Pump-Priming Project Awards - Round 7 Awardees

Research Professor Bente Børud

Norwegian Institute of Public Health (NIPH)
Norway

Collaborators:

Professor Myron Christodoulides, University of Southampton, UK

Jan Haug Anonsen, Researcher, Climate & Environment, NORCE Norwegian Research Centre, Norway

Professor Afework Kassu Gizaw, Director-General of AHRI, Armauer Hansen Research Institute (AHRI), Ethiopia

Summary

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, represents an emerging global health problem with increasing antibiotic resistance. Vaccine development is complicated by the fact that gonorrhoea infections fail to engender protective immunity. A successful vaccine against gonorrhoea should therefore include multiple antigens and induce a protective immune response beyond that generated through natural infections.

Identification of suitable vaccine targets remains a bottleneck and identification of conserved antigens through comparative genomics overlooks post-translational modifications. Here, we aim at providing proof-of-concept for protein glycans as important vaccine antigens against gonorrhoea. The glycoproteins identified in N. gonorrhoeae are mainly lipoproteins or transmembrane proteins localized in the periplasm or on the cell surface, and we have established that the attached glycans are antigenic and immunogenic.

We have interesting results that protein glycans are protecting the gonococci against bactericidal activity and indirect results that antibodies against these glycans might be bactericidal. We will confirm and validate these results in different strains and investigate the molecular mechanisms behind these observations by using a panel of mutant strains that express different glycans or glycoproteins. Recent studies have shown decreased rates of gonorrhea after vaccination with meningococcal outer membrane vesicle (OMV) vaccines, and we will identify the glycoproteins and attached glycans within these OMV vaccines.

Project outcomes

Neisseria gonorrhoeae represents a global health problem with significant morbidity and mortality and increasing number of antimicrobial resistant isolates. Surveillance remains limited in several regions, and here we present the first WGS study from Ethiopia with seventy N. gonorrhoeae isolates. Endocervical and urethral swabs were collected among symptomatic patients at STI clinics of health facilities in Bahir Dar, Ethiopia. Among the isolates, multi-locus sequence typing ST-1587 was dominant (54%) and the most common NG-STAR sequence types were ST-5621 (18.6%), ST-1203 (14.3%) and ST-5666 (10%). All isolates were resistant to Tetracycline, Ciprofloxacin, and most isolates (95.7%) were resistance to Penicillin-G, while all isolates were susceptible for Cefixime, Ceftriaxone, Azithromycin and Spectinomycin. The genome-based AMR prediction was matched with phenotypic observations for all six antimicrobials and the genotypic AMR determinants was identified. This study contributes to increased knowledge on antimicrobial susceptibility pattern and mechanisms in Ethiopia.

Vaccine development has been difficult, and even repeated gonorrhoea infections fail to engender protective immunity. An effective vaccine should therefore include several antigens to induce a strong and protective immune response.

Here, we have investigated the potential for protein glycans as vaccine antigens. Species of Neisseria encompass broad-spectrum O-linked protein glycosylation where the glycoproteins are mainly transmembrane proteins or lipoproteins localized on the cell surface or in the periplasm^1-4,6. Several of the N. gonorrhoeae glycoproteins elicit bactericidal or functional blocking antibodies and have previously been suggested as vaccine candidates: PilE⁷, Mip^8,9, AniA¹⁰, PilQ^11,12, MetQ^13,14, PorB¹⁵, ZnuD^16,17 and PotF3¹⁸. We suggest that since glycans impact on the immunogenicity, as shown for the PilE protein¹, glycan function and diversity ought to be considered when including glycoproteins in future vaccines.

Multiple studies have reported decreased rates of gonorrhoea after vaccination with meningococcal serogroup B (MenB) outer membrane vesicles (OMV) – based vaccines^19-21. However, we found that meningococcal OMV vaccination did not significantly induce bactericidal activity against N. gonorrhoeae. Meningococcal OMV vaccines contain various outer membrane proteins that are known to be glycosylated, and we are identifying these glycoproteins and glycans. In addition, we examined the potential impact of bacterial protein glycosylation on susceptibility to complement-mediated killing of bacteria (serum bactericidal activity) elicited by human antibodies. We observed significantly higher bactericidal titres against glycosylation null mutant N. meningitidis versus the wild type strains conceivably through masking protein epitopes important for bactericidal killing. This effect was less evident for the N. gonorrhoeae strains studied.

An effective gonorrhoea vaccine will control this epidemic and reduce the need for antibiotic treatment and it is therefore important to explore new vaccine antigens. We believe that protein glycans fulfil key criteria for an optimal vaccine antigen in that they are surface-exposed, immunogenic, and possess limited antigenic variability in N. gonorrhoeae. However, further studies are necessary to understand the complex molecular mechanisms involved, as well as the involved regulatory mechanisms.

Professor Susanna Dunachie

Professor of Infectious Diseases 
University of Oxford, UK 

Collaborators:

Dr Duy Pham, Head of Molecular Microbiology and Wellcome Fellow, Oxford University Clinical Research Unit, Vietnam

Professor Christine Rollier, Professor of Vaccinology, University of Surrey, UK

Dr Victoria Ward, Academic Clinical Fellow in Infectious Diseases, University of Oxford, UK

Summary

Escherichia coli and Klebsiella pneumoniae are among the top three pathogens responsible for global deaths related to antimicrobial resistance (AMR). In Vietnam, a low and middle-income country (LMIC) with high rates of AMR, these two pathogens are leading causes of bloodstream infections (BSIs), with mortality rates exceeding 35%. Developing vaccines against E. coli and K. pneumoniae is challenging due to the presence of many disease-causing bacterial components (virulence factors) to target, significant variability between bacterial strains (serotypes), and the potential impact on the balance of healthy bacteria people carry in their gut (commensal strains). There are several vaccine candidates currently in clinical trials, and pre-clinical studies have highlighted virulence factors of interest, such as adhesins and iron acquisition proteins. We will use clinical cohorts of patients with BSIs and build on our experience in vaccine target discovery for other pathogens to define target proteins for E. coli and K. pneumoniae for development into vaccine candidates by our collaborating team. We will assess the immune responses (T-cell and antibodies) in patients with BSIs in Vietnam and the UK compared to responses in healthy individuals.

Project Outcomes

Each year many people around the world die from bacterial infections and increasing rates of antimicrobial resistance (AMR) indicate an emerging crisis where infections are becoming harder to treat and may become untreatable. Escherichia coli and Klebsiella pneumoniae are among the top bacteria responsible for global deaths related to AMR. In low and middle-income countries (LMIC) with high rates of AMR, access to antibiotics to treat these infections is difficult. The World Health Organization has recommended research on developing vaccines to prevent or reduce the impact of these infections as an important priority.Developing vaccines against these bacteria is challenging due to the presence of many disease-causing bacterial components (virulence factors) to target, significant variability between bacterial strains, and the potential impact on the balance of healthy bacteria people carry in their gut (commensal strains). There are several vaccine candidates currently in clinical trials, and studies in the bacteria themselves and in mouse models have highlighted virulence factors of interest. However, there is a crucial gap in knowledge around how humans respond to infection – especially which bacterial components are targeted during infection, and the contribution of the cellular arm of the immune response (T-cells) to protection.In this project we recruited patients and healthy volunteers from both UK and Vietnam. We used blood samples from patients with bloodstream infections (BSIs) and built on our experience in vaccine target discovery for other infections to define target proteins for E. coli and K. pneumoniae for development into vaccines and monoclonal antibody therapies. Our goal is to design “anti-disease” vaccines that stop people getting severely ill and reduce the chance of needing antibiotics or allow shorter courses of antibiotics. We assessed the immune responses in patients with BSIs in UK and Vietnam compared to responses in healthy individuals. We looked at antibody and T cell responses to 6 proteins from E. coli and 5 proteins from K. pneumoniae. We were able to identify antibody responses to all the proteins in both patients and healthy volunteers, in both UK and Vietnam, with higher antibody responses seen in patients compared to healthy controls in Vietnam but not in UK. T cell immune responses to the bacterial proteins were particularly interesting, because healthy volunteers in both UK and Vietnam had higher responses than patients with BSI. This supports the concept that boosting T cell immunity to key bacterial proteins in people at risk of infection could be helpful. In the future we plan to scale up the work to include bigger numbers of people. This will allow us to see if higher antibody and T cell responses are associated with better outcomes during BSI. This will allow us to select the most promising bacterial targets and make candidate vaccines using adenovirus vector and mRNA technologies established during the COVID-19 pandemic.

Dr Malick Gibani

Clinical Lecturer in Infectious Diseases
Imperial College London, UK

Collaborators:

Dr Sean Elias, Public Engagement Lead (Gilbert Group), University of Oxford, UK

Dr Brama Hanumunthadu, Dphil Student, University of Oxford, UK

Dr Maheshi Ramasamy, Associate Professor, Malawi Liverpool Wellcome Programme, Malawi

Professor Melita Gordon, Professor of Clinical Infection, Microbiology & Immunology, Malawi Liverpool Wellcome Programme, UK

Dr Helen Dale, PhD Clinical Fellow/Paediatric Registrar, Malawi Liverpool Wellcome Programme, Malawi

Dr Esmeda Chirwa, PhD Clinical Fellow/Clinician, Malawi Liverpool Wellcome Programme, Malawi

Professor Constantino III Roberto López-Macías, Head of Unit, Medical Research Unit of Immunochemistry, Mexico

Dr Tonney Nyirenda, Associate Professor of Immunology, Kamuzu University of Health Sciences (KUHeS), Malawi

Summary

Non-typhoidal Salmonellae are a group of bacteria that typically cause diarrhoeal disease (dNTS). This group of bacteria are particularly harmful to individuals who are HIV-positive, malaria-infected, or malnourished in sub-Saharan Africa. In these individuals, the bacteria can spread throughout the body and cause a more severe illness known as invasive non-typhoidal Salmonella (iNTS). There is a growing interest in developing vaccines to prevent iNTS, particularly with the rise of antibiotic-resistant infections, and several vaccines are in early-stage development.

To develop effective vaccines, we need to better understand the way the immune system protects against NTS infection. Secretory IgA (sIgA) is a type of common mucosal antibody known to provide protection against some intestinal (gut) bacteria. Compared with other bacteria, the role of sIgA in preventing NTS infection in humans has been relatively understudied.

Our research project aims to develop and validate a way to measure sIgA targeted at Salmonella Typhimurium (STm) in stool and saliva samples. We aim to do this by standardised enzyme-linked immunosorbent assay (ELISA) to measure sIgA against STm. We then aim to use this to measure the strength, durability and targets of the sIgA response in healthy volunteers challenged with STm and individuals exposed to STm in a high-burden setting. These will be linked with antibody measures in blood to see how closely they are related. We propose to use these methods in future vaccine studies to better understand immunity to STm and NTS.

Project Outcomes

Non-typhoidal Salmonella (NTS) bacteria often cause diarrheal diseases but can lead to a severe condition called invasive non-typhoidal Salmonella (iNTS). This condition disproportionately affects vulnerable populations, such as individuals with HIV, malaria, or malnutrition, particularly in sub-Saharan Africa. With rising antibiotic resistance, Salmonella has been identified as a priority pathogen by the WHO. There is currently significant interest and momentum in developing vaccines to protect against this infection. This study focuses on a type of antibody in the gut called secretory IgA (sIgA), which may help prevent the Salmonella bacteria from crossing the gut barrier.

Our research project analysed sIgA levels in saliva and stool samples from a human challenge study in the UK, where healthy volunteers were deliberately exposed to two strains of Salmonella Typhimurium (STm): 4/74 and D23580. We developed a method to measure sIgA using an in-house ELISA test, which allowed us to measured IgA that targeted specific molecules on the surface of the bacteria. We went on to measure levels before infection and at several timepoints afterward (14, 28, and 90 days). This allowed us to measure how the immune response develops over time. We measured these antibodies in salvia and stool samples, and compared how well these correlated with one another.Our key findings were:• sIgA Levels over time: Salmonella specific IgA levels in stool varied significantly between individuals. The levels started off low and generally peaked 2–4 weeks after infection before returning to baseline by three months. IgA levels in saliva followed a similar pattern, with levels increasing significantly between 14 and 28 days and returning to baseline by three months.• Strain-specific responses: One of the Salmonella strains, D23580, triggered lower IgA responses to a key bacterial protein, OmpD, compared with the 4/74 strain. This may reflect differences in how well these strains survive in the gut.• Impact of pre-existing immunity: Participants with higher baseline IgA levels showed smaller increases in antibody levels post-infection, suggesting that prior exposure to Salmonella – or related bacteria – influences the immune response.• Antibodies in saliva correlate with antibodies in blood: IgA in saliva – but not stool – correlated strongly with Salmonella antibody levels in blood.

Overall, the study highlights the variability in immune responses among individuals and emphasizes the importance of factors such as the infecting Salmonella strain and pre-existing immunity. Our ongoing studies will help us to determine whether the presence of strong antibody response correlates with protection from disease. We are currently measuring the same antibodies in patients from Malawi with a history of iNTS infection. This will help us to better understand the relationship between natural infection in a high-burden country and healthy UK volunteers. Together, these findings will help us to understand how natural immunity develops, which in turn could inform the design of vaccines for Salmonella.

Dr Alex Keeley

Wellcome Clinical PhD Fellow in Global Health
MRC Unit The Gambia at London School of Hygiene
and Tropical Medicine, The Gambia/UK

Collaborators:

Dr Claire Turner, Royal Society/Wellcome Trust Sir Henry Dale Research Fellow, University of Sheffield, UK

Professor Thushan de Silva, Professor of Infectious Diseases, University of Sheffield, UK

Fatoumata Camara, Scientific officer, MRC Unit The Gambia at LSHTM, The Gambia/UK

Dr Gabrielle de Crombrugghe, F.R.S - FNRS Clinical PhD Fellow, Université Libre de Bruxelles (ULB), Belgium

Professor Pierre Smeesters, Professor of Paediatric Infectious Diseases, Université Libre de Bruxelles (ULB), Belgium

Professor Anne Botteaux, Associate Professor, Université Libre de Bruxelles (ULB), Belgium

Summary

Streptococcus pyogenes, also known as Strep A, is a bacterium that causes severe infections in humans. Approximately half a million people die from Strep A infections each year, predominantly in low resource settings. The creation of a vaccine to protect against Strep A is therefore a top priority. However, progress is slow in part due to our limited knowledge of exactly how the immune system responds to Strep A and provides protection against infection. One of the challenges in creating a vaccine is knowing which parts of the bacteria to target. There are two main strategies in the development of a vaccine; 1) targeting parts of the bacteria that would be present in all strains and 2) targeting the parts of the bacteria (the M protein) which make each strain unique.

Our aim is to understand how antibodies develop with increasing age and following exposure to unique strains of bacteria, and to explore the extents that antibodies generated to unique strains protect from infection.

In our research, we will use samples collected over one year from 442 people in The Gambia where rates of infection were high. We will measure antibodies by expanding a technique we have already developed at the MRC Unit The Gambia. We will explore if antibodies to particular strains are associated with reduced risk of infection from that strain, from similar strains and from unrelated strains. This understanding will help guide the strategy to eventually role out the most effective vaccine against Strep A in high burden settings like The Gambia.

Project Outcomes

This project aimed to better understand how the body’s immune system protects against Streptococcus pyogenes (Strep A), a bacterial infection responsible for many deaths worldwide. Our focus was on studying specific immune responses to different types of Strep A proteins, known as M proteins, which vary greatly across different strains of the bacteria. These proteins might be important targets for vaccines, but we need to know more about how our immune system responds to them to develop effective vaccines.

To investigate this, we developed and optimized a specialised test called a multiplex assay, which can measure immune responses (IgG antibody levels) to many different M proteins at once. We selected 22 M proteins for the test, ensuring they represented the most common and important types found in our study population in The Gambia. We also included some proteins that are part of current vaccine candidates.

Our test successfully measured antibody levels in samples from participants in a Gambian study on Strep A infections. We found that antibody levels generally increased with age, suggesting that repeated exposure to different Strep A strains helps build immunity over time. We also observed that participants who had higher antibody levels to multiple M proteins were less likely to have a confirmed Strep A infection in the future.

However, we faced challenges with the specificity of our test—meaning that in some cases, the test could not clearly distinguish between antibodies targeting different M proteins. Despite these challenges, our findings support the idea that natural immunity to Strep A develops as people are repeatedly exposed to various strains of the bacterium. This insight is valuable for designing vaccines that could mimic this natural immunity and protect against a wide range of Strep A strains.

Our ongoing work aims to refine our understanding of these immune responses and contribute to the development of safe, effective, and widely accessible Strep A vaccines, particularly for regions like The Gambia, where the disease burden is high.

Dr Sreeja Lakshmi

Postdoctoral Research Scientist
King Nandhivarman College of Arts and Science, India 

Collaborators:

Dr. Wang Tiehui, CSO, EpitogenX Limited, UK

Dr. Punnadath Preetham Elumalai, Associate Professor (Biochemistry), Cochin University of Science and Technology Cochin, India

Dr. Eakapol Wangkahart, Associate Professor, Mahasarakham University, Thailand

Summary

Aquaculture is the fastest growing animal food production sector globally and very important to food security. Tilapia is a fast growing fish and a very important aquaculture species for many low to middle income countries (LIMCs), since they provide an important source of protein and essential revenue for many low-income families. Intensification of tilapia farming has promoted severe disease outbreaks, resulting in high mortalities and economic hardship for tilapia farmers. Animal husbandry in LMICs is threatened by the increase in AMA resistance and if not properly managed would drive the increase in zoonotic infections.

Streptococcosis is one of the major bacterial diseases resulting in severe economic losses for tilapia farmers. There is no commercial vaccine against Streptococcus infection till date, and farmers resort to using antibiotics to control this disease. To solve this problem, a novel bacterin vaccine, using molecular adjuvants flagellin and tilapia interferon-γ, will be developed and tested in this research project, using tilapia streptococcosis as a disease model. Flagellin is a potent immune activator in fish. It induces the expression of the Th17 cytokine IL-17A/F1 and Th2 cytokine IL-4/13, but not the Th1 cytokine interferon-γ in fish. IFN-γ is a pleiotropic cytokine with immunostimulatory and immunomodulatory effects. A balanced Th1/Th2 and Th17 immune response is important for promoting protective immunity and eliminating tissue injury (pathological inflammation).

Therefore, novel injection vaccine formulation by incorporation of flagellin and interferon-γ as molecular adjuvants may enhance the immune response to the vaccine leading to long-lasting protection. This work will lead to the development of a cost-effective, easily administered vaccine for use in a large scale tilapia-production in Low and Middle-Income Countries (LMICs).

Project Outcomes

Streptococcus agalactiae causative agent of Streptococcosis result in massive mortalities and thereby huge economic loss in the tilapia aquaculture industry. The current study focussed to develop a novel bacterial vaccine used molecular adjuvants- flagellin and tilapia interferon gamma (IFNγ), against S. agalactiae infections in Nile tilapia. The fish were vaccinated with 100 μl formalin-killed S.agalactiae together with either flagellin or IFNγ and both together by intraperitoneal injection. A booster vaccination was carried out at 22 days post vaccination using the same procedure.

The vaccinated fish were challenged with a virulent strain of S. agalactiae on day 36 and monitored three weeks for cumulative mortality and survival rates. Samples were collected at set time points during the trial to assess specific IgM titres, histopathology, and immune gene expression using RT-qPCR and several immune parameters. The vaccine conferred significant protection with relative percentage survival (RPS) of 59.37%, 71.87% and 81.25% was observed for bacterin vaccine adjuvanted with flagellin, IFNγ and both respectively with an RPS of 15.62% for the unadjuvanted bacterin control group after challenge with S. agalactiae. Similarly, the levels of specific IgM antibodies of fish vaccinated with molecular adjuvants were significantly enhanced post-vaccination.

An upregulation of the immune related gene expression was observed in head kidney and spleen. Furthermore, the measured immune parameters were significantly enhanced in fish administered with vaccine along with molecular adjuvants. The fish vaccinated with both the adjuvants showed significant upregulation in Lysozyme, Catalase, superoxide dismutase and myeloperoxidase activities when compared to the unvaccinated control group which indicated the efficacy of the booster vaccine increasing the antioxidant profiling in fishes. The highest bactericidal percentage of 76% was noticed in vaccinated fishes compared to the control group.

A decreased melanomacrophage cells with decreased inflammation was observed in the spleen tissues by histopathological examinations. In conclusion, this study showed that both flagellin and IFNγ are promising candidates as molecular adjuvants to enhance the efficacy of fish vaccines against S.agalactiae infections in tilapia aquaculture. The findings emphasize safe potential approach for vaccination strategies employing novel vaccines incorporating molecular adjuvants, providing protection to fishes and inducing protective immunity against infectious bacterial diseases.

Dr Boon Lim

Chief Technology Officer
Oxford SimCell Ltd, UK 

Collaborators:
Assoc. Professor. Dr. Mohammad Noor Amal Azmai, Head, Aquatic Animal Health and Therapeutics Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Malaysia

Summary

Aquaculture is a rapidly growing food-producing sector, accounting for 52% of the fish consumed globally in 2020 and generating over £200 billion in economic value. Low-and-middle income countries (LMICs) have become increasingly reliant on fishery trade, with an estimated 56.6 million people employed in the industry, with the vast majority (94%) located in Asia.

One of the most significant challenges facing the aquaculture industry is bacterial disease, which has costed the industry over £4.8 billion in loss-of-yield each year. One of the most common bacterial diseases affecting cultured aquatic organisms is motile Aeromonas septicaemia (MAS), caused by Aeromonas spp. MAS usually infecting cultured tilapia, catfish and carps causing up to 50-70% of mortality, where these species are highly cultured and consumed in LMICs.

Antimicrobial resistance (AMR) further complicates the treatment of this bacterial disease, as Aeromonas spp. is among the most reported AMR organisms in various fish species. As a result, treating these pathogens is becoming increasingly challenging and expensive, which puts the livelihoods of many aquafarmers in LMICs at risk.

To address this issue, this project aims to develop whole-cell bacterial vaccines using the SimCell technology. SimCells are genome-free bacterial cells produced by enzymatically shearing the bacterial genome, rendering the bacterium replication-deficient while preserving immunogenic cell-surface features. Developing SimCell vaccines against MAS could offer an effective solution for protecting fish from the pathogen, potentially reducing economic losses and supporting the livelihoods of small-scale farmers and fishing communities in LMICs.

Project outcomes

Aquaculture, a vital component of global fish consumption and contributing £200 billion annually to the economy, faces substantial threats from bacterial diseases like motile Aeromonas septicaemia (MAS). Particularly impactful in low- and middle-income countries (LMICs), these diseases, compounded by antimicrobial resistance, present challenges to both aquafarmers' livelihoods and global food safety.

This study explored the immuno-protective efficacy of a novel Aeromonas hydrophila-based SimCells vaccine against MAS in tilapia. Engineered via a switchable restriction pathway using a homing endonuclease, A. hydrophila SimCells were produced in quantities of up to 10¹² cells through a proprietary manufacturing process. These SimCells were then incorporated into commercial fish feed using palm oil as an adjuvant.

Tilapia (Oreochromis sp.) were divided into three groups: negative control, A. hydrophila SimCells vaccine, and formalin-killed A. hydrophila vaccine. Oral vaccination was administered on three consecutive days on day 0, with a booster on day 14. On day 21, fish were intraperitoneally challenged with virulent A. hydrophila. Various parameters and samples were collected for growth performance and immune response analysis, with protective efficacy was assessed by bacterial challenge.

No significant differences (p ≥ 0.05) in fish weight and length were observed post-vaccination across all treatment groups. A. hydrophila SimCells provided a 40% RPS, compared to 30% with formalin-killed A. hydrophila and 0% with the control following bacterial challenge. Serum lysozyme production and IgM antibody levels were significantly higher (p ≤ 0.05) in vaccinated fish than controls.

Gene expression analysis revealed differing patterns between the two vaccines: formalin-killed A. hydrophila elicited higher peak expression of IL-1β, MHCII, CD4, IgT, and IgM in the fish hindgut, while A. hydrophila SimCells induced higher peak expression of immune-related genes in the head kidney. In addition, A. hydrophila SimCells were found to induce a longer-lasting immune genes expression in both organs when compared to formalin-killed A. hydrophila. Histopathological assessment demonstrated fewer pathological changes in vaccinated fish organs compared to controls.

The study underscores the efficacy of feed-based A. hydrophila SimCell vaccination in tilapia, with potential enhancements such as an additional booster to further improve protective efficacy throughout the fish culture period.

Professor Constantino III Roberto López-Macías

Head of Unit
Medical Research Unit of Immunochemistry IMSS
Mexico 

Collaborators:

Dr Natalia Martin Orozco, Chief Science Officer, R&D Providence Therapeutics Holdings, Inc, Canada

Dr Tania Rivera Hernández, CONAHCYT Fellow-IMSS, Medical Research Unit on Immunochemistry IMSS, Mexico

Professor Adam Cunningham, Professor of Functional Immunity, University of Birmingham, UK

Summary

Infections caused by Salmonella represent a major public health problem globally, even more so with the raise in antimicrobial resistance. In this project we take advantage of mRNA vaccine technology to test several vaccine candidates based on Salmonella porins. The vaccine protection will be evaluated in mice challenged with different Salmonella serovars.

We aim to obtain proof-of-concept evidence that a multivalent Salmonella mRNA vaccine could provide cross protection against endemic, typhoid and non typhoid strains that cause systemic and gastrointestinal manifestations.

Project outcomes

We successfully designed and synthesized mRNA constructs encoding the Salmonella proteins OmpC, OmpF, and OmpD. These constructs were efficiently translated in an in vitro system, expressed in Huh-7 cells, and recognized by antigen-specific antibodies generated against purified Salmonella proteins.Immunogenicity assessment of the OmpD mRNA construct revealed no detectable antibody responses, prompting the need for further evaluation of antigen-specific T cell responses. In initial challenge studies using a model primarily reliant on antibody-mediated protection, no significant protection against infection was observed. However, in an intragastric challenge model extending over 10 days, we noted a higher percentage of survival in mRNA-immunized mice, although this difference did not reach statistical significance. These findings suggest the need for further optimization and investigation of the immune mechanisms elicited by the mRNA constructs.

Professor Calman MacLennan

Professor of Vaccine Immunology
University of Oxford, UK

Collaborators:

Mr Iason Vichos, Senior Project Manager, Jenner Institute, UK

Dr Paul Stickings, Head of Toxins Group, National Institute for Biological Standards and Control, UK

Dr Seanette Wilson, Senior Project Manager, Biovac, South Africa

Professor Mariagrazia Pizza, Professor, Imperial College London, UK

Summary

Gonorrhoea is a sexually transmitted disease that disproportionately affects women in low- and middle-income countries and threatens to become resistant to all available antibiotics. Globally, there are over 82 million episodes of gonorrhoea a year and the UK has recently recorded its highest level ever of confirmed gonorrhoea cases. An effective vaccine would be of enormous global public health benefit and have a major impact on the silent pandemic of antimicrobial resistance. Although no vaccine against gonorrhoea is currently available, several promising candidate vaccines are in development and about to enter clinical trials. Alongside vaccine development, it is essential to have robust assays that can accurately and effectively measure the immune response induced in humans by these vaccines. Such assays will ultimately be necessary for the licensing of gonococcal vaccines, but it takes many years to develop an internationally accepted assay, and this often involves the creation of a standard serum which can be used to ensure results are comparable between different laboratories.

This project will prepare and convene a workshop which brings together key stakeholders from high-, middle- and low-income countries in the field of gonococcal vaccine and assay development, along with regulators, policymakers, and funders. The meeting will set a pathway forward for the development of such an assay and the preparation of an international standard serum which will be suitable for acceptance and endorsement by the World Health Organisation. Findings and recommendations will be compiled in a report for publication and public dissemination.

Project outcomes

A workshop was organised in Oxford, United Kingdom to discuss harmonisation and standardisation of assays for assessing gonococcal immunity. This is important for assessing the immunogenicity of vaccines against Neisseria gonorrhoeae, many of which are starting to enter clinical trials.

The workshop brought together representatives from vaccine developers, regulators, assay harmonisation specialists and from across the gonorrhoea research field. It was agreed that it is vital to be able to compare vaccine responses from across clinical trials of gonorrhoea vaccines. Furthermore, an international gonococcal serum standard is necessary for standardising assays used to measure immunogenicity of these vaccine candidates. It was also agreed that a pathway should be laid out to create an accessible panel of diverse gonorrhoea strains which could be used as the targets for these assays. Smaller working groups will be established to advance these goals.

Dr Amirul Islam Mallick

Associate Professor Indian Institute of Science Education and
Research Kolkata (IISER Kolkata), India

Collaborator:

Dr. Ozan Gundogdu, Assistant Professor, London School of Hygiene and Tropical Medicine, UK

Summary

Campylobacter jejuni (C. jejuni) is one of the major causes of diarrhoeal diseases in humans and is highly prevalent in Low and Middle-Income counties (LMICs). Although C. jejuni has a broad host range, chickens remain the primary source of human infection. Intriguingly, C. jejuni naturally inhabits chicken’s gut with little or no sign of diseases or illness, while in the case of humans, C. jejuni causes acute gastroenteritis and several autoimmune disorders. Except for bio‐security measures, currently, no vaccine is available for humans or chickens. Moreover, with the high prevalence of antibiotic-resistant C. jejuni strains in developing countries, including India, Campylobacteriosis has become a more pressing global health issue. In search for effective measures to control C. jejuni, our group recently identified multiple vaccine targets which are generally associated with bacterial attachment to host cells and facilitate disease progression.

For this study, we modified a food-grade probiotic bacteria Lactococcus lactis (L. lactis), a commonly used bacterial strain to ferment dairy products, to express the multiple target proteins of C. jejuni. In the proposed research, we intend to use a combination of these modified recombinant bacteria (rL. lactis) as an oral vaccine against C. jejuni in commercial chickens. We expect that in addition to the general health benefits of probiotics, the combinatorial application of multiple vaccine targets will (combination of CadF, JlpA, and Hcp of C. jejuni) prevent C. jejuni colonization in vaccinated chickens, ensuring better poultry health and reduce the risk of human infection via the food chain.

Project outcomes

Diarrhoeal diseases are the second leading cause of preventable death, especially among children under five in developing countries, with 2-3 million deaths per year 1-4. The major bacterial diarrhoeal illness is mediated by the faecal-oral transmission of Escherichia coli, Salmonella, Shigella, Vibrio cholerae, Yersinia enterocolitica, and Campylobacter jejuni (C. jejuni). Among them, C. jejuni infection (campylobacteriosis) is considered the major bacterial cause of diarrhoeal disease in humans and is highly prevalent in Low- and Middle-Income Countries (LMICs) 4–6. Although C. jejuni has a broad host range, poultry, particularly chickens, remain the primary source of human transmission 7,8. Recently, we have demonstrated the role of major surface-expressed colonization protein (SECPs) of C. jejuni in mediating host cell adhesion, invasion, and subsequent pathogenesis in both avian and non-avian hosts 2,3. Considering our central hypothesis to reduce the number of C. jejuni in poultry intestines, we have presented here a novel mucosal immunisation strategy using a food-grade engineered live vector (such as Lactococcus lactis). Results from this study altogether advocate the current modality of oral vaccine as a safe and cost-effective approach to reduce pathogen load in the primary host (chickens). Since systemic immunisation would be time-consuming, labor-intensive, and costly, we propose that mucosal immunisation using a live vectored-based delivery platform could be a promising alternative when large numbers of flocks are needed to be immunised against C. jejuni. 

Dr Ina Salwany Md Yasin

Deputy Director Institute of Bioscience
Universiti Putra Malaysia
Malaysia

Collaborators:

Professor. Dr. Mohd Zamri-Saad, Technical Advisor, Bio-Angle Vacs Sdn. Bhd., Malaysia

Professor. Dr. Alim Isnansetyo, Lecturer, Universitas Gadjah Mada, Indonesia

Dr. Tharangani Herath, Senior Lecturer, Harper Adams University, UK

Summary

Vaccination provides a sustainable alternative to the unsustainable use of antibiotics in aquaculture for the treatment of bacterial diseases, thereby reducing the incidence of antimicrobial resistance (AMR). Our project focuses on the development of a feed-based vaccine against infection by Vibrio spp. (MyIPO Malaysia, patent filing number: PI2021000105). This vaccine is unique in term of its cost-effectiveness, ease of administration, and environmental safety. Moreover, it reduces the reliance on trained personnel for vaccine administration and minimizes fish stress during vaccination. The oral vaccine stimulates both mucosal and systemic immune responses that prevent infections by multiple Vibrio species, resulting in an average survival of 80% to reach market weight. For optimal protection, it is recommended that this vaccine be administered orally according to the prescribed schedule.

The promising efficacy calls for commercialization efforts, including upscaling and field validation of the upscaled product on efficacy and suitability in cage cultured marine farms in low- and middle-income countries (LMICs). In collaboration with Bio-Angle Vacs Sdn. Bhd. (co-applicant industry), we plan to optimize the manufacturing process in a certified GMP facility. The upscaled vaccine will undergo field studies in farms in Malaysia and Indonesia (co-applicant LMIC, Indonesia) to validate its efficacy, elucidate vaccination mechanisms (co- applicant UK partner), and conduct a cost-benefit analysis of vaccination. These goals contribute to the development of an effective oral vaccine for marine fish, addressing the challenges that small- to medium-scale farmers in LMICs are facing when utilising injectable vaccines and eventually, reduce AMR for sustainable aquaculture production.

Project outcomes

This project focused on developing and testing the ViVac feed-based vaccine, a novel solution to combat vibriosis in aquaculture, particularly targeting marine fish species. The vaccine was designed to be incorporated into fish feed, enabling easier administration and widespread application in aquaculture farms. Key laboratory studies demonstrated the vaccine's stability under conditions mimicking fish digestion and industrial feed production. Acid stability tests confirmed the vaccine's ability to remain effective in the highly acidic environment of the fish gut (pH 2), ensuring efficient delivery of antigens to trigger immune responses. However, heat sensitivity tests revealed that the vaccine's effectiveness declined at high temperatures, with 40 °C identified as the safe upper limit for maintaining its integrity during feed production.

Field trials conducted at a farm in Indonesia showcased the vaccine's potential under real-world aquaculture conditions. Over a 16-week trial, vaccinated fish achieved notable improvements in feed efficiency, consuming less feed compared to unvaccinated fish while maintaining comparable growth rates. Vaccinated fish also exhibited a better feed conversion ratio (FCR), an essential metric for cost-effective fish farming. Survival rates were high for all fish, but vaccinated fish demonstrated enhanced disease resilience.

Immunological assessments revealed significantly higher antibody levels (IgM titers) in vaccinated fish, peaking at four weeks after the initial vaccination. However, the immune response did not show further enhancement following a second booster dose, suggesting that adjustments to the vaccination schedule may further improve long-term immunity. Advanced genetic analyses confirmed the upregulation of critical immune-related genes, such as IL-1β, TNFα, and IgM, indicating that the vaccine stimulates both innate and adaptive immune responses.

Gut health and microbiota diversity also improved in vaccinated fish. Metagenomic analyses showed a more balanced and diverse gut microbiota in vaccinated fish, with lower levels of harmful pathogens like Vibrio vulnificus and Photobacterium damselae. Importantly, traces of the vaccine's bacterial antigen (V. harveyi strain VH1) were detected in the fish gut, demonstrating that the vaccine remains intact through digestion.

In summary, the ViVac feed-based vaccine offers promising benefits for marine aquaculture by improving feed efficiency, enhancing immune responses, reducing harmful pathogens, and promoting gut health. While these findings highlight the vaccine's potential, further refinements in the vaccination schedule and production processes are recommended to maximize its effectiveness.

Professor Juan Mosqueda

Professor
Autonomous University of Queretaro
Mexico

Collaborators:

Dr Tharangani Herath, Senior Lecturer, Harper Adams University, UK

Dr J. Gustavo Ramirez-Paredes, Aquaculture Business Manager, Ridgeway Biologicals Ltd. a Ceva Santé Animale Company, UK

Dr Ariadne Hernández-Pérez, Associate Professor, National Autonomous University of Mexico, Mexico

Summary

Vaccines can enhance sustainability in tilapia farming by reducing the environmental impact of disease outbreaks and the resultant antimicrobial use. Current practices of widespread antimicrobial use in aquaculture, particularly in low and middle-income countries (LMICs) have a negative consequence for water quality, ecosystem health, and the surrounding environment increasing the risk of AMR development and transmission. The use of vaccines could diminish these issues, but there's a lack of vaccines effective against multiple strains of pathogens.

This project proposes to develop and test a vaccine against conserved antigens in tilapia serotypes of Streptococcus agalactiae, a major pathogen in tilapia farming. Also known as Group B streptococcus (GBS) in humans, GBS has serious impacts on infants, and pregnant women, and can cause foodborne illness in adults. GBS's genetic diversity in tilapia makes the development of a conventional cross-protective vaccine challenging. Utilizing state-of-art immune-bioinformatics, the project will develop a chimeric vaccine with multiple pathogen signatures to stimulate B and T cells in fish.

Led by an LMICS scientist, in partnership with UK and Mexico scientists from academia and industry, this project forms a skilful interdisciplinary team that has expertise in immunoinformatic, vaccine development, efficacy testing, aquaculture, and aquatic veterinary sciences. This vaccine will protect tilapia from deadly GBS and humans from serious foodborne illnesses with serious long-term consequences. The cross-disciplinary platform that the project develops can be adopted to control diseases and AMR across a number of pathogens causing diseases in fish and other animals.

Project outcomes

Tilapia aquaculture is vital for food security and protein supply in low- and middle-income countries. However, its sustainability is increasingly threatened by infectious diseases, particularly Group B Streptococcus (GBS), caused by Streptococcus agalactiae. This pathogen affects tilapia through multiple serotypes, leading to significant biomass loss, economic hardship for producers, and the risk of foodborne disease transmission. Compounding the issue, GBS is also known to cause severe infections in humans. In the absence of commercial vaccines, disease control in tilapia farming relies heavily on antibiotic use, which contributes to the global challenge of antimicrobial resistance (AMR).

To address this urgent need, our project focuses on designing a novel, immunoinformatics-guided multi-epitope vaccine that targets conserved surface antigens of S. agalactiae. This strategy aims to reduce dependence on antibiotics and enhance the sustainability of tilapia aquaculture.

Using advanced immunoinformatic tools such as BCEPred, ABCpred, BepiPred 2.0, and IEDB MHC I and II predictors, we screened the proteome of S. agalactiae to identify peptides containing predicted B-cell and T-cell epitopes. A total of 113 peptides were initially selected for further analysis. Each candidate peptide underwent additional screening to ensure: (1) 100% conservation across global S. agalactiae sequences; (2) no homology with the tilapia proteome; and (3) absence from the proteomes of commensal microbiota. These rigorous filters narrowed the list down to 19 highly specific and conserved peptides.

These selected peptides were synthesized either in linear forms or in a multi-antigenic peptide (MAPS 8) structure to enhance immunogenicity. They were solubilized and emulsified using Montanide 763, a commercially available fish adjuvant known for its efficacy in aquatic vaccine formulations.

For the in vivo immunization trial, 80 adult tilapias (800–1000 g) were divided into groups of four and immunized intraperitoneally three times. Each dose consisted of 1 mL containing 20 µg of peptide emulsified with adjuvant. Following immunization, serum samples were collected to evaluate the presence of IgM-specific antibodies using indirect ELISA. Immunogenic peptides were identified and selected for subsequent in vitro assays to evaluate their capacity to neutralize S. agalactiae.

Promising peptide candidates were encoded into a synthetic multi-epitope gene designed to produce a recombinant multi-antigenic protein. Although a challenged experiment was planned, was not delivered due to time constrains and failing request for extension.

The goal of this project is to develop a safe, effective, and sustainable epitope-based recombinant vaccine that can stimulate strong and durable immune responses in tilapia. This innovative approach addresses the pressing need for antibiotic alternatives in aquaculture, contributes to the global fight against AMR, and supports food security in vulnerable regions.

We organized three workshops, participated in three national conferences in Mexico and participated in three international meetings presenting our results.

Dr Nopadon Pirarat

Professor
Chulalongkorn University
Thailand

Collaborators:

Dr Sirikorn Kitiyodom, Researcher, Chulalongkorn University, Thailand

MrJakarwan Yostawonkul, PhD student, Chulalongkorn University, Thailand

Ms. Pimwarang Sukkarun, PhD student, Chulalongkorn University, Thailand

Dr Kim Thompson, Principal Investigator, Moredun Research Institute, UK

Summary

Striped catfish, scientifically known as Pangasianodon hypophthalmus, has gained significant recognition as a primary whitefish species in aquaculture worldwide. Its production has seen a remarkable increase to meet the global demand for seafood. This species holds crucial importance for many low to middle income countries (LIMCs), especially South-East Asia, as it serves as a vital protein source and generates essential income for numerous low-income families. However, the necessity for intensive farming methods to meet the rising demand has been impeded by several challenges, including substandard fish farming practices, water quality issues, and a surge in infectious disease outbreaks within aquaculture systems. Among these challenges, Bacillary necrosis of Pangasianodon (BNP), a recently identified disease caused by Edwardsiella ictaluri, has emerged as a significant infectious disease concern in intensive striped catfish production. E. ictaluri can affect fish of all ages, but it particularly leads to high mortality rates among fingerling and juvenile fish. During the initial stages of infection, BNP exhibits minimal external symptoms. Clinical signs become apparent shortly before death occurs. Affected fish swim slowly near the water's surface and, upon examination, display pale discoloration on their skin and gills. Additionally, internal examination reveals white spots on the liver, kidney, and spleen. Outbreaks attributed to BNP lead to complete mortality, reaching up to 100%, resulting in significant economic implications. This poses a threat to food security and undermines sustainability in aquaculture. Consequently, the primary approach currently employed to treat BNP is the administration of antibiotics. However, relying on drug application to combat disease outbreaks is expensive and raises significant concerns for both the environment and consumers. It is not a sustainable long-term solution to address the problem. Vaccination is a preferred strategy for managing infectious diseases in aquaculture, as it can effectively prevent disease, reduce economic losses caused by high levels of mortality, and decrease the reliance on antibiotics. Hence, researchers will develop a mucoadhesive cationic lipid-based nano-immersion vaccine for striped catfish (Pangasianodon hypophthalmus) against Edwardsiella ictaluri.

Project outcomes

Striped catfish (Pangasianodon hypophthalmus) is a key whitefish species in global aquaculture, particularly vital for low- and middle-income countries (LMICs) in Southeast Asia. It serves as an essential source of protein and income but faces significant challenges due to intensive farming practices, poor management, water quality issues, and infectious diseases like Bacillary necrosis of Pangasianodon (BNP), caused by Edwardsiella ictaluri. BNP affects fish of all ages, leading to high mortality rates, especially in fingerlings and juveniles, with symptoms such as pale skin and white internal spots. Outbreaks of BNP can result in up to 100% mortality, posing severe threats to food security and sustainability. Given the high cost and unsustainability of antibiotic treatments, vaccination has emerged as a preferred strategy. This study aimed to develop a mucoadhesive cationic nanoemulsion vaccine, characterize its properties, assess its mucoadhesive abilities, evaluate skin penetration, and determine the efficacy of the vaccine against E. ictaluri infection in striped catfish. Healthy striped catfish were immersed in either formalin-killed sonicated cells (FK-SC), used as a positive control, or in chitosan nanoemulsion (CS-NE) and cetyltrimethylammonium bromide CTAB nanoemulsion (CAT-NE) for 30 minutes, followed by a challenge with E. ictaluri at 30 and 45 days post-vaccination (dpv). Fish survival was monitored for 15 days after each challenge, and specific IgM antibody levels were measured in serum and mucus up to 28 dpv, along with the expression of inflammatory genes. The CS-NE vaccine demonstrated promising characteristics, including a nano-sized (261 nm), positively charged (32.9 mV) structure with a PDI of less than 0.3, ensuring stability at 25°C for 90 days. CS-NE exhibited superior gill fluorescence intensity in striped catfish post-immersion and was the only vaccine to achieve a significant, time-dependent increase in skin penetration, reaching depths of 80 µm and 100 µm after 1 and 5 minutes, respectively. Additionally, CS-NE provided higher relative percent survival (RPS) at 30 and 45 days dpv compared to FK-SC and CAT-NE. Vaccination with CS-NE and CAT-NE also resulted in significantly elevated serum and mucus IgM levels. Significant Differences in the expression of MHC-2 and IL-1 key immune gene marker were observed in gill, kidney and splenic tissues post-vaccination in CS-NE vaccines, highlighting the great adjuvant potential of cationic polysaccharide chitosan potentiating mucosal immune response by triggering and induction of IL-1b secretion in antigen presenting cells.

Dr Hina Raza

Research Associate Bahauddin Zakariya University
Pakistan

Collaborators:

Dr Sudaxshina Murdan, Reader / Associate Professor in Pharmaceutics, University College London, UK

Dr Mubashir Aziz, Associate Professor, Bahauddin Zakariya University Multan (DMMG), Pakistan

Dr Ahsan Sattar Shiekh, Associate Professor, University of Lahore, Pakistan

Mr Asim Raza Khosa, CEO, POULVET Pharmaceutical Pvt. Ltd, Pakistan

Summary

Avian pathogenic Escherichia coli (E. Coli), an etiologic agent of colibacillosis in poultry, is responsible for great economic losses worldwide with a special focus on middle-income countries. It has been an abusive practice globally to use antibiotics prophylactically to prevent E. coli outbreaks. Antibiotics are routinely added in bulk to poultry diets globally, particularly in LMIC, to prevent various diseases and as growth promotors. The situation is worst in LMIC countries such as Pakistan due to lack of regulatory bodies, awareness, and facilities, the only available vaccine is not in reach of local farmers due to vaccine cost and availability issues. A holistic approach is required to manufacture a vaccine against E.Coli in Pakistan using local strains. In this project, we are aiming to encapsulate killed inactivated local E. Coli strain in novel pH-responsive polymeric carriers to elicit an immune response in the chick gut to provide maximum protection to birds. We will conduct vaccination and challenge studies and evaluate the immune response induced by our prototype vaccine. This vaccine will prevent the release of antigens in the environment. The project will be led by an initial female researcher from LMIC and Pakistani mid-career researchers, a UK academic scientist, and a veterinary pharmaceutical industry from LMIC (Pakistan). These all will bring together a unique combination of expertise in Pharmaceutics, immunology, Poultry, and veterinary sciences to advance a novel approach to poultry vaccination with a special focus to control E. Coli outbreak particularly in low and middle-income countries.

Project outcomes

Chicken is one of the most popular and consumed sources of protein in the world. Good poultry farming ensures a healthy supply of chicken. However, poultry is susceptible to many diseases such as E. coli and other bacterial outbreaks. Poultry farmers rely heavily on using antibiotics to combat such problems, which is an unsustainable practice, and also poses the continuation of the global AMR problem.In this project, an oral vaccine against E. coli was developed based on inactivated bacteria encapsulated in polymeric microparticles.  The latter can be mixed with feed or drinking water. This will release maximum antigen to the chick gut and boost local immunity of the gut results in increased protection from the infection. To evaluate the vaccine’s efficacy, groups of chicks were immunized, by injection or orally (via feed or drinking water), and then exposed to E.Coli.  We found survival was higher for chicks immunized orally than those immunized by injection or controlled non-vaccinated chicks.  Our findings confirm the potential of oral vaccines to prevent infections in poultry and reduce antibiotic use, the risk of worsening antimicrobial resistance, and economic loss to farmers.