Vibrio cholerae O139 and ETEC pathogens, not just symptoms.

• Cost-Effective and Accessible: It is cold-chain free and remains stable at room temperature (25°C ±2°C and RH 60% ± 5%) for 180 days. This simplifies production and administration, reducing costs by 40% compared to existing options and making it accessible to resource-limited settings.

• Safety and Efficacy Proven: Extensive testing in three animal models (mice, rats, and rabbits)  confirms the safety and effectiveness of MyChol^TM, in preventing weight loss and diarrhea.

• Poised for Impact: MyChol^TM holds a Malaysian patent (MY-190074-A) and has filed in other countries. 

• GLP and GMP: Ongoing GLP studies paved the way for Scaling up GMP production.

• Phase O clinical trials: being funded by the Strategic Research Fund from MOSTI, Malaysia.

B) 3-in-1 Vaccine Innovation: A Powerful Combination of Key Technologies:

Prototyping a 3-in-1 vaccine development utilizes three core technologies:

• Recombinant technology (used to develop VCUSM14P, a live-attenuated  bacterial vaccine candidate in MyChol^TM), 

• Reverse Vaccinology (used to develop a plasmid DNA vaccine for Rotavirus A), and

• Cold-chain free live vaccine formulation platform (used for MyChol^TM vaccine development).

Strategy: MyChol^TM (2-in-1) + Rotavirus DNA vaccine = 3-in-1 Vaccine for Cholera, ETEC & Rotavirus:

•  A live attenuated bacteria (VCUSM14P) in the MyChol^TM platform delivers the DNA rotavirus vaccine at mucosal entry points. This enables oral delivery of DNA vaccine and enhances the feasibility of a combined 3-in-1 diarrheal vaccine development.

C) Global Collaboration for Fast-Track Development of Rotavirus DNA Vaccine:

• Multi-Epitope DNA Vaccine for Rotavirus: Our collaborator, the National University of Sciences & Technology (NUST), Pakistan designed an in silico multi-epitope DNA vaccine targeting Rotavirus VP4 and VP7 genes and validated it.

• Development of a Plasmid DNA vaccine: Dual promoter vectors were chosen for co-expressing this DNA vaccine in VCUSM14P, evaluating the safety and immune response of 3-in-1 vaccines in mice and rabbits.

• Licensing 3-in-1 Vaccine Technology: Upon completing the ongoing studies, we will proceed with the GLP regulatory studies to license the 3-in-1 vaccine technology to a vaccine manufacturer. 

D) Impact and Benefits:

• Critical Solution: The 3-in-1 vaccine tackles co-infections, a major barrier in diagnosing and preventing the three major diarrheal diseases in vulnerable 2 billion children under five in LMICs.

• Global Reach: Cold-chain free 3-in-1 vaccination simplifies logistics and increases daily vaccination rates and coverage for underprivileged children under five.

• Cost-effectiveness potential: The 3-in-1 vaccine reduces capital for cold-chain logistics and operating costs by 40% compared to administering three separate vaccines in resource-poor areas, and improves adherence to global vaccination programs. 

Target Population: Prioritizing the Bottom Two Billion:

• Primary: Diarrheal diseases threaten 2 billion children under five in 69 LMICs, especially in Sub-Saharan African countries. Over 65% of diarrheal diseases globally are caused by three major co-infectious pathogens (Rotavirus A, ETEC, and Vibrio cholera). Limited access and expensive vaccines make these children highly susceptible.

• Secondary: One-fifth of adults in LMICs, including travellers, aid workers, and military personnel (around 40% infected with Cholera, ETEC within 3 days). 

How They Are Currently Underserved:

• Limited access to clean water and sanitation: A staggering 780 million lack clean water, and 2.2 billion lack proper sanitation creating breeding grounds for diarrheal pathogens.

• Weakened Immune Systems: Childhood wasting, unsafe water, and poor sanitation weaken immune systems, contributing to 80% of diarrheal deaths in LMICs. Frequent diarrhea can worsen malnutrition, creating a vicious cycle. Hence, the effectiveness of existing vaccines is reduced in LMICs.

• High prevalence of diarrheal diseases: Rotavirus and ETEC are especially prevalent, leading to severe illness and death. Most diarrheal patients are concurrently infected with V. cholerae and ETEC, resulting in severe diarrhea. The pathogenesis of both pathogens is quite similar.

• Surging Child Deaths from Rotavirus, Cholera, and ETEC: These diseases collectively cause 370,000 child deaths yearly, with a 145% surge in cholera fatalities over the past five years (WHO, 2024).

• No Single Vaccine for All Three Diarrheal Diseases simultaneously: WHO-approved live-attenuated rotavirus and killed cholera vaccines address only individual pathogens separately.

• High Cost for Cold-Chain Supply: Cholera vaccination costs around $6/person, rotavirus $2/dose. A significant portion (32%) is for cold chain requirements and 68% for multiple doses.

• Price Disparity and Unequal Access to Rotavirus Vaccines:  The prices of rotavirus vaccines vary based on income level. LMICs pay between USD 2.85 and USD 18.00 per course through UNICEF, while UMICs can pay up to USD 27.00 per course. 

How Our Solution Impacts the Bottom Two Billion:

• Why We Need a Combined Vaccine: Similar watery diarrhea symptoms hinder diagnosis (Rotavirus, ETEC, Cholera) accurately for effective intervention and treatment. The single 3-in-1 vaccine addresses this issue by protecting against individual pathogens or their co-infections simultaneously.

• 3-in-1 Comphrenesive Vaccine Protection: The vaccine offers simultaneous protection against Rotavirus, ETEC, and Cholera, reducing morbidity and mortality rates for children in LMICs.

• Simplified Vaccine Delivery; Eliminating Cold Chain Constraints: Oral delivery and room-temperature stability (compared to existing vaccines) potentially reduce vaccination costs by 40% through streamlined logistics, improving access in remote areas.

 MyChol^TM: A Game-Changing, Transformative Vaccine:

• Cost-Effective Production: Live, combined (3-in-1) vaccine production does not require sophisticated downstream processing, hence it costs less than existing vaccine options.

• 40% Low for Wider Reach: The combined vaccine reduces costs by 40%, is free from cold-chain logistics and saves time compared to existing options of administering individual vaccines separately, thereby improving access for wider underserved children. 

Benefits extend beyond children:

- Adults in LMICs: Safeguards adults in LMICs from ETEC and Cholera. 

- Reduces Traveler's Diarrhea for tourists and aid workers in high-risk areas. 

It is a community-led development supported by Malaysian government agencies (MOSTI, MOHE, IPHARM, and NIBM), a national university partner USM, an international partner NUST, and a startup (VAX BIOTECH Sdn Bhd) in Malaysia. This ensures that our 3-in-1 diarrheal vaccine development aligns with community needs, ideas, and priorities.

Team Lead and Team's Deep Commitment to the Communities We Serve:

Visionary Techno-Commercial Leader: The Project Leader is a techno-commercial professional with 30 years of expertise in Vaccine Development, Veterinary Drugs, and Industrial Enzymes. Managed six commercial projects, executed one international 'turnkey' project, and led Malaysia's first Cholera Vaccine development. He is supported by a professional team with a startup for vaccine production and scale-up validation at a cGMP facility.

National & International Collaborative Efforts in 3-in-1 Innovation:

A)  2-in-1 Innovation (a Dual-Use Vaccine for Cholera and Travellers' Diarrhea):

• Universiti Sains Malaysia (USM) developed the live attenuated Vibrio. cholerae O139 (VCUSM14P), the vaccine candidate, and AIMST University developed the live, cold-chain-free, oral dual-use vaccine formulation.

• Two Decades of Progress in Vaccine Development: It has been a 20-year journey in developing the Dual-use Vaccine (MyChol^TM) for Diarrheal Diseases (Cholera and ETEC diarrhea). Funded by Ministry of Science, Technology, and Innovation (MOSTI) and the Ministry of Higher Education (MOHE) grants.

• Advanced Development: MyChol^TM is undergoing Good Laboratory Practice (GLP) studies at the Malaysian Institute of Pharmaceutical and Nutraceutical (IPHARM) supported by NIBM. The proposed GMP studies are at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia. 

• Strategic Research Funding: The project secured Strategic Research funding from MOSTI, supporting progress towards Phase O clinical trials.

• Forging a Strategic Partnership with VAX BIOTECH to compile the preclinical dossier for MyChol^TM and its submission to the National Pharmaceutical Regulatory Agency (NPRA), Malaysia for further clinical development. 

B)  3-in-1 Vaccine Innovation (Combined Vaccine for 3 Major Diarrheal Diseases): 

• We collaborate with the National University of Sciences & Technology (NUST), Pakistan for the Rotavirus DNA vaccine. NUST designed and validated an in silico multi-epitope DNA vaccine targeting VP4 and VP7 genes of Rotavirus (RVA).

C) Community-Informed Design and Implementation Strategy:

In Malaysia, Rotavirus is causing around 6% of hospital admissions for children under five. The Ministry prioritizes the inclusion of the Rotavirus vaccine in the National Immunization Program, supported by two key initiatives that support this goal are:

• National Vaccine Development Roadmap (NVDR): Aims to achieve self-sufficiency in human vaccine production in Malaysia within ten years.

• Malaysian Genome and Vaccine Institute (MGVI): Plays a crucial role in establishing an ecosystem for vaccine development and manufacturing by supporting R&D efforts. 

• MOSTI, being a key stakeholder, supports validating GMP production and Phase O clinical trials, ensuring the highest quality and safety standards throughout our vaccine development journey.

In brief, driving collaborative innovation through partnerships with diverse stakeholders and communities to develop and transfer the 3-in-1 diarrheal vaccine with a vision of “Vaccinating the Last Child, Last Mile, at Low Cost for Diarrhoeal Diseases”.

Integrative 3-in-1 Diarrheal Vaccine for Rotavirus, ETEC, and Cholera:

The project's core objective is to prototype a 3-in-1 diarrheal vaccine that targets Rotavirus, ETEC, and Cholera and license. These three diseases are the primary causes (>65%) of childhood diarrhea and often co-infect children, especially in resource-limited settings. 

The combined vaccine development involves the integration of a multi-epitope DNA vaccine for Rotavirus A into the existing MyChol^TM, a Prototype 2-in-1 oral, cold-chain free, live attenuated vaccine designed for Cholera and ETEC. The ultimate goal is to mitigate the complex issue of co-infections of these diarrheal diseases among the world's most vulnerable children with a focus on accessibility and feasibility in implementation, as explained below.

1) MyChol^TM: A Transformative Prototype 2-in-1 Diarrheal Vaccine for Cholera and ETEC 

Advanced Development Stage (TRL 7):

• Pre-clinical Development and Testing: The existing cold-chain free, prototype 2-in-1 vaccine (MyChol^TM) is in the advanced development stage at Technology Readiness Level (TRL) 7.

• GLP Regulatory Studies: Currently, the dual-use vaccine is undergoing Good Laboratory Practice studies as per OECD guidelines at IPharm, Malaysia, which is a crucial step for regulatory approval.

• GMP validation: The scale-up validation of the vaccine production process is planned for August 2024 in a WHO-compliant GMP facility, ensuring adherence to the highest quality standards.

• Failure Mode Effects Analysis (FMEA): is now the standard Technology Transfer tool, allowing risk-based acceleration. FMEA follows the guidelines outlined in ICH Q8, Q9, and Q10. Additionally, the current QS 9000 certification program mandates the incorporation of FMEA in product design. Therefore, we are implementing FMEA for our 2-in-1 diarrheal vaccine production validation in the cGMP facility in association with the Commonwealth Scientific and Industrial Research Organisation (CSIRO), an Australian Government agency. 

• Patent Protected: MyChol^TM is patent protected (MY-190074-A).

• Funding Secured: The project has secured Strategic Research Funding from The Ministry of Science, Technology and Innovation (MOSTI), Malaysia supporting progress towards Phase O clinical trials. 

• Target Product Profile (TPP): We will focus on the TPP framework, the strategic planning tool to achieve the key features of the commercial vaccine product based on FDA guidance. Towards this, we are associating with CSIRO through MGVI and NIBM.

This innovation positions MyChol^TM as a transformative and economically viable solution in combating the diarrheal diseases associated with Cholera and ETEC as highlighted in the below figures.

Leveraging FMEA to Enhance Risk-Based Acceleration in 2-in-1 Diarrheal Vaccine Development and GMP Production Validation 

Implementing Failure Mode Effects Analysis (FMEA) is the standard practice for technology transfer. It allows for risk-based acceleration based on the Rating System. FMEA adheres to the guidelines of ICH Q8, Q9, and Q10, and is a requirement in the current QS 9000 certification program for product design. Therefore, we are implementing FMEA for our 2-in-1 diarrheal vaccine production validation in a cGMP facility through CSIRO, Australia. 

2) Salient Features of MyChol^TM, 2-in-1 Diarrheal Vaccine

• Natural Mimicry for Superior Immunity: MyChol^TM mimics natural infection by colonization, eliciting immune responses that are highly specific, effective, and long-lasting.

• Dual Protection: It prevents infection by both the V. cholerae O139 and ETEC pathogens, not just disease symptoms.

• Thorough Evaluation: MyChol^TM has been evaluated for safety and efficacy in three distinct animal models, including mice, rats, and rabbits.

• Stability: It is cold-chain free and remains stable at room temperature (25°C ±2°C and RH 60% ± 5%) for 180 days.

• Colonization Potential: MyChol^TM demonstrated higher colonization potential in the infant mouse colonization assay.

• Nonreactogenicity: It was nonreactogenic in the ligated rabbit ileal loop assay.

• Antibody Response: MyChol^TM showed a 275-fold increase in anti-CT IgG antibodies, a 31-fold increase in vibriocidal antibodies, and higher antibody titers against anti-CT, anti-LT, anti-CT-B, and anti-LTB IgG in mice.

• 100% protective efficacy against Wild Type V.cholerae O139 challenge in rabbits.

• 100% protection in prototypical ETEC H10407 strain challenge studies due to neutralization of LT toxin, preventing weight loss and diarrhea.

• Cost Savings: MyChol^TM costs around 40% less than comparable vaccines due to simple downstream unit operations and cold-chain free supply.

• VCUSM14P: is the live bacterial vaccine candidate in MyChol^TM. Therefore, we are leveraging the VCUSM14P platform for the oral delivery of the rotavirus DNA vaccine. 

In brief, the salient features are summarized in the below Figure:

3) Development of a 3-in-1 Diarrheal Vaccine for co-infections of Rotavirus, ETEC and Cholera 

Current Status: 

Rotavirus DNA Vaccine Development Strategy: 

On prototyping 3-in-1 vaccine development utilizes three core technologies: Recombinant technology, Reverse Vaccinology, and Cold-chain free live vaccine formulation for 3-in-1 vaccine development. Leveraging the success of MyChol^TM, a pioneering Prototype 2-in-1 vaccine, this project advances the development of a revolutionary 3-in-1 vaccine for diarrheal diseases addressing the complex issue of co-infections among the world's most vulnerable children as depicted in the below Figures 1 & 2.

In brief, the 3-in-1 vaccine is developed in three stages/parts and the details are as follows: 

Part A: Development of DNA Vaccine for Rotavirus A 

• Insilco DNA Vaccine for Rotavirus Designed: A multivalent vaccine was designed using VP4 and VP7 proteins of RVA.

• Multi-Epitope Screened: Epitopes were screened for antigenicity, allergenicity, homology with humans, and anti-inflammatory properties.

• Population Coverage Analysis Completed. Regional Coverage: Population coverage analysis in South Asia (99.0%) and worldwide (98.47%).

• Genotype Protection: Potential protection against all RVA genotypes.

• Dual Promoter Vectors Chosen: Dual promoter vectors (pDUALgc and pET expression system) for the DNA vaccine for rotavirus were selected for efficient co-expression of VP4 and VP7 genes.

• Progress in Plasmid DNA Vaccine Construction: Plasmid DNA vaccine development is underway with the intention of protein expression in both prokaryotic and eukaryotic cells.

Part B: Fast Track Development of 3-in-1 Vaccine

Upon completion of the ongoing studies, safety and immune response will be evaluated in mice and rabbits for a 3-in-1 combined vaccine incorporating a rotavirus DNA component. The MyChol^TM platform (VCUSM14P, the live attenuated bacterial vaccine candidate) will be used to deliver the rotavirus DNA vaccine, as illustrated in the accompanying figures.

Part C: Advancing Towards Regulatory GLP Studies for 3-in-1 Vaccine and Licensing

Upon completion of the ongoing studies, we will proceed with the GLP regulatory studies as per ICH guidelines to license the 3-in-1 vaccine technology to a vaccine manufacturer.

4) Funding for the past 10 years (2015 to present): Total USD 1.6 Million

Our progress in developing vaccines for diarrheal diseases is evident in securing a total funding of USD 1.6 million over the past five years. We express our gratitude to the Ministry of Higher Education (MOHE) and the Ministry of Science, Technology and Innovation (MOSTI), Malaysia for their funding and support of this national initiative.

Details about Research Grants:

Total USD 1.6 Million

• FRGS Grant for the value of RM 146,000 from MOHE to research on “Evaluation of the Cross Protection of O1 and O139 Cholera Vaccines in Different Animal Models Challenged with Serogroup-specific Vibrio cholerae Toxigenic Strains for the Development of a Dual-Purpose Vaccine” in the year 2023. 
• MOSTI SRF Grant for SP1 research on “Towards the development of the first-in-human trial for an oral vaccine against traveler’s diarrhea and cholera caused by the O139 strain of Vibrio cholerae” in the year 2021. Grant Amount: RM 5.8 Million
• MOSTI SRF Grant for SP6 research on “Preclinical Toxicity Study for Cholera Vaccine (OECD GLP Regulatory Study)” in the year 2021.Grant Amount RM 800,000
• PRGS Grant for the value of RM 479,000 from the Minister of Higher Education (MoHE), Malaysia to research “ICH GLP-Compliant Pre-Clinical Safety Studies to Support the Licensure of Prototype Cholera Vaccine for Clinical Development” in the year 2019.
• FRGS Grant for the value of RM 185200 from (MoHE), Malaysia to research on “Evaluation of Prototype Cold Chain Free Live Attenuated Oral Cholera Vaccine for its Cross-ProtectiveImmunity against Enterotoxigenic Escherichia Coli (ETEC) as a Dual-Use Vaccine forDiarrheal disease” in the year 2018.
• PRGS Grant for the value of RM 250,000 from (MoHE), Malaysia to research on “Development of Cold Chain free live attenuated Cholera Vaccine formulation” in the year 2015.

5) List of Publications in the past 5 years:

• Hui Xian, T., Parasuraman, S., Ravichandran, M., & Prabhakaran, G. (2022). Dual-Use Vaccine for Diarrhoeal Diseases: Cross-Protective Immunogenicity of a Cold-Chain-Free, Live-Attenuated, Oral Cholera Vaccine against Enterotoxigenic Escherichia coli (ETEC) Challenge in BALB/c Mice. Vaccines, 10(12), 2161. https://doi-org.ezproxyberklee.flo.org/10.3390/vaccines10122161

• Ravichandran, M., Tew, H. X., Prabhakaran, G., Parasuraman, S., & Norazmi, M. N. (2022). Live, Genetically Attenuated, Cold-Chain-Free Cholera Vaccine-A Research and Development Journey: Light at the End of a Long Tunnel. The Malaysian journal of medical sciences: MJMS, 29(2), 1–7. https://doi-org.ezproxyberklee.flo.org/10.21315/mjms2022.29.2.1

• Xian, T. H., Sinniah, K., Yean, C. Y., Krishnamoorthy, V., Bahari, M. B., Ravichandran, M., & Prabhakaran, G. (2020). Immunogenicity and protective efficacy of a live, oral cholera vaccine formulation stored outside-the-cold-chain for 140 days. BMC immunology, 21(1), 29. https://doi-org.ezproxyberklee.flo.org/10.1186/s12865-020-00360-1

• 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(5), 711–720. https://doi-org.ezproxyberklee.flo.org/10.1016/j.vaccine.2018.12.027

•  Usman M, Ayub A, Habib S, Rana MS, Rehman Z, Zohaib A, Jamal SB, Jaiswal AK, Andrade BS, de Carvalho Azevedo V, Faheem M, Javed A. Vaccinomics Approach for Multi-Epitope Vaccine Design against Group A Rotavirus Using VP4 and VP7 Proteins. Vaccines (Basel). 2023 Mar 24;11(4):726. doi: 10.3390/vaccines11040726. PMID: 37112638; PMCID: PMC10144065.

Why We Are Applying to MIT Solve?

A) Leveraging the Existing 2-in-1 Diarrheal Vaccine for Success and Collaboration:

• We are applying to Solve to accelerate the prototyping and licensing of our innovative 3-in-1 oral, live, single-dose, cold-chain-free Diarrheal Vaccine.
• Our project builds upon the success of MyCholTM, a pioneering 2-in-1 oral vaccine for Cholera and ETEC co-infections developed in collaboration with USM.
• The prototype is patented. Ongoing GLP regulatory studies will be completed in June 2024. The regulatory GMP production validation studies will commence in August 2024.
• We have secured a Strategic Research Fund for Phase O clinical trials from the Ministry of Science, Technology, and Innovation (MOSTI), Malaysia. In brief, the technology has reached TRL7.
• 3-in-1 Vaccine Project Progress: By incorporating a DNA vaccine for Rotavirus (developed by NUST) into the MyCholTM platform, we are developing a comprehensive prototype 3-in-1 oral Diarrheal Vaccine against co-infections of Rotavirus, Cholera, and ETEC pathogens and their devastating impact. 

B) Specific Barriers and How Solve Can Help

1) Limited GMP Facilities:

Malaysia lacks cGMP facilities for scale-up validation and large-scale production of our oral live vaccine meeting international standards. Solve's network can connect us with suitable industrial partners to bridge this gap.

2) Regulatory Navigation:

Our innovative vaccine uses a novel DNA component for Rotavirus delivered through a live bacterial vector. This requires navigating complex regulatory hurdles for 3-in-1 vaccine clinical trials and licensure. Solve's support can be invaluable in guiding us through this process by connecting us with suitable regulatory clinical trial experts/partners for our unique 3-in-1 vaccine, ultimately speeding up licensure.

3) Affordability and Accessibility:

Market analysis shows a significant demand for the three targeted diarrheal vaccines (Rotavirus, Cholera, and ETEC) in 2030, amounting to USD 3.1 billion.

Rotavirus Vaccine Market: While the global rotavirus vaccine market is growing from the current USD 2.2 billion, ensuring affordability and accessibility in low-income countries remains a challenge.

A few key players including Bharat Biotech, Merck & Co., Inc., GSK, and Lanzhou Institute of Biological Products dominate the market. Solve's networking with industry leaders can help us navigate the complexities of technology transfer and cost-effective production and distribution.

Cholera & ETEC Vaccine Demand:

The global demand for oral cholera vaccines exceeds available stockpiles (16 million doses against the needed 30 million, valued at USD 297 million in 2029). No exclusive vaccine is licensed for ETEC. However, the revenue projections are USD 600 million in 2030. This emphasizes the need for innovative solutions and increased production capacities. Collaborating with industry leaders like Sanofi, GSK, and EuBiologics, presents a valuable opportunity to advance our project and address global health challenges. This collaboration will help us tackle urgent needs and increase production capacities.

C) Collaborative Opportunity:

We believe that partnering with industry leaders and organizations such as Gavi, the Vaccine Alliance, PATH, and MIT Solve demonstrates significant interest and support from influential global health organizations. We are seeking guidance and support from Solve to expedite these collaborations and overcome obstacles related to regulatory barriers, technology transfer, and licensing complexities.

How Our 3-in-1 Vaccine Revolutionizes Diarrheal Disease Treatment:-

1) 3-in-1 Oral Vaccine: Innovative Solution to Prevent Child Diarrhea

Our single-dose, cold-chain-free, live, 3-in-1 oral vaccine would provide comprehensive protection against three major childhood watery diarrheal diseases (Rotavirus, ETEC, and Cholera), addressing the problem of co-infections that cause over 65% of diarrheal diseases in 69 LMICs. This will be the first combined vaccine of its kind, addressing several key challenges and offering significant improvements in treating diarrheal diseases, which are urgently needed.

- Cold-chain-free formulation:

The prices of diarrheal vaccines needed to be lowered. There was no way someone earning less than USD 2 per day could afford a vaccine that costs USD 6 to 10. In this direction, our vaccine does not require a cold-chain supply (2-8°C). This simplifies logistics, reduces costs (by 40% compared to existing options), and improves vaccine accessibility and coverage to the "Last Mile - Last Child" in resource-limited regions with unreliable power supplies.

- Simplifying diagnosis, treatment, and outbreak response:

By targeting all three major diarrheal pathogens individually or their co-infection that cause similar watery diarrhea, our 3-in-1 vaccine eliminates the need for complex diagnostic procedures. This enables prompt intervention, minimizes the risk of misdiagnosis, and allows healthcare workers to focus on treatment and implementing public health measures during outbreaks in resource-limited settings.

- Beyond live vaccines: A 3-in-1 solution with enhanced Rotavirus safety:

By integrating a DNA vaccine for Rotavirus into our existing 2-in-1 vaccine platform, we can avoid the risks associated with live Rotavirus vaccines, such as intussusception and viral shedding. This is a fast-track approach to developing an oral DNA vaccine for Rotavirus.

2) Potential for broader positive impacts:

This project has the potential to have positive impacts across the diarrheal vaccine landscape:

- Encouraging the development of combination vaccines:

Our success in developing a 3-in-1 vaccine can inspire other researchers to explore similar combination approaches for different enteric infectious diseases such as Shigella and Helicobacter pylori. This can lead to the development of more comprehensive and efficient cold-chain-free vaccination strategies.

- Promoting innovation in oral DNA vaccines:

By incorporating a DNA Rotavirus vaccine into our live attenuated bacterial platform, we can lay the foundation for further development of other Oral DNA vaccines based on this safe and effective technology for other enteric diseases in veterinary animals.

3) Potential market transformation:

This solution has the potential to significantly transform the diarrheal vaccine market by: 

- Expanding vaccine coverage:

Increased affordability, accessibility, and ease of administration in resource-limited settings can result in a substantial increase in vaccination rates among the bottom billion vulnerable children in Sub-Saharan African countries and LMICs.

- Reducing healthcare burden:

By preventing diarrheal diseases and their detrimental impact on childhood physical growth and cognitive development, the vaccine can significantly decrease healthcare costs and improve overall health outcomes for children.

- Creating a new market segment:

The success of a 3-in-1 vaccine can create a new market for combination vaccines, diversifying options for infectious diseases and improving global health outcomes.

Impact Goals:

MyChol^TM's innovative approach directly addresses SDGs 3, 9, and 11 by reducing childhood deaths caused by three major diarrheal diseases (Rotavirus, ETEC, Cholera) in children under five, particularly in resource-limited settings, fostering innovation in vaccine development, and promoting sustainable vaccination campaigns to create healthier communities.

Aligned with SDG 3 (Ensure healthy lives), MyChol^TM aims to reduce the global burden of diarrheal diseases. Diarrheal diseases cause one out of nine children's deaths and also affect one-fifth of adults, (around 900 million) tourists, and travelers visiting LMICs.

Targets and Indicators:

1) Addressing SDG 3: Target 3.2

• MyChol^TM directly addresses Target 3.2: End preventable deaths of newborns and children under 5 years of age.

• MyChol^TM contributes to reducing childhood mortality rates associated with Rotavirus, Cholera, and ETEC infections (Indicator 3.2.1). Improved child health outcomes directly contribute to better physical growth and cognitive development.

• Lower the incidence of diarrheal diseases in children (Indicator 3.2.1: Under-five mortality rate) from the current rate of 500,000 deaths per annum. And, to promote the health and well-being of children, it is crucial to address potential complications from these diarrheal diseases, including stunted growth and their impact on cognitive development.

• Live, Single dose & Cold-chain free supply. Storage stability at room temperature (25°C ± 2°C, 60% RH ± 5% RH) for 180 days.

• No wastage of vaccine from heat & freeze-shocks (~US$ 15-40 million/year across GAVI) and improves accessibility.

• 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).

2) SDG 9: Industry, Innovation, and Infrastructure: 

MyChol^TM fosters innovation in vaccine development. Eliminating cold-chain requirements significantly reduces infrastructure costs for storage and transportation of a live, oral 3-in-1 diarrheal vaccine from the manufacturing point to the immunization point.

3) SDG 11: Sustainable Cities and Communities:

The long-term protection offered by MyChol^TM allows for efficient vaccination campaigns, leading to broader coverage and improved community health.

• Long-term protection
• Vaccinate more people/day & coverage.

Current Progress Indicators:

• Preclinical Testing: Initial testing of MyChol^TM in animal models has demonstrated its safety and efficacy.

• DNA Rotavirus Design: A multi-epitope DNA vaccine for Rotavirus has been designed and validated.

• GLP Studies Underway: MyChol^TM is currently undergoing Good Laboratory Practice (GLP) studies.

• The regulatory GMP studies to validate the vaccine production process will commence in August 2024.

• Funding Secured: Initial funding has been secured to support progress towards Phase 0 clinical trials.

MyChol^TM's unique features and positive impact on SDGs 3, 9, and 11 make it a compelling solution. With our vision, we aim to improve global vaccination rates and combat diarrheal diseases effectively. We can significantly impact public health by reducing costs by 40%, ensuring stability, and reaching the last mile, particularly in underserved regions.

A Novel 3-in-1 Diarrheal Vaccine Powered by Live Attenuated Bacteria and DNA Technology 

This project aims to develop an innovative 3-in-1 vaccine for diarrheal diseases in children. The vaccine targets Rotavirus, ETEC, and Cholera, utilizing the existing advanced platform of MyChol^TM, the 2-in-1 vaccine for Cholera and ETEC.  The goal is to improve accessibility, address co-infections, and have a significant global impact on children's health.

The Core Technology:

The project utilizes three core technologies: Recombinant technology, Reverse Vaccinology, and Cold-chain free live vaccine formulation.

A Powerful Combination of 3 Key Technologies for 3-in-1 Vaccine Development namely:

1) Live Attenuated Bacteria Platform (MyChol^TM): 

This prototype platform developed by Recombinant technology acts as the delivery system for the combined vaccine. It utilizes a rationally attenuated (weakened) live bacterium (VCUSM14P) to stimulate immunity against Cholera and ETEC.

• Safety and Efficacy: Extensive testing in three distinct animal models demonstrates the safety and ability of MyChol^TM to elicit a strong immune response.
• Cost-Effective Production: Lower production by 40 % by simple downstream unit operations and cold-chain-free vaccine formulation compared to existing options.

2) DNA Vaccine for Rotavirus A:

• An in silico Multi-Epitope DNA Vaccine targeting VP4 and VP7 genes of Rotavirus (RVA) has been designed and validated by Reverse Vaccinology technologies. 

• The plasmid DNA vaccine is transformed and expressed in the VCUSM14P, offering its convenient oral delivery, allowing the body's cells to produce antigens and build immunity. 

• Integrating DNA Vaccine technology into the existing 2-in-1 vaccine platform, a 3-in-1 vaccine has evolved.

3) Cold-chain-free formulation Platform: 

• We have developed a unique cold-chain-free, live, oral dual-use vaccine formulation for Cholera and ETEC. 

• This first-of-its-kind vaccine remains stable at room temperature (25°C ± 2°C, 60% RH ± 5% RH) for 180 days, resulting in a significant cost reduction and removing the barriers in cold-chain logistics in resources-limited LMICs. 

• MyChol^TM is protected by a patent (MY-190074-A) and a PCT filed in 6 countries.

Development Stage: Advancing Towards a Licensable Solution:

This project is currently in an advanced stage, with a powerful combination of recombinant technology, reverse vaccinology and cold-chain free live vaccine formulation platform technologies:

• MyChol^TM: It is in an advanced stage of development (TRL7), undergoing GLP regulatory studies.

• Ongoing GLP studies: supporting the progress towards GMP validation and Phase O clinical trials. This signifies significant progress towards licensure.

• 3-in-1 Vaccine:  The dual promoter vectors were chosen to co-express the DNA vaccine of rotavirus in the MyChol^TM (VCUSM14P) platform. 

• Safety and immune response testing in animals is planned for the combined vaccine. After animal testing, the GLP regulatory studies for the 3-in-1 vaccine will be pursued.

Overall, the project demonstrates a clear roadmap for further ongoing regulatory GLP and GMP approvals to license the vaccine technology.

This 3-in-1 vaccine innovation revolutionizes the prevention of 65% of diarrheal diseases by addressing co-infections by three major pathogens, improving accessibility, and reducing vaccination costs. This benefits the health of children and travellers in resource-limited settings.

Our team, which is working on the development of the 3-in-1 vaccine technology, consists of seven full-time academic staff members: one Professor specializing in medical molecular microbiology at Universiti Sains Malaysia (USM), two Senior Associate Professors (toxicologist and bioprocess technologist), and one Lecturer specializing in molecular biology and vaccines at AIMST University. Moreover, one Associate Professor of molecular medicine (specializing in virology and immunology) and two Postdoctoral researchers on vaccines at the National University of Sciences & Technology (NUST) are contributing their expertise to this multidisciplinary project.

3-in-1 Diarrheal Vaccine Development: A Scientific Journey of Over 20 Years

This collaborative project between AIMST University, USM, and the NUST Universities aims to develop the 3-in-1 Diarrheal Vaccine for Rotavirus, ETEC, and Cholera over 20 years. 

2-in-1 Vaccine

The journey began in 2000 at USM, and the vaccine candidate VCUSM14P was developed. AIMST University formulated a cold-chain-free version of VCUSM14P (MyChol™) and demonstrated cross-protective efficacy against ETEC in animal models in 2022. 

3-in-1 Vaccine

NUST developed a multi-epitope DNA vaccine for Rotavirus. The plasmid DNA vaccine will be incorporated with VCUSM14P, and tested in animals for clinical development and licensure.

Fostering a Diverse and Inclusive Team

The development of the 3-in-1 diarrheal vaccine is guided by our team's core value of building a diverse, equitable, and inclusive (DE &I) environment. We recognize the importance of a variety of perspectives and backgrounds in this digital era in achieving scientific excellence and ensuring our vaccine reaches those who need it most. Speed is the essence of timely project success. Therefore, the expertise and networks of our team members complement each other, contributing significantly to our progress on the 2-in-1 diarrheal vaccine and advancing towards the 3-in-1 prototype.

Our Diverse Team:

Composition: We have seven full-time academic staff members with diverse expertise:

- Medical molecular microbiology (Professor, Universiti Sains Malaysia - USM)

- Toxicology & Bioprocess Technology (Senior Associate Professors, AIMST University)

- Molecular Biology & Vaccines (Lecturer, AIMST University)

- Molecular Medicine (Virology & Immunology) (Associate Professor, National University of Sciences & Technology - NUST)

- Vaccine Research (Postdoctoral Researchers, NUST)

Team Balance: Our team consists of three women and four men scientists.

Members' Experience: Our team members range in age from 35 to 65 years old.

Ethnic Background: We have two Malaysians, two Indians, and three Pakistanis on our team.

Benefits of Diversity:

Multi-Institutional Collaboration: We leverage the expertise of two public universities (USM & NUST, one national and one international) and a private institution (AIMST), fostering a broader range of perspectives.

Disciplinary Expertise: Our team combines specialists in microbiology, toxicology, bioprocessing, molecular biology, bioinformatics, reverse vaccinology, and immunology, leading to a more comprehensive approach to vaccine development.

Gender Balance: We strive to create an environment that values and empowers all team members, ensuring a balanced gender representation to contribute their unique ideas in problem-solving at different stages of the project.

Fostering an Inclusive Environment:

Shared Values: Our team aligns with the values of USM (quality, equity, accessibility), AIMST (integrity, respect, excellence, and service), and NUST (honesty and ethical behaviour) and believes in empowering researchers to make a positive impact on people's lives.

Open Communication & Collaboration: We actively encourage open discussions and teamwork to ensure all voices are heard and valued, as demonstrated in the development of this project proposal.

Continuous Improvement:

Building a truly inclusive team is a continuous journey. Driven by our passion for serving underprivileged children with affordable, accessible diarrheal vaccines for Rotavirus, ETEC, and Cholera (the "bottom billion"), we aim to further diversify our team backgrounds and experiences in future endeavours.

Strength in Diversity:

This team structure allows us to combine the research strengths of public and private institutions with diverse nationalities and scientific backgrounds in this digital era. This fosters innovation and ensures a well-rounded approach to developing a life-saving vaccine, as evidenced by our success in securing USD 1.6 million in government grants.

Inspired by DE and I:

We draw inspiration from GAVI, PATH, UNICEF and MIT Solve's commitment to DE&I, firmly believing that a diverse and inclusive team is crucial in achieving global health impact through our 3-in-1 diarrheal vaccine project.

Business Model: Assessing the Desirability, Feasibility, and Viability of the 3-in-1 Diarrheal Vaccine

This project addresses the gaps in existing licensed vaccines for three major diarrheal diseases (Rotavirus, ETEC, and Cholera) among underprivileged children under five in 69 LMICs, one-fifth of adults, and tourists and travelers to these regions. The 3-in-1 vaccine is designed to be cold chain-free, accessible, and convenient, providing an affordable single-dose option.

The business model of the 3-in-1 diarrheal vaccine prototype, depicted in the Figure below, demonstrates a compelling case of success in terms of desirability, feasibility, and viability.

The details of the 3-in-1 Diarrheal Vaccine Business Model are summarized below.

1) Desirability:

- Addresses a Critical Need:

Diarrheal diseases pose a significant health threat to children in LMICs, causing one in nine deaths (500,000 deaths per year), particularly in Sub-Saharan Africa, Haiti, Cameroon, Malawi, Lebanon, Somalia, Pakistan, Bangladesh, India, and Central America. The 3-in-1 vaccine offers a comprehensive solution by targeting three specific pathogens (Rotavirus, ETEC, and Cholera), possibly addressing co-infections involving any combination of these pathogens.

- Superior Value Proposition:

The single-dose, cold chain-free vaccine simplifies administration, reduces costs by 40%, and improves accessibility, making it highly desirable for resource-limited settings.

- Benefits Multiple Segments:

The vaccine caters to several customer groups: 2.2 billion underprivileged children under five (primary beneficiaries), approximately 900 million tourists/travelers, and one-fifth of adults in the LMICs (secondary beneficiaries).

2) Feasibility: - Strong Technology Foundation:

The business model leverages a 2-in-1 vaccine (MyChol^TM) for Cholera and ETEC technology, which is supported by 20 years of developmental research.

- Prototyping 3-in-1 vaccine development:

Utilizes 3 Core Technologies: Recombinant technology, Reverse Vaccinology, and Cold-chain free live vaccine formulation for 3-in-1 vaccine development.

- Experienced Global Partners:

This collaborative project is between 3 universities for developing a DNA vaccine for Rotavirus A. And, collaboration with an established vaccine manufacturer for contract manufacturing and regulatory bodies would facilitate the cGMP production and market access.

- Financial Backing:

The Strategic Research Funding secured from MOSTI demonstrates initial financial support for GMP validation of the production process and Phase 0 clinical trials. This signifies significant progress towards licensure.

3) Viability:- Cost-Effective Vaccine:

Lower production costs of live vaccines due to simple unit operations compared to existing vaccine options and elimination of cold chain expenses translate to a 40% cost reduction and enhance its affordability and broader program adoption.

- Diversified Revenue Streams:

Primary vaccine sales are accompanied by potential secondary revenue streams like licensing and royalties, creating financial sustainability.

- Addressing Global Health Priorities:

Alignment with the goals of organizations like GAVI, WHO, and PATH increases the product's marketability and potential for large-scale adoption in vaccination programs.

Overall, the business model demonstrates strong potential for success. It addresses a critical need with a desirable product, leverages existing prototype 2-in-1 vaccine technology and partnerships for feasibility, and utilizes a viable revenue strategy for long-term sustainability.

Financial Sustainability Plan for the 3-in-1 Diarrheal Vaccine

This plan outlines the financial sustainability strategy for the 3-in-1 diarrheal vaccine project. The primary goal is to transfer the technology to an established manufacturer to ensure efficiency and faster global reach. The plan provides details on revenue streams, past funding successes, and future funding strategies to ensure the long-term financial viability of the project and its global health impact.

1) Market Opportunity and Revenue Streams

Market analysis shows a significant demand (USD 3.1 billion in 2030) for the three targeted diarrheal vaccines (Rotavirus, Cholera, and ETEC). The estimated 5% market share in the first three years by the manufacturer translates to a potential market value of approximately USD 310 million.

Our revenue model combines primary and secondary streams:·

Primary Revenue: 

The core revenue stream is technology transfer royalties. We aim for a 2% royalty rate from vaccine sales by licensed manufacturers in the first three years, generating an estimated USD 6.2 million based on the projected market share.

Secondary Revenue Streams:

Technology Transfer: Partnerships with established manufacturers will focus on licensing the "MyChol^TM" technology and exploring potential co-development of future products. This can generate upfront payments or royalties on future sales. This provides crucial funding to support the continued research and development (R&D) of the more complex vaccines for humans and veterinary animals.

2) Past Funding Successes and Strategic Partnerships

Past Funding:

Over the past ten years, we have secured USD 1.6 million in funding from prestigious organizations like MOHE and MOSTI in Malaysia. Details were already provided for each grant received between 2015 and 2023, highlighting research focus and funding sources.

Current Funding:

We have received initial funding of USD 1.06 million from MOSTI in Malaysia for preclinical development activities, including GMP validation and Phase 0 clinical trials for the 2-in-1 version of the vaccine.

Strategic Partnerships:

Collaborations with Gavi, the Vaccine Alliance, PATH organizations and MIT Solve showcase strong interest and support from prominent global health organizations. These partnerships open avenues for future funding, procurement opportunities, and global reach.

3) Future Funding Strategies

Grant Pursuit: 

We will continue seeking grants from government agencies and philanthropic organizations dedicated to global health initiatives to support advanced clinical trials, regulatory approvals, and market expansion.

Impact Investment: 

We will explore attracting investors seeking social impact alongside financial returns. This will bolster our long-term financial sustainability and growth. 

Overall, this plan underscores our commitment to financial prudence, strategic partnerships, and leveraging diverse revenue streams for the successful development, commercialization, and global impact of our 3-in-1 Diarrheal Vaccine. Developing detailed financial projections and securing additional funding through grants and impact investments will be crucial for long-term success.