Pump-Priming Project Awards - Round 11.1 Awardees

Professor Kannan Ganapathy

Professor of Poultry Infection, Immunity, and Vaccinology
University of Liverpool, UK

Collaborators:

Professor Nicholas Evans, Professor, University of Liverpool, UK

Dr M.C Harish, Assistant Professor, Thiruvalluvar University, India

Dr Emily Herschell-Kelly, Research Assistant, University of Liverpool, UK

Summary

Mycoplasma gallisepticum (MG), a bacterium lacking a cell wall, is classified under the class Mollicutes, within the Mycoplasmataceae family. In poultry, it causes respiratory and reproductive diseases and has been identified as the fifth most economically significant disease by the World Organisation for Animal Health. The disease leads to increased mortality, decreased body weight, a higher feed conversion ratio, heightened severity of respiratory diseases, reduced egg production, and a decline in hatchability.

Co-infection with respiratory viruses and other bacteria leads to increased antibiotic use and a higher risk of antimicrobial resistance. The most effective way to prevent infections, diseases, and losses from MG is through vaccination. Current vaccines are not fully effective at inducing mucosal, cellular, and humoral immunity, nor in preventing MG infections, diseases, or losses.

We propose the development of a multiepitope vaccine targeting the cytoadherence protein molecules GapA, CrMA, HLP3, and PLPA in MG. These proteins are involved in colonisation and immunopathogenesis. Thus, the cytoadherence proteins GapA, CrMA, HLP3, and PLPA can serve as potent candidates for the development of a multiepitope vaccine against MG. Host immune responses are primarily directed against these proteins.

We intend to synthesise these proteins as multiepitopes in Escherichia coli at the University of Liverpool (UoL) and in a plant at Thiruvaluvar University (India), respectively. The immunogenicity of the above multiepitope will be assessed in chickens at the Uol. Based on the preliminary results, in conjunction with research and industry collaborators, we will apply for additional funding.

Project Outcomes

 Mycoplasma gallisepticum (MG) is an important bacterial pathogen of poultry that can cause respiratory disease, reduced productivity and economic losses in chicken production. Current vaccines do not always provide consistent protection and may have limitations associated with the use of live or non-replicating vaccine approaches. This project, therefore, investigated two multiepitope vaccine candidates designed for expression in plant (TU vaccine) and Escherichia coli (UoL vaccine) systems to stimulate an immune response.

Multiepitope vaccine candidates were successfully produced in the E. coli-based expression system. Successful production of the vaccine proteins provided the basis for comparing their ability to stimulate immunity and protect chickens against MG infection.

The second phase of the project evaluated the vaccines in chickens. Birds were vaccinated intramuscularly, alongside appropriate control groups. Twenty-one days after vaccination, a proportion of the birds were challenged with an MG reference strain using an aerosol challenge model. Following the challenge, birds were monitored for clinical signs for ten days. Samples collected before and after challenge included serum and respiratory swabs, while tracheal and air-sac tissues were examined for gross and microscopic changes.

Analysis of the pre-challenge serum samples using an in-house indirect ELISA demonstrated that both vaccine formulations (TU and UoL vaccine) elicited a measurable, significantly increased humoral immune response (antibodies) compared with the control groups. The UoL vaccine showed a higher antibody-associated response, as indicated by absorbance measurements, indicating strong immunogenicity of the vaccine candidate.

The study's challenge component also successfully established the MG infection model. MG was recovered from respiratory swab samples following challenge using culture-based methods, including confirmation through re-isolation, demonstrating that the challenge resulted in detectable MG infection and shedding.

Although some indications of improved clinical outcomes and reduced respiratory lesions were observed in vaccinated groups compared with controls, the relatively small number of birds used in this preliminary study limited the ability to conclusively determine protective efficacy. Therefore, the findings should be considered encouraging preliminary evidence rather than definitive proof of protection.

Overall, the project successfully demonstrated that the multiepitope MG vaccine candidates designed both in plant and E. coli expression systems can stimulate a significant immune response in chickens. The findings support further investigation of this vaccine approach as a potential alternative to existing MG vaccination strategies. Future studies using larger numbers of birds, carefully controlled challenge conditions, and optimisation of the challenge dose will be important for determining the level and consistency of protection provided by these promising vaccine candidates.

Dr David Jarvis

Chief Technical Officer
Liselo Labs, South Africa; Lesotho

Collaborators:

Professor Giuseppe Stefanetti, Associate Professor, University of Urbino, Italy

Professor Faith Osier, Professor of Immunology and Vaccinology, Imperial College London, UK

Summary 

Conjugate vaccines are highly effective but expensive solutions for preventing diseases caused by bacteria such as Klebsiella pneumoniae, a major cause of severe infections worldwide. This project proposes an innovative, affordable alternative: antibodies designed to mimic cognate vaccines, significantly reducing cost of production and enhancing accessibility for low- and middle-income countries (LMICs).

The biomimetic antibodies will be produced using a two-phase immunization protocol. In the first phase, quails will be immunized with a K. pneumoniae capsular polysaccharide conjugate vaccine (building on prior BactiVac-funded work in Klebsiella glycoconjugate design) and vaccine-specific antibodies will be purified from egg yolks. In the second phase, a new set of quails will be immunized with the purified antibodies to elicit an anti-anti-vaccine (vaccine mimicking) antibody response. These antibodies are expected to act as molecular mimics of key regions on the vaccine. By structurally resembling vaccine surface features recognized by the immune system, these antibodies could serve as functional surrogates, enabling the development and validation of diagnostic assays and potentially stimulating similar immune responses as the original vaccine antigen.

The biomimetic antibodies will be characterised to assess their binding properties, specificity, and functional similarity to the original vaccine. If validated, these antibodies could serve as cost-effective tools to evaluate vaccine candidates and enable affordable preclinical testing, especially in LMIC settings. This project is designed to deliver proof-of-concept data for follow-on translational funding and contributes to global efforts to tackle antimicrobial resistance through scalable vaccine technologies.

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-Hernandez, Secihti Fellow-IMSS, Medical Research Unit on Immunochemistry IMSS, Mexico

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

Summary

Salmonella infections remain a major global public health concern, exacerbated by the rising threat of antimicrobial resistance. In a previous BactiVac-funded project (BVNCP7-29), we successfully designed and synthesised mRNA constructs encoding the porins OmpC, OmpF, and OmpD, which were effectively translated in vitro and recognised by porin-specific antibodies.

Although the OmpD mRNA construct failed to elicit detectable antibody responses and initial challenge models showed no protection, a 10-day intragastric challenge demonstrated improved survival in immunised mice, albeit without reaching statistical significance. These findings underscore the need for further optimisation to obtain a good conformation in eukaryotic cells and in-depth characterisation of the immune responses elicited. 

The current project aims to refine the porin mRNA vaccine prototypes, evaluate their expression in different eukaryotic cells and test their immunogenicity. The results obtained on the vaccine design could impact the field of bacterial vaccines made with mRNA technology, which can be applicable to other transmembrane bacterial proteins present in antimicrobial resistance strains. These advances can be leverage in an emergency where no other treatment is available taking advantage of the rapid production of mRNA vaccines.

Project Outcomes

Salmonella enterica causes substantial disease worldwide, particularly in low- and middle-income countries (LMICs), and the emergence of antimicrobial-resistant strains reinforces the need for new preventive approaches. This project investigated whether messenger RNA (mRNA) technology could be adapted to develop vaccines targeting Salmonella porins, important bacterial outer-membrane proteins.

Building on our previous BactiVac-funded work, we designed three new mRNA vaccine candidates based on OmpD, the dominant porin of Salmonella Typhimurium. The constructs were engineered to improve expression and antigen presentation in mammalian cells. All three mRNAs were successfully produced and translated, and their encoded proteins were detected following expression in mammalian cells, including at the cell surface.

When evaluated in mice, the mRNA candidates did not induce detectable OmpD-specific antibody responses. Importantly, however, one construct, OmpD-2, induced a significant OmpD-specific T-cell response, measured by IFN-γ production. OmpD-2 contains a furin cleavage site designed to promote secretion of the expressed antigen, suggesting that antigen processing and secretion may be important determinants of the immune response generated by bacterial antigens delivered using mRNA technology.

The project therefore identified both a major challenge and a promising route forward for bacterial mRNA vaccines. These findings are now informing the next generation of vaccine constructs and formulation strategies, including next-generation lipid nanoparticle/liposomal technologies. The project has also generated follow-on funding activity aimed at further optimising the platform and evaluating its potential for protection against Salmonella infection.

Dr Claudia Ivette Juarez Molina

Postdoctoral research scientist
University of Oxford, UK

Collaborators:

Dr Elizabeth Jones, Postdoctoral Research Scientist, University of Oxford, UK

Dr Anna Rydlova, Research Associate, Imperial College London, UK

Prof Farah Qamar, Professor and Associate Dean Research, Aga Khan University, Pakistan

Dr Young Chan Kim, Sir Henry Wellcome Fellow, Principal Investigator (PI) at OVG, University of Oxford, UK

Dr James Meiring, Senior Clinical Research Scientist, University of Liverpool, UK

Summary

Enteric fever and invasive non-typhoidal Salmonella (iNTS) cause hundreds of thousands of deaths each year. Current vaccines only protect against one serovar at a time. In South and South-East Asia, both S. Typhi and S. Paratyphi A circulate; in sub-Saharan Africa, S. Typhimurium and S. Enteritidis dominate, but licensed vaccines cover only S. Typhi leaving vulnerable populations partly unprotected. This project aims to bridge that gap by identifying bacterial proteins recognised by our immune system that are cross-reactive across these major Salmonella serovars.

Using Controlled Human Infection Models (CHIMs), volunteers are deliberately exposed to one of three serovars (S. Typhi, S. Paratyphi A or S. Typhimurium) under strict clinical supervision. Blood samples are collected from those who either develop disease or remain well. From these samples, antibodies (IgG, IgA and IgM) will be purified and immobilised onto columns. Bacterial extracts from each serovar are then passed over the antibody-loaded columns, capturing only those proteins recognised by the immune system. The bound proteins are eluted and identified by high-resolution mass spectrometry.

By comparing which antigens bind across different serovars and in protected versus susceptible individuals, the study aims to generate a ranked list of promising targets for a multivalent or “pan-Salmonella” vaccine. This evidence-based roadmap will inform formulations that combine two or three antigens tailored to regions where multiple serovars co-circulate.

We aim to help deliver broadly protective vaccines that reduce Salmonella outbreaks, curb antibiotic use and save lives, especially in low- and middle-income countries that bear the greatest burden.

Project Outcomes

Enteric fever and invasive non-typhoidal Salmonella together cause a huge burden of illness worldwide, yet current vaccines protect against only one Salmonella serovar at a time. This project set out to identify bacterial proteins recognised by the immune system across three major disease-causing serovars – S. Typhi, S. Paratyphi A and S. Typhimurium – using blood samples from volunteers who took part in Controlled Human Infection Model (CHIM) studies.

The team successfully grew and characterised all three challenge bacteria, and purified IgG and IgM antibodies from CHIM participant plasma collected before and after challenge, from both participants who developed disease and those who remained protected. These antibodies were coupled to affinity columns and used to try to capture the bacterial proteins they recognise for each serovar.

IgA antibody yields recovered from the plasma pools were too low to support reliable capture and mass spectrometry analysis, so this isotype was not progressed further. Subsequent quality checks also showed that the IgM antibody-capture step had not successfully captured bacterial antigens above background levels, so IgM was also excluded. Work instead focused on generating a robust IgG dataset, which is ready to be submitted for high-resolution mass spectrometry pending funding. Generating the resulting antigen lists and cross-serovar antigen ranking requires further laboratory and analysis time beyond the funded project period. These findings, once completed, are expected to directly inform the design of next-generation multivalent Salmonella vaccine candidates, particularly benefiting regions of Africa and Asia where more than one serovar co-circulates.

Professor Tatiana de Castro Abreu Pinto

Associate Professor
Universidade Federal do Rio de Janeiro, Brazil

Collaborators:

Dr Elita Jauneikaite, Assistant Professor in Microbiome and Bacterial Genomics, Imperial College London, UK

Professor Shiranee Sriskandan, Professor of Infectious Diseases, Imperial College London, UK

Summary

Group A Streptococcus (Strep A) is a common type of bacteria responsible for mild to severe infections, like sore throat and flesh-eating disease. Strep A can also lead to long-term complications, like rheumatic heart disease. Recently there was a sharp rise in serious Strep A infections in several countries, including Brazil, which was linked to specific Strep A variants. This new concerning trend called out the need of further investigation in Strep A, especially in low- and middle-income countries like Brazil, where we still have very limited information available.

The World Health Organization established Strep A vaccine as a top priority to prevent deaths and long-term health problems, and to reduce the use of antibiotics. Global research efforts are underway to better understand the regional traits of Strep A and inform vaccine developers; however, in South America there are no studies being conducted. This project will analyse Strep A from Brazil, helping us to better understand which variants may be spreading, and how they compare to those found in other countries. We will also look back in time, comparing new isolates with older ones, giving us clues about how the bacteria have changed over the years.

Finally, we will build a system in the Brazilian laboratory aimed at monitoring Strep A variants circulating in the hospitals, as recommended by international and regional health authorities. The data gathered will allow estimation of the impact of the proposed leading strepA vaccine candidates while delivering epidemiological insights and hopefully leading to beneficial changes.

Professor John Tregoning

Professor
Imperial College London, UK

Collaborators:

Professor Mariagrazia Pizza, Professor, Imperial College London, UK 

Dr Giuseppe Stefanetti, Associate Professor, University of Urbino, Italy

Summary 

Acinetobacter baumannii is one of the six major antibiotic-resistant bacterial threats identified by the WHO. It causes severe hospital-acquired infections, particularly in intensive care units, and has a high burden in low- and middle-income countries (LMICs). Despite this, no vaccine currently exists.

Bacterial surfaces carry two main vaccine targets: proteins and polysaccharides. Polysaccharides alone often generate weak immune responses, especially in young children, but their effectiveness increases when chemically linked to a protein (glycoconjugation). This approach has revolutionized bacterial vaccinology and underpins successful vaccines against Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis and Salmonella Typhi. Licensed conjugate vaccines use a narrow set of carrier proteins, such as tetanus toxoid, diphtheria toxoid, a non-toxic diphtheria derivative (CRM197), or Haemophilus protein D. However, recent studies suggest that using proteins from the same pathogen may improve vaccine immunogenicity.

We have previously identified protective proteins from A. baumannii and, through ongoing BactiVac-funded studies, are evaluating novel protein antigens from K. pneumoniae. This project will generate conjugate vaccines pairing polysaccharides from one pathogen with proteins from the same or the other species—allowing us to explore the concept of syndromic vaccines. Such vaccines aim to protect against multiple pathogens that cause the same clinical syndrome (e.g. hospital-acquired pneumonia), rather than targeting one organism at a time.

If successful, this approach could form the basis of a next-generation vaccine strategy to prevent polymicrobial, drug-resistant infections, with transformative potential in LMIC healthcare settings.