Pump-Priming Project Awards - Round 8 Awardees

Professor Stephen Baker

Professor of Molecular Microbiology
University of Cambridge, UK

Collaborators:

Dr Timothy Scott, Research Associate, University of Cambridge, UK

Professor Brendan Wren, Professor of Microbial Pathogenesis, London School of Hygiene and Tropical Medicine, UK

Summary

There are currently no licensed vaccines against non-typhoidal Salmonella (NTS), which is associated with significant burden of invasive (iNTS) and diarrheagenic (dNTS) disease in LMICs, particularly those in sub-Saharan Africa. We aim to generate a series of low-cost experimental glycoconjugate vaccines against one of the principal NTS serovars, Typhimurium (STm), using the Wren group’s Protein Glycan Coupling Technology (PGCT). Immunogenic protein antigens identified by the Baker group from LMIC clinical studies and conserved across NTS, typhoid and paratyphoid, will be biologically conjugated with STm O-antigen, and high-throughput screening will be exploited to optimise protein glycoconjugate vaccine production. Functional immunogenicity and protection of the prototype vaccines will be assessed in an established iNTS mouse model. This project aims to provide proof of principal that novel protein antigens can serve as the carrier proteins for broad-coverage, standalone NTS or combination NTS/typhoid/paratyphoid conjugate vaccines. Additionally, we aim to demonstrate that PGCT can be deployed to create NTS vaccines and provide initial preclinical data that would facilitate follow-on funding and clinical studies.

Project outcomes

This study aimed to produce prototype biconjugate vaccines cheaply and efficiently against a neglected enteric bacterial disease, invasive non-typhoidal Salmonella (iNTS), which causes a significant burden of disease and mortality across LMICs and are a leading cause of bacteraemia in sub-Saharan Africa, particular affecting children under five. We used engineered bacterial cells to assemble the vaccine components and used a variety of methods and expression strategies aimed at enhancing vaccine production, with the goal of producing sufficient vaccine for animal trials.

Dr Rochelle Da Costa

Postdoctoral Research Fellow 
The University of Queensland, Australia

Collaborators:

Professor Ian Henderson, Director - Institute for Molecular Bioscience, The University of Queensland, Australia

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

Summary

Salmonella enterica is a leading cause of food poisoning and typhoid disease. Compounding this issue is the emergence of multi-drug resistant strains, posing grave risks to human health. Combatting the escalating threat of antibiotic resistance necessitates novel intervention methods, notably vaccines. Yet, existing Salmonella vaccines offer only fleeting protection against typhoid fever and lack efficacy in safeguarding young individuals. Previously, our research pinpointed a set of four highly conserved Salmonella antigens. We demonstrated that immunization with these antigens conferred protection in a murine model against lethal Salmonella challenges. Notably, our findings extended to providing defence against heterologous challenges with distinct serovars. Fuelled by the COVID-19 pandemic, mRNA vaccines have emerged as potent alternatives to conventional protein subunit vaccines. Their attributes include heightened potency, safety, efficacy, rapid clinical development, and potential for cost-effective manufacturing. Building upon this momentum, we hypothesize that harnessing mRNA technology will serve to complement and potentially enhance the outcomes achieved thus far in Salmonella vaccine development. Here we will benchmark mRNA versions of our vaccine components against the protein vaccines, establishing if they are effective at reducing bacterial loads in an acute model of infection, and if they prolong survival against a lethal challenge.

Project Outcomes

Salmonella enterica infects a broad range of hosts and is a leading cause of food poisoning and typhoid diseases. The rise of antibiotic-resistant Salmonella strains has restricted treatment options. There is an urgent need for a vaccine that provides long lasting protection against all strains of Salmonella. We have previously identified four highly conserved antigens in Salmonella and demonstrated that immunisation with these antigens induces robust antibody responses and provides protection against lethal challenges with distinct serovars of Salmonella. mRNA vaccine delivery systems present a promising alternative to conventional protein-based vaccines. Here, we benchmarked mRNA sequences encoding the four Salmonella antigens against the protein vaccines. The mRNA versions of our vaccines elicited antibody responses in mice which were comparable to the protein-based vaccine; however, they did not provide similar protective efficacy at early time points of infection. Furthermore, in the survival assay, the mRNA vaccines alone exhibited similar mortality rates to the non-immunised group. Although, combining the mRNA vaccines with OMVs extended survival in mice, the four antigen protein-based vaccines outperformed all other groups.

Professor Jeremy Derrick

Professor of Molecular Biology
The University of Manchester, UK

Collaborators:

Dr Anja Saso, Paediatric Registrar & Wellcome Trust Global Health Clinical Research Fellow, Vaccine & Immunity Theme MRC Unit The Gambia (MRCG), Department of Clinical Research London School of Hygiene & Tropical Medicine (LSHTM), Gambia & UK

Professor Beate Kampmann, Professor of Paediatric Infection & Immunity (LSHTM), Professor of Global Health (LSHTM, Charité), Department of Clinical Research, London School of Hygiene & Tropical Medicine, Centre for Global Health Charité, UK & Germany

Summary

Despite the availability of vaccines, whooping cough (pertussis) remains a leading cause of vaccine-preventable deaths, particularly in children under five and Sub-Saharan Africa. One reason for this is the switch that occurred from the whole-cell (wP) to the subunit acellular (aP) pertussis vaccine within childhood immunisation schedules. It is widely accepted that aP vaccination, while protecting against severe disease, has a shorter duration of protection and requires frequent booster doses. Importantly, boosters are recommended in pregnant women to reduce deaths in newborn infants. Our previous work conducting vaccine trials in The Gambia suggests that levels of antibody to vaccine antigens may be significantly reduced in infants born to mothers who received the pertussis vaccine in pregnancy. In this study we will use a method, termed microarray antigen profiling, to compare the antibody responses in mothers and infants. This method produces a ‘fingerprint’ response for each vaccine recipient, providing information on the targets for the antibodies produced. We can use machine learning methods (a type of AI) to map antibody responses to various parts of the pertussis toxin which is used in the aP vaccine. This is a small scale, proof of principle study which could lead to more detailed investigations into the longer-term immunological impact of pertussis immunisation during pregnancy. It may help to design more effective maternal and infant vaccine strategies, particularly in poorer countries where pertussis vaccines are often not routinely given in pregnancy.

Project Outcomes

Whooping cough is caused by the bacterium Bordetella pertussis and is one of the leading causes of vaccine-preventable deaths worldwide. The infection particularly affects children under five and populations from Sub-Saharan Africa; it is a leading cause of vaccine-preventable deaths. One reason for this is the switch that occurred from the whole-cell (wP) to the subunit acellular (aP) pertussis vaccine within childhood immunisation schedules. It is widely accepted that aP vaccination, while protecting against severe disease, has a shorter duration of protection and requires frequent booster doses. Importantly, boosters are recommended in pregnant women to reduce deaths in newborn infants. Our previous work conducting vaccine trials in The Gambia suggests that levels of antibody to vaccine antigens may be significantly reduced in infants born to mothers who received the pertussis vaccine in pregnancy. In this study we have used a method, termed microarray antigen profiling, to compare the antibody responses in mothers and infants. The aP and wP vaccines are complex, consisting of multiple protein components. Following vaccination, antibodies are produced against these components- in the case of this project, our focus is on protein antigens. To analyse the antibody responses, we prepared a dedicated microarray, which contains fragments of protein antigens, in the form of peptides, which are immobilised onto a glass slide. Antibodies from the serum of vaccinated individuals are then bound to the peptides. In this way, the antibody responses to these individual components form a ‘fingerprint’-type response for each vaccine recipient. We can then compare these fingerprint profiles against each other: machine learning methods (a type of AI) are particularly powerful at picking out these patterns. Using this approach, we were able to show that there was clear differentiation between antibodies from mothers and infants up to 9 months after birth. Shortly after birth, at the 2-month timepoint, profiles from the infants suggest that they originate from transmission of maternal antibodies. 9 months after delivery, however, the pattern associated with maternal antibody faded and the remaining profiles were attributable to antibody responses from the infants. This study represents proof of principle that peptide microarrays can play a useful role in understanding how vaccination of mothers may help transmit immunity to their children. This could lead to more detailed investigations into the longer-term immunological impact of pertussis immunisation during pregnancy. It may also help to design more effective maternal and infant vaccine strategies, particularly in poorer countries where pertussis vaccines are often not routinely given in pregnancy.

Dr Helge Dorfmueller

Principal Investigator/Senior Lecturer
University of Dundee, UK

Collaborators:

Dr Kishore Alugupalli, CEO, TurboVax Inc, USA

Summary

Group A Streptococcus (GAS) is a human-exclusive bacterial pathogen killing more than 650,000 people annually, and no licensed vaccine exists. GAS bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. We engineered the Group A Carbohydrate biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within E. coli. Purified RhaPS-glycoconjugates elicited specific antibodies in mice and rabbits and bound to the surface of multiple GAS strains of diverse M-types, confirming the recombinantly produced RhaPS-glycoconjugates as valuable vaccine candidates. An efficient immunogenicity of antigens/vaccines requires suitable adjuvants. We found immunogenicity of polysaccharide vaccines that lack adjuvants is primarily due to the presence of Toll-like receptor (TLR) ligands, and that both antigen receptor and TLR signalling are essential for the antibody responses in vivo. Based on this rationale, we have developed a monophosphoryl lipid A (a TLR4-ligand)-based adjuvant formulation named Turbo. When adjuvanted with Turbo, FDA-approved or WHO-prequalified vaccines induced high levels of IgG of all isotypes across all ages of mice, minimised booster requirements, and prolonged the antibody responses for at least one year in mice, suggesting that Turbo as an adjuvant promotes durable and polyfunctional antibodies and maximise the efficacy of a vaccine. The goal of this proposal is to test the immunogenicity of RhaPS-glycoconjugates adjuvanted with Turbo or Alum, a widely used adjuvant as a comparator in infant and adult mice.

Project Outcomes

This study evaluated the immune response generated by different formulations of a glycoconjugate vaccine in mice. The vaccine contained a key protein (Protein1-RhaPS) and was tested with two different adjuvants—Alum and Turbo—to assess their effectiveness in enhancing immunity. The main focus was to investigate the generation of antibodies against the vaccine candidate and the activation of cytokines in spleen restimulation assays. In summary, both adjuvants produce vaccine specific antibodies, and our data revealed that the Turbo adjuvant may induce cell-mediated immunity, thereby potentially triggering the activation of memory cells.

Professor Mohamed Elhadidy

Professor of Biomedical Sciences and
Director of Centre for Genomics 
Zewail City of Science and Technology, Egypt

Collaborators:

Dr Sherif Abouelhadid, Deputy Director, AMR Centre- London School of Hygiene & Tropical Medicine LSHTM, Infection Biology, London School of Hygiene & Tropical Medicine LSHTM, UK

Dr Eman Badr, Associate Professor of Biomedical Sciences, Biomedical Sciences Program, Zewail City of Science and Technology, Egypt

Summary

Staphylococcus aureus (S. aureus) is one of six nosocomial ESKAPE pathogens, showing concerning levels of multidrug resistance (MDR). It causes Staphylococcus aureus bacteremia (SAB), a predominant cause of bloodstream infection, leading to 20-30 cases per 100,000 per year. While SAB causes a global health burden, its impact on low- and middle-income countries (LMIC) remains underrepresented. Currently, there is no vaccine effective against this major pathogen. The objective of this project is to pinpoint novel protein vaccine targets effective against both local and global S. aureus clinical isolates, with the goal of providing cross protection against diverse strains, including antibiotic-resistant variants. This will provide the initial steps of effective broad-spectrum vaccine through obtaining reliable candidates directly from clinical isolates. We will implement pan-genome reverse vaccinology to screen and characterize staphylococcal vaccine targets. Our preliminary data reveals six primary categories of vaccine candidates identified through our reverse vaccinology approach, conducted on a limited number of isolates. These categories include cell-surface adhesins, secretion systems, siderophores, exotoxins, cell wall-localized proteins, and proteins involved in immune evasion. This approach will establish a solid foundation for us to seek additional significant funding to clone promising candidates and to evaluate the immunogenicity and protective efficacy, thereby enabling the proposed workflow to be further implemented to screen potential vaccine candidates among a wide range of MDR pathogens and prevent the dissemination and emergence of novel MDR pathogens in LMIC.

Project Outcomes

The current project aims to address the issue of multidrug-resistant (MDR) S. aureus infections by applying a reverse vaccinology approach. The study addressed the reasons behind the failure of previous S. aureus vaccine trials by implementing a comprehensive workflow, called AntiPan. Here we represent the in-silico vaccine discovery pipeline, AntiPan, which augments pangenome estimation, reverse vaccinology, and immuno-informatics to predict subunit vaccine antigen candidates against MDR S. aureus. By considering S. aureus genomic diversity, AntiPan offers a tailored approach and lay the foundation for global vaccine design utilizing broader genome datasets. The AntiPan tool suite is a command-line interface designed to offer a high throughput and user-friendly platform for discovering vaccine targets. It is applicable to multidrug-resistant pathogens, and is publicly available at https://github.com/ComputationalBiologyLab/AntiPan.

AntiPan highlights antigens central to Staphylococcus aureus pathogenesis, immune evasion, and virulence, while addressing several limitations of previous subunit antigen prediction methods. In this project, three distinct S. aureus genome datasets were analyzed:

1. A set of 198 Egyptian clinical isolates previously deposited in GenBank;
2. A newly generated dataset of 156 Egyptian S. aureus isolates, sequenced specifically for this project.
3. A collection of 572 global S. aureus genomes, also publicly available in GenBank.

This diverse genomic representation enhances the predictive power and global applicability of the AntiPan pipeline.

The reverse vaccinology analysis of the first set of 198 Egyptian clinical S. aureus isolates yielded 29 antigen candidates, 24 non-toxin and 5 toxin antigens, which were implicated in host invasion, nutrient acquisition, and immune evasion. Epitope mapping revealed overlapping B- and T-cell epitopes. The functional annotation of these antigens showed their role in secretion systems, adhesion, and cytolytic activity. The top surface-exposed/secreted and highly-immunogenic potential antigen candidates (PACs) included EbpS, AmiA, TagH, SspB, IsdC, EssA, SirA, EsxA, HlgC, and HlgB were shortlisted for future experimental validation. Molecular docking analysis showed strong binding of candidate antigens to critical residues in TLR1/TLR2 and TLR4/MD-2 heterodimer complexes. Notably, IsdC, AmiA, and TagH demonstrated strong and complementary binding to both TLR heterodimer complexes, marking them as top vaccine candidates.

The second set of 354 Egyptian S. aureus genomes, which included the abovementioned 198 genomes in addition to 156 newly sequenced genomes, showed 33 antigen candidates after reverse vaccinology filtration, 28 non-toxin and 5 toxin antigens involved in the abovementioned virulence mechanisms including bacterial pathogenesis, host cell invasion, nutrient acquisition, and hemolysins. The immune-informatics analyses shortlisted 10 surface-exposed/secreted and highly-immunogenic antigens comprising the previously mentioned PACs with IsdD replacing AmiA antigen. These results underline the significance of iron-regulated surface determinant proteins (Isd family) and TagH as promising subunit vaccine targets.

The reverse vaccinology analysis of the third set of 572 global S. aureus genomes showed 39 antigen candidates, 33 non-toxin and 6 toxin antigens involved in various virulence mechanisms including bacterial pathogenesis, nutrient acquisition, adherence, host invasion, and immune evasion. The functional annotation of the beforementioned antigens highlighted key functions and domains such as heme uptake, ABC transporters, iron transport, proteolytic activity, hydrolases, lipase activity, anchorage and adherence, fibrinogen binding, immunoglobulin binding, secretion system VII, LPXTG cell wall anchor, etc. A total of 14 surface-exposed/secreted highly-immunogenic antigen candidates were predicted from the immune-informatics analyses. The data showed a consensus of 8 PACs between the three analyzed S. aureus genomic datasets as represented in the venn diagram below. These global antigens, which included SirA, TagH, EsxA, EbpS, HlgB, HlgC, IsdC, and EssA, are promising targets for formulating an effective vaccine against S. aureus with global protection. Future work would entail protein expression followed by in vitro and in vivo validation of the immunogenic characteristics and safety of the identified PACs for the purpose of developing a successful vaccine formula.

Dr Michael A Jarvis

CSO (TVG) and Associate Professor (Reader)
University of Plymouth/The Vaccine Group Ltd (TVG)
UK

Collaborators:

Professor Hoa Thi Ngo, Head of Bacterial Zoonosis and Associate Professor, Oxford University Clinical Research Unit (OUCRU), Vietnam and Nuffield Department of Medicine (NDM), University of Oxford, Vietnam

Professor Alexander Tucker, Professor and Vice President, University of Cambridge & UK Pig Veterinary Association, UK

Professor Matthew Upton, Professor and Associate Head of School, School of Biomedical Sciences, University of Plymouth, UK

Professor Zhiyong Ma, Director General and Professor, Department of Swine Infectious Diseases, SHVRI, China

Professor Jianchao Wei, Associate Professor at SHVRI, Department of Swine Infectious Diseases (DSID), SHVRI, China

Summary

The COVID-19 pandemic has, without doubt, established the effectiveness of mRNA-based vaccines in fighting microbial pathogens. However, this technology's use in low- and middle-income countries (LMICs) is limited due to high production and formulation costs and the need for ultra-cold temperatures during distribution.

Our BactiVac pump-priming proposal aims to demonstrate proof-of-principle for a new technology called 'RNA-amplification following DNA viral delivery (RADD).' This technology can provide the benefits of mRNA-based vaccines without the limitations of expensive production, transportation and administration.

We will construct RADD-versions of our conventional bovine herpesvirus-4 (BoHV-4) viral-vectored vaccine against the bacterial pathogen Streptococcus suis (S. suis). We will then compare the conventional and RADD versions in vitro for relative S. suis antigen expression levels, stability of antigen expression, and vector genome stability. The comparator S. suis BoHV-4 vaccines have been shown to be immunogenic in pigs and provide moderate protection against S. suis challenge. We anticipate that the RADD version will be genetically stable and express 20-30-fold higher levels of the S. suis target antigen than the conventional viral-vectored version in vitro.

The aim of the current project is to provide proof-of-principle (TRL-2) in vitro data to support subsequent translation of a RADD-based S. suis vaccine into pig immunogenicity/challenge proof-of-concept (TRL-3) studies for a S. suis vaccine. Additionally, data from this project will be used to demonstrate the potential of RADD as a new, innovative technology capable of providing the benefits of mRNA-based vaccine technology without the associated costs, making it suitable for LMIC environments.

Project Outcomes

Our BactiVac pump-priming proposal aimed to demonstrate proof-of-principle equivalent to testing the new technology in initial in vitro studies) for a new enhanced viral vector-based technology. We hypothesised that this technology would be able to provide the benefits of high antigen levels normally associated with mRNA-based vaccines, but without the significant production costs and need for ultracold temperatures during transportation of mRNA vaccines. Such an enhanced viral-vectored vaccine platform would be suited for use in low and middle-income countries (LMICs), where environmental conditions and limited infrastructure severely impact vaccine access.Two different enhancement strategies were used to modify our conventional bovine herpesvirus-4 (BoHV-4) viral vector vaccine against the bacterial pathogen Streptococcus suis (S. suis) (called BoHV-4/S. suis). The primary approach, called RADD, was based on RNA-amplification; a secondary approach, called ENH, was used as an independent alternative strategy to ensure successful completion of the project. RADD and ENH versions of the conventional BoHV-4/S. suis vaccine were designed and constructed. Our conventional BoHV-4/S. suis vaccine, which had been shown to be immunogenic in pigs and to provide protection against S. suis challenge, was used as a comparator for in vitro studies. The primary comparison was measuring S. suis protein levels produced by the different vaccines in vitro using a western immunoblot technique. Before starting the study, we set as a goal a ≥10-fold increase in S. suis protein levels by either approach compared to the conventional version as a 'Go' signal for future advancement of the technology into studies to support translation into TRL-3 (complete in vitro demonstration of the technology and initial in vivo immunogenicity/efficacy studies in pigs).

We successfully constructed RADD and ENH versions of the BoHV-4/S. suis. However, additional development of the RADD-based approach will be required before testing whether the vaccine can achieve the desired (≥10-fold) increase in S. suis protein levels. This unexpected outcome for RADD showed the utility of including the alternative ENH-based enhancement strategy as built-in redundancy for the project. Quantitation of the relative level of S. suis protein produced by the ENH compared to our standard conventional version was >20-fold. We tested the level of protein production multiple times and using separate versions of the ENH-BoHV-4/S. suis vaccine, which showed that the ability of ENH to increase protein levels produced by the BoHV-4 vector was reproducible.

These exciting results demonstrate proof-of-principle at the TRL-2 level for enhancement by ≥10-fold (actually >20-fold for the S-ABC version) by ENH compared to the standard BoHV-4-based viral vector S. suis vaccine, which was the indicated goal of the project. Separate patent applications were filed to protect the RADD and ENH ideas, and commercial vaccine companies have shown substantial interest in the ENH-based technology. We are in the process of signing a contract with one of these companies, which will fund the studies to move the project beyond TRL-3.

Dr Dominic F Kelly

BRC Consultant and Honorary SeniorLecturer in Paediatrics and Vaccinology
Department of Paediatrics and Oxford Vaccine Group
University of Oxford, UK

Collaborators:

Professor Shrijana Shrestha, Professor of Paediatrics, Department of Paediatrics, Patan Academy of Health Sciences, Nepal

Professor Sir Andrew J Pollard, Ashall Professor of Infection and Immunity, Department of Paediatrics and Oxford Vaccine Group, University of Oxford, UK

Summary

Pneumococcal infection is a major cause of pneumonia in children, particularly in low- and middle-income countries (LMICs). Pneumococcal conjugate vaccines (PCVs) have been introduced widely over the past two decades, including Nepal in 2015. However, currently used diagnostic tests, such as culture of pneumococci from blood, are not sensitive for confirming pneumococcal or most other infections. This makes assessment of the impact of PCV against pneumococcal pneumonia difficult, and limits data to support the effectiveness and ongoing need for PCV in LMICs.

Current estimates of the incidence of pneumococcal pneumonia in different populations rely on proxies for pneumococcal infection, such as nasopharyngeal carriage of pneumococcal serotypes, or “consolidation” (opacities) on chest radiographs. This project will use a new type of test to demonstrate how the cause of pneumonia could be more accurately diagnosed by looking at the child’s response to the infection, rather than detecting the pathogen itself. This test measures the expression levels of genes (RNA) in a small volume of blood from children with a microbiologically-confirmed cause of pneumonia.

We propose to use the RNA multiclass pneumonia signature (MPS) to describe the cause of infection in children with pneumonia recruited to Patan Hospital in Nepal between 2015–2017, before and following introduction of 10-valent PCV. This project may enable an assessment of PCV impact against pneumococcal pneumonia in Nepal. It may also establish the principle of using a RNA MPS for diagnosing the cause of infection in a large cohort of children.

Professor John S Tregoning

Professor in Vaccine Immunology
Imperial College London, UK

Collaborators:

Dr David Peeler, Marie Skłodowska-Curie Postdoctoral Research Fellow, Imperial College London, UK

Professor Patrick Stayton, Distinguished Career Professor, University of Washington, USA

Professor Robin Shattock, Chair in Mucosal Infection and Immunity, Imperial College London, UK

Professor Dame Molly Stevens, Professor of Biomedical Materials and Regenerative Medicine, Imperial College London, UK

Dr Mohommad Mainul Ahasan, Head and Assistant General Manager, Incepta Pharmaceuticals LTD., Bangladesh

Summary

The COVID-19 pandemic demonstrated the potential of RNA vaccines for the prevention of viral infections. However, this exciting platform has yet to be proven for use against bacterial pathogens with high levels of antibiotic resistance, such as Acinetobacter baumannii. Further optimisation is needed to ensure the success with SARS-CoV-2 can be replicated with AMR bacteria. Improvements can be made to increase protective immune responses of RNA vaccines while reducing unwanted side effects.

RNA must be formulated with nanomaterials (e.g., lipids, polymers) to enter cells and produce vaccine antigen. Careful engineering of formulation materials can also improve the immunogenicity vs. adverse effect profile of RNA vaccines. Our team’s strength in biomaterial engineering enables us to independently control the biodistribution and innate stimulating effects of RNA itself (mRNA/saRNA; Tregoning/Shattock), the materials used to deliver it (LNPs/polymers; Peeler/Stevens), and targeted adjuvants (Stayton), all from a foundation of LMIC vaccine manufacturing expertise (Incepta).

We hypothesise that specifically activating danger-signalling pathways in antigen presenting cells with exogenous adjuvant can fine tune the adaptive response to RNA vaccines. This project will provide a proof-of-concept application of next generation adjuvants in combination with state-of- the-art RNA vaccines, unlocking the potential of adjuvanted RNA vaccines for immunity against AMR pathogens and beyond.

Project Outcomes

This study explored how adding a special immune stimulant called polySTING affects different types of RNA vaccines against Acinetobacter baumannii (AB), a dangerous antimicrobial-resistant bacteria that infects hospital patients through their lungs. We compared different RNA vaccine nanomaterial delivery formulations (made from fats and plastics), different RNA vaccine types (short- and long-expressing), and different delivery locations (intramuscular and inhaled), as well as the timing of polySTING delivery relative to RNA. We proposed that adding the adjuvant polySTING would enable us to change the type of immune response generated to AB RNA vaccines without compromising safety, and that we would learn more about how the immune system responds to vaccines regardless of whether we made a better vaccine in the process.
We found that changing the RNA delivery method matters significantly. When using lipid nanoparticles (tiny fat bubbles), adding polySTING boosted protective immune responses. However, when using a safe plastic-based delivery system called polyplexes, adding polySTING reduced vaccine effectiveness unless it was given a few days after the vaccine. This is because of the types of immune cells these delivery systems attract, and where they attract them to. Polyplex vaccines primarily activated immune cells in muscle tissue, while lipid nanoparticles and polySTING activated cells in lymph nodes. Inhaled vaccines involved different cell types than those that respond to intramuscular vaccines, but it’s not clear if delivering polySTING separate from the RNA was a poor choice in this setting. Better inhalable RNA delivery formulations are needed to make these questions addressable.

When comparing different RNA types, regular mRNA vaccines that only make vaccine protein for a short time worked better on their own than when combined with polySTING. However, self-amplifying RNA vaccines (which can copy themselves inside cells and make vaccine protein for weeks) were better at preventing AB infection in the lung when they were combined with polySTING. Surprisingly, this combination worsened the ability to control bacteria in the spleen, which might be related to a polySTING-induced shift in the type of protective immune response generated.

These findings highlight how the timing and location of immune stimulation critically impact vaccine effectiveness. Vaccine designers need to carefully consider not just what goes into a vaccine, but exactly how and when each component is delivered to create the most effective immune response without compromising safety. Most critically, early inflammatory events right after vaccine delivery seem to have a long-lasting impact on the protective immune responses that follow, even for RNA types that make protein for a long time. Adding safe, targeted adjuvants (like polySTING) to self-amplifying RNA thus represents an attractive, but complex strategy for optimizing immune responses to prevent bacterial infection.

Dr Prerna Vohra

Lecturer in Microbiology
Institute of Immunology and Infection Research
University of Edinburgh, UK

Collaborators:

Professor Ian Henderson, Institute Director, Institute for Molecular Bioscience, Australia

Professor Mark Stevens, Chair of Microbial Pathogenesis & Deputy Director – Research, The Roslin Institute & Royal (Dick) School of Veterinary Studies, University of Edinburgh, UK

Summary

Salmonella is a key global cause of diarrhoea causing over 94 million cases of foodborne infections annually. It is the second most common cause of bacterial gastroenteritis in Europe and the USA and high incidence of salmonellosis have been reported in the developing world. Poultry are a major reservoir of Salmonella. On farms, Salmonella can be transmitted from infected chickens to their offspring, between birds via the environment and to humans working in close contact with infected poultry. The key poultry-associated serovars pose distinct problems: S. Typhimurium, which causes gastroenteritis in humans, colonises the chicken intestines for long periods of time with few or no symptoms leading to undetected contamination of eggs and meat, while S. Gallinarum does not affect humans but causes fowl typhoid with a high fatality rate. The currently available live-attenuated vaccines offer serovar-specific protection, that is an S. Typhimurium vaccine protects mainly against S. Typhimurium. Moreover, the S. Gallinarum live-attenuated vaccine can revert to being virulent. Therefore, there is an urgent need for safe pan-Salmonella vaccines with efficacy against multiple serovars to reduce the overall burden of Salmonella in poultry and improve animal and human health. We have developed novel inactive vaccines containing four Salmonella proteins shared between serovars that together significantly reduced Salmonella colonisation in mouse models of salmonellosis and were even more effective when combined with outer membrane vesicles, which themselves are promising low-cost vaccines and adjuvants. Here, we will test the efficacy of these vaccines against S. Gallinarum and S. Typhimurium in poultry.