Pump-Priming Project Awards - Round 4 Awardees

Dr Tatiana de Castro Abreu Pinto

Associate Professor
Federal University of Rio de Janeiro (Brazil)

Collaborator:
Dr Stephen Taylor, Public Health England (UK)

Summary

Group B Streptococcus (GBS) is a leading cause of neonatal disease. Mothers are the main source for newborn colonization since this microorganism can be found in the anovaginal tract of 40% of pregnant women. The only currently available tool to prevent GBS neonatal infections is the intrapartum antibiotic prophylaxis. However, this approach may contribute to the emergence of antimicrobial-resistant GBS isolates and/or multidrug-resistant bacteria (MDR) such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and carbapenem-resistant enterobacteria (CRE), which can also colonise the anovaginal tract of pregnant women.

Although GBS and MDR can share this habitat, it is still unknown if they can present mutual interference. GBS vaccines based on capsular polysaccharides are currently in advanced development, but their impact in the anovaginal microbiota, especially in MDR, and potential coverage in many LMIC, including Brazil, are still unknown. There are 10 different GBS capsular types, but vaccines are being developed against 5 or 6; thus, estimating vaccine coverage in different countries is important.

Considering the imminence of a GBS capsular-based vaccine, the lack of information on GBS-related characteristics in Brazil, and the potential interrelationship between GBS and MDR in the anovaginal microbiota, this proposal aims to evaluate coverage and impact of a future GBS vaccine by collecting data on capsular types and antimicrobial resistance of GBS isolates circulating in Brazil, estimating correlate of protection (with opsonophagocytic killing assay) of current vaccine proposals, and predicting the potential impact in the interrelationships between GBS and major MDR in the anovaginal tract of pregnant women.

Project outcome

We have determined anovaginal colonization rates of group B Streptococcus (GBS) and three groups of multidrug resistant (MDR) bacteria (namely methicillin-resistant Staphylococcus or MRS, vancomycin-resistant Enterococcus or VRE, and carbapenem-resistant gram-negative bacilli or CRGNB) among pregnant women attended at a maternity in Rio de Janeiro, Brazil between 2019 and 2021. We have also evaluated correlates of protection of a maternal GBS vaccine by opsonophagocytic killing assays (OPKA), and assessed the impact of the pandemic by comparing characteristics before (Jan 2019 to Feb 2020) and after (Mar 2020 to Mar 2021) the onset of COVID-19. GBS was detected in 10.8% of clinical samples, while MDR bacteria were detected in 25.9%. GBS colonization rate significantly decreased after the onset of the pandemic while MDR anovaginal colonization rate increased. No difference in the population profile of pregnant women attended at the maternity was detected between before and after the onset of COVID-19; likewise, there was no correlation between GBS and MDR, suggesting that changes in GBS and MDR incidence could not be attributed to changes in population profile and that increase of MDR was probably not due to reduction in GBS or vice versa. However, the use of different antibiotics during pregnancy was associated with presence or absence of certain MDR.

Distribution of GBS serotypes and antimicrobial susceptibility profiles was also different between before and after the onset of COVID-19, highlighting serotype Ib that increased in occurrence and is mostly associated with antibiotic resistance. Nevertheless, all recent GBS isolates from pregnant women in Brazil belong to serotypes comprised by frontrunner GBS vaccine proposals. Additionally, all isolates were susceptible to killing using standard human homologous vaccine reference sera in an opsonophagocytosis assay, implying efficacy of polysaccharide vaccines against all isolates.

Among MDR detected, MRS was the most frequent followed by CRGNB. VRE was not detected. More than half of MRS strains were also resistant to other three or more antibiotics, and harbored SCCmec V, a type that is usually associated with community-acquired staphylococcal strains, corroborating the hypothesis that our findings were not due to hospital (maternity) cross-contamination. Five different extended-spectrum beta-lactamase (ESBL) and carbapenemase genes were detected among CRGNB strains, highlighting bla_TEM (68%), and their distribution varied between pre- and post-COVID onset.

Our results show a remarkable reduction in GBS anovaginal colonization with a concomitant increase in MDR colonizing pregnant women in Brazil after the onset of COVID-19. Our data suggest an expected positive impact in our setting of GBS vaccines currently in development, but also highlight the possible rise of new threats in maternal and child health, with a special attention to antibiotic-resistant variants. Novel personal habits and changes in clinical practices due to the onset of COVID-19 may have an impact in the broad scenario of infectious diseases. Continued and sustained surveillance in our setting is required to evaluate if these changes are only temporary and to keep tracking these potential post-pandemic bacterial threats in the perinatal population.

Dr Nopadon Pirarat

Associate Professor
Chulalongkorn University (Thailand)

Collaborators:
Dr Sirikorn Kitiyodom, Chulalongkorn University (Thailand)

Dr Teerapong Yata, Chulalongkorn University (Thailand)

Dr Channarong Rodkhum, Chulalongkorn University (Thailand)

Dr E P Preetham, Kerala University of Fisheries and Ocean Studies (India)

Dr Kim Thompson, Moredun Research Institute (UK)

Dr Janina Costa, Moredun Research Institute (UK)

Summary

Aquaculture is the fastest growing animal food production sector globally and very important to food security. Reaching harvest size in just 6 months, tilapia is 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. Columnaris disease, caused by Flavobacterium columnare, has emerged as one of most important bacterial diseases for farmed tilapia, for which there is no commercial vaccine, and farmers resort to using antibiotics to control this disease.

With increasing concerns about using antibiotics in aquaculture, attention had focused on vaccination for disease control. Many tilapia farmers will not vaccinate by injection once the fish have been moved onto the farm and would prefer fish to be vaccinated in the hatchery before they are moved to their grow-out site; however, injection of small fish is difficult. Alternative vaccine delivery methods are needed, such as oral or immersion delivery, but vaccines delivered by these routes tend to provide low efficacy and short duration of protection. We will investigate a novel and innovative immersion vaccine for columnaris disease in tilapia. We will deliver inactivated Flavobacterium columnare to tilapia in nanoparticles, coated with a mucoadhesive biopolymer, to provide "pathogen-like" properties that enhance binding of the nanoparticles to fish mucosal membranes and we will assess the immune response and level of protection elicited by these in vaccinated tilapia.

Project outcomes

Columnaris, a highly contagious bacterial disease caused by Flavobacterium columnare, is recognized as one of the most important infectious diseases in farmed tilapia, especially during the fry and fingerling stages of production. The disease is associated with characteristic lesions in the mucosa of affected fish, particularly their skin and gills. Vaccines delivered via the mucosa are therefore of great interest to scientists developing vaccines for this disease. In the present study, we characterized field isolates of F. columnare obtained from clinical columnaris outbreaks in red tilapia to select an isolate to use as a candidate for our vaccine study. This included characterizing its colony morphology, genotype and virulence status. The isolate was incorporated into a mucoadhesive polymer chitosan-complexed nanovaccine (CS-NE), the efficacy of which was determined by experimentally infecting red tilapia that had been vaccinated with the nanoparticles by immersion. At 30 days post-vaccination, the fish were infected F. columnare. The relative percentage of protection was 78% in vaccinated fish relative to control fish. Histology of the mucosal associated lymphoid tissue (MALT) showed a significantly higher presence of leucocytes and a greater antigen uptake by the mucosal epithelium in CS-NE vaccinated fish compared to control fish and whole cell vaccinated fish, respectively, and there was statistically significant up-regulation of IgT, IgM, TNF α, IL1-β and MHC-1 genes in the gill of the CS-NE vaccinated group. Overall, the results of our study confirmed that the CS-NE particles achieved better adsorption onto the mucosal surfaces of the fish, elicited great vaccine efficacy and modulated the MALT immune response better than the conventional whole cell-killed vaccine, demonstrating the feasibility of the mucoadhesive nano-immersion vaccine as an effective delivery system for the induction of a mucosal immune response against columnaris disease in tilapia.

Professor Imran Saleem

Professor in Nanomedicine
Liverpool John Moores University (UK)

Collaborators:
Dr Viviane Maimoni Gonçalves, Instituto Butantan (Brazil)

Dr Eliane Namie Miyaji, Instituto Butantan (Brazil)

Dr Richard Broadhead, Precision Nanosystems (UK)

Summary

Infection by Streptococcus pneumoniae, which can result in development of pneumonia, meningitis, sepsis and bacteraemia, is a leading cause of ill-health and death in children, elderly and immunocompromised worldwide. The high prevalence of antimicrobial resistant pneumococci has prompted further emphasis on the importance of pneumococcal vaccines. There are over 95 serotypes, and current licenced vaccines, based on capsular components, only protect against 13. These vaccines are expensive to produce which limits the affordability and availability, especially in low and middle-income countries where the burden of the disease is the highest. In addition, there is potential for serotype replacement, which can hinder efforts to control colonisation and the disease.

To address these limitations, we propose a spray dried nanoparticle (NP) vaccine, which incorporates multiple pneumococcal protein antigens, to ensure coverage against multiple strains. The formulation will be administered through the mucosal route in order to induce mucosal immune responses. A mucosally administered pneumococcal vaccine which has complete coverage of serotypes, would not only provide a single vaccine which could be used all over the world, but also offers a needle-free means of administration to promote use. Additionally, the formulation of the vaccine will be prepared using readily available scale-up production methods, addressing a large hurdle in pre-clinical translation and further reducing costs. The dry powder form of the vaccine also addresses accessibility through and negation of cold chain requirements. The project seeks to produce and evaluate spray dried NPs, incorporating pneumococcal protein antigens, as a mucosal vaccine against all pneumococcal serotypes.

Project outcomes

Our aim was to produce a dry-powder NPs vaccine formulation incorporating pneumococcal protein antigens for lung delivery. In order to induce optimal cellular and protective immunity, recombinant pneumococcal proteins will be used as the antigenic component of the vaccine. The pneumococcal proteins are found in virtually all pneumococcal strains, unlike, polysaccharides found in current pneumococcal vaccines, which are specific for 13-serotypes. We have successfully produced and purified multiple pneumococcal protein antigens, without the use of polyhistidine-tagging, and with the minimal number of purification steps, which both simplify and reduce the cost for large scale production. The proteins were produced with very high purity (> 94% after multimodal chromatography).

The protein antigens were successfully incorporated within nanoparticles (NPs) produced using an optimised microfluidic mixing method. The NPs were produced from combining two polymers (core and coating). Experiments were carried out to ensure the suitability of the mixing ratio, reproducibility of results, and reliability of the method for NPs formulations and antigen incorporation. The optimum NPs using combination polymers demonstrated in vitro compatibility and adjuvant immunogenicity with upregulation of cell surface markers CD40 and CD86 compared. Furthermore, the protein antigens were successfully incorporated achieving, size <400 nm, PDI <0.3 and positively charged. Moreover, the NPs and protein antigens were stable, and the protein integrity maintained following nebulisation as determined by SDS-PAGE and lactoferrin-binding western blot assays.

We have successfully produced a spray dried nanocomposite microcarrier system incorporating NPs-antigens with high yield and low moisture content and NPs characteristics following dispersion achieving similar physicochemical characteristics to NPs prior to spray drying. The spray dried formulation generated upregulation of cell surface markers which was higher than NPs alone. Furthermore, the protein stability and integrity were maintained following spray drying and nebulisation as indicated by SDS-PAGE and lactoferrin-binding western blot assays. The results were maintained following 3 weeks of storage as a dry powder at room temperature.

Dr Seanette Wilson

Group Leader & Program Manager
The Biovac Institute (South Africa)

Collaborators:
Dr Gaurav Kwatra, University of the Witwatersrand (South Africa)

Dr Fatme Mawas, National Institute for Biological Standards and Control (UK)

Summary

Streptococcus agalactiae or group B Streptococcus (GBS) bacterial infection is a major health concern and a leading cause of sepsis and meningitis in infants, particularly in Africa.

A promising prevention for GBS infection in newborns is maternal immunization with a GBS vaccine. However, there is currently no vaccine for GBS available. Biovac is developing a traditional conjugate GBS vaccine using polysaccharides expressed on the surface of the GBS bacterium as antigens linked to tetanus toxoid as a carrier protein.

As part of WHO’s target product profile for a GBS vaccine, a one-dose vaccination regimen has been recommended. It remains to be ascertained whether a single dose of a traditional maternal GBS conjugate vaccine will elicit a sufficiently protective immune response in newborns. To enhance the response to a vaccine, we are proposing a novel vaccine design using GBS surface proteins conjugated to GBS polysaccharides. GBS proteins common to all serotypes have been identified as inducers of immune responses in infected individuals, making them potential candidates for novel GBS vaccines.

In this proof of concept project, we will conjugate a single GBS common protein to a single GBS polysaccharide with the aim to not only providing superior protection compared to traditional conjugate vaccines but to potentially provide protection against those serotypes not included in the vaccine. If successful, this will allow for the development of an affordable and cost-effective monovalent vaccine that protects against all GBS serotypes.

Project outcomes

In a collaborative effort between RMPRU, NIBSC and Biovac, a novel GBS glyco-protein conjugate using GBS polysaccharide linked to a GBS membrane protein as the carrier protein was generated. This conjugate will be further investigated towards a vaccine for maternal immunization to prevent GBS infections in newborns, while advancing African vaccine development and manufacturing capability.

RMPRU identified genes for several conserved GBS surface proteins and one of these proteins was cloned into vectors that were subsequently transformed into an Escherichia coli BL21 strain for protein expression ^1-3. This strain was expanded into a working cell bank at Biovac.

To produce sufficient quantities of this surface protein, Biovac completed a proof of concept (POC) for antigen production and characterisation. To this end, approximately 1 g of protein was produced using a recombinant protein production process.

The physio-chemical characterisation of the novel GBS conserved protein was confirmed at NIBSC and Biovac utilising analytical tools that enables quantification of the protein as well as elucidating secondary and tertiary structure.

Biovac generated a novel GBS glyco-protein conjugate at small scale (25 mg) using GBS polysaccharide linked to a GBS membrane protein as the carrier protein.

The availability of these novel conjugate vaccines is a requirement for future studies to test the hypothesis that the GBS membrane protein-GBS polysaccharide conjugate is a superior vaccine candidate compared to traditional polysaccharide-Tetanus Toxoid conjugate vaccines. After successful generation of the GBS protein-CPS conjugates we are planning to assess their efficacy in producing an enhanced immune response in a mouse model compared to a conjugate utilizing a traditional carrier protein.

References:

1. Sonwabile Dzanibe (2017), Natural Induced Antibodies Against Group B Streptococcus Surface Proteins and Capsular Polysaccharides; Department of Clinical Microbiology and Infectious Diseases, School of Pathology, Faculty of Health Sciences, University of Witwatersrand.
2. S. Dzanibe et al., (2016), Natural acquired group B Streptococcus capsular polysaccharide and surface protein antibodies in HIV-infected and HIV-uninfected children; Vaccine 34 (Issue 44), pages 5217-5224.
3. S. Dzanibe et al., (2017), Reduced Transplacental Transfer of Group B Streptococcus Surface Protein Antibodies in HIV-infected Mother–Newborn Dyads; Journal of Infectious Diseases (215), pages 415-419

https://www.uniprot.org/uniprot/Q8E2L6

Dr Qibo Zhang

Senior Lecturer in Immunology
University of Liverpool (UK)

Collaborator:
Rene Raeven, Intravacc (Institute for Translational Vaccinology) (The Netherlands)

Summary

Whooping cough, caused by the bacterium Bordetella pertussis, is a vaccine-preventable respiratory disease with high risk for respiratory failure, encephalopathy and death in unvaccinated or incompletely vaccinated infants. Although pertussis vaccination resulted in a dramatic decrease of whooping cough cases, pertussis resurged the last decades even in the vaccinated population, pinpointing the need for improved pertussis vaccines or vaccination strategies. Whereas parenteral vaccination with current acellular pertussis vaccines prevents disease, it fails to block nasopharyngeal carriage and transmission of B. pertussis.

Intranasal vaccination could be a promising strategy to overcome these shortcomings as this allows activation of local mucosal immune tissue in the nasopharynx such as nasopharynx-associated lymphoid tissues (NALT), comprising adenoids and tonsils, which are potent induction sites for natural immunity against upper respiratory pathogens. Located in the strategic anatomical site, NALT are likely to play a critical role in mediating bacterial carriage and its clearance in the nasopharynx. Recent studies in mice showed that intranasal immunization with a non-replicating pertussis vaccine candidate based on outer membrane vesicles (omvPV) prevents B. pertussis colonization in the nose and that the induction of mucosal T-helper 17 cells and IgA antibodies are thought to play an important role.

In this project, we aim to evaluate the capacity of omvPV to induce these mucosal Th17 and IgA responses in an established ex vivo mucosal immune tissue model from humans and thus provide an indicator for this vaccination strategy to induce the desired immunity to block transmission in humans.

Project outcomes

Whooping cough, caused by the bacterium Bordetella pertussis, is a vaccine-preventable respiratory disease with high risk for respiratory failure, encephalopathy and death in unvaccinated or incompletely-vaccinated infants. Although pertussis vaccination resulted in a dramatic decrease of whooping cough cases, pertussis resurged the last decades even in the vaccinated population, pinpointing the need for improved pertussis vaccines or vaccination strategies. Whereas parenteral vaccination with current acellular pertussis vaccines prevents disease, it fails to block nasopharyngeal carriage and transmission of B. pertussis. Intranasal vaccination could be a promising strategy to overcome these shortcomings as this allows activation of local mucosal immune tissue in the nasopharynx such as nasopharynx-associated lymphoid tissues (NALT), comprising adenoids and tonsils, which are potent induction sites for natural immunity against upper respiratory pathogens. Located in the strategic anatomical site, NALT are likely to play a critical role in mediating bacterial carriage and its clearance in the nasopharynx. Recent studies in mice showed that intranasal immunization with a non-replicating pertussis vaccine candidate based on outer membrane vesicles (omvPV) prevents B. pertussis colonization in the nose and that the induction of mucosal T-helper 17 cells and antibodies are thought to play an important role. In this project, we aim to evaluate the capacity of omvPV to induce these mucosal Th17 and anti-pertussis antibody responses in an established ex vivo mucosal immune tissue model from humans and thus provide an indicator for this vaccination strategy to induce the desired immunity to block transmission in humans.

We have shown omvPV, as a candidate pertussis vaccine induce significant T cell response including Th17 cell activation and anti-pertussis antibody responses in human nasopharynx mucosal tissue. This suggests that omvPV may become an effective intranasal spray vaccine preventing pertussis infection in humans.