Using General Modules for Membrane Antigens (GMMA) as combined multivalent ETEC-Shigella vaccines for Low- and Middle-Income Countries (LMIC) and travellers 

Summary 

Background 

Bacterial diseases are a grave threat for humankind causing approximately six million deaths per year. Antibiotic resistance is increasing. Hygiene measures are failing. Global travel makes disease control increasingly difficult. Enterotoxigenic Escherichia coli (ETEC) and Shigella are enteropathogens that cause significant global mortality and morbidity, particularly in low- and middle-income countries (LMIC). Development of new and improved vaccines against diarrheal infections is a fundamental element of the strategy towards reducing deaths from diarrhea in children under 5 years of age. No vaccines are currently widely available for both residents and travellers to endemic areas.

Aim

The aim of the proposed project is to use Generalised Modules for Membrane Antigens (GMMA) as a means to generate a multivalent and low cost vaccine to protect humans against diarrheal diseases caused by Shigella and ETEC. GMMA are outer membrane vesicles naturally shed by Shigella and other Gram-negative bacteria genetically manipulated to increase blebbing and decrease reactogenicity. GMMA present multiple antigens in the context of a membrane and have optimal size for immune stimulation plus self-adjuvanting properties. These vesicles are easy and cheap to produce, are strongly immunogenic and protective. GMMA can also be genetically or chemically modified to present heterologous antigens from other pathogens, supporting the development of multivalent vaccines. 

Work plan

Here, we will test the feasibility of using the GMMA approach for the development of a vaccine covering both Shigella and ETEC. As proof of concept we will use Shigella sonnei GMMA as scaffold and test different strategies for the delivery of LTK63, a not toxic derivative of the Heat-labile enterotoxin (LT) antigen of ETEC, together with the immunodominant O-Antigen (OAg) of Shigella sonnei. LTK63 is a powerful immunogen, non-toxic but maintaining all immunogenic and adjuvant properties of the wild-type LT toxin and is expected to play a Key role as strong mucosal immunogen and adjuvant. S. sonnei GMMA presenting LTK63 will be fully characterised and tested in robust and tractable preclinical animal models to investigate the immunogenicity and protective activity of these multivalent vaccine preparations. 

This project will represent a valuable starting point for the development of a low cost and effective multivalent GMMA-based vaccine protecting against both Shigella and ETEC, that have a high incidence worldwide and coexist in many geographical areas, especially LMIC.

Project Outcomes

Shigella and Enterotoxigenic E. coli (ETEC) have a high global incidence and are major causes of bacterial diarrhoea worldwide, especially in children under five years of age in low and middle income countries (LMICs). Various studies have indicated that antibodies generated against the O-Antigen (OAg) portion of Shigella lipopolysaccharides (LPS) correlate with protection from disease and also that the effects of ETEC infection can be mitigated by the action of antibodies against the bacterial toxins. We combined the ETEC toxin and Shigella OAg LPS to make a vaccine which aimed to target these pathogens, producing an immune response with the potential to reduce infection and disease.

We used a Shigella sonnei strain, that express the OAg and are already mutated to increase outer membrane blebbing and to make generalised modules for membrane antigens (GMMA). These are tiny virus sized non-infectious vesicles that are ideally suited to generate an immune response. We then engineered this Shigella strain to express the LTK63 toxoid antigen, that would either be expressed on the surface of GMMA or could be directly bound to their surface. This approach therefore aimed to generate immune responses to both bacteria in a single vaccination. We also looked at different routes of vaccination with the aim to induce both blood responses and mucosal responses, particularly the gut as the main site of entry that these bacteria use to get into the body.

The project successfully generated vaccines that expressed both the Shigella OAg and the ETEC LTK63 antigen and these were characterised and then used in animal studies to investigate the immune responses. The two components of the vaccine were used either embedded within the same GMMA vesicle or the LTK63 was physically chemically attached to the GMMA vesicle. Both worked very well with the physical linkage generating slightly higher levels of specific antibodies, however the differences were not significantly different. In these studies we also measured the effect of administering the vaccines to the mice via different immunisation routes. These animal studies demonstrated that the preparations were very potent, generating high levels of antibody responses that were functionally able to neutralize LT and had bacteriocidal activity against Shigella.

Due to the COVID pandemic the animal studies were delayed but by the end of the program the intended mouse studies that aimed to examine the effect of different routes had been completed, with the only remaining work being the assessment of the immune responses, the mucosal response and sera neutralization / bacteriocidal activity. These final analyses are in progress and will be completed within 3 months after the end date of the project, at which time the results will be published in a suitable peer reviewed journal.