The iNTS-GMMA vaccine: a promising step in non-typhoidal Salmonella vaccine development

KEYWORDS:

• Invasive non-typhoidal Salmonellosis
• Salmonella Typhimurium
• Salmonella Enteritidis
• Vaccine

1. Introduction

Non-typhoidal Salmonellae (NTS) most commonly cause a self-limiting gastroenteritis that is clinically indistinguishable from that caused by many other enteric pathogens. However, these bacteria can also cause an invasive syndrome (invasive non-typhoidal Salmonellosis, iNTS) with bacteremia, high fever and metastatic infection which, if untreated, can lead to septicemia and death. iNTS has an incidence of 51 per 100,000 persons per year in Africa with the highest incidence rates in infants and children under 5 years of age [1]. Over 90% of iNTS infections are caused by Salmonella enterica serovars Typhimurium and Enteritidis with a global pooled case fatality rate of 15% [2]. Host-pathogen factors appear to determine whether bacteria cause localized or systemic disease; NTS strains causing invasive disease in Africa are associated with genome degradation and the acquisition of multi-drug resistance [3]. Malnutrition, malaria, immature humoral responses in infants, and impaired T-cell immunity in people living with human immunodeficiency virus (HIV) infection predispose to invasive disease [4]. Animal vaccines based on attenuated organisms are in widespread use in domestic livestock and have been successful at reducing food-borne gastroenteritis, but there is clear need for a safe and effective vaccine to prevent iNTS in vulnerable populations.

2. NTS vaccine-derived protective immunity

Whilst no immune correlate of protection against iNTS has been confirmed, protection likely depends on a combination of T cell-dependent and -independent pathways. Humoral immunity appears to be vital, and the acquisition of NTS-specific antibodies in young African children correlates with a decline in incidence of iNTS [5]. Mechanisms of protection by antibodies include direct complement-mediated killing of bacteria as well as opsonization with subsequent facilitation of oxidative burst-mediated killing and phagocytosis [5,6].

The antigen targets of protective antibodies are potentially varied. Antibodies specific to NTS lipopolysaccharide (LPS) have been shown to be bactericidal in African populations, although when present in excess in adults infected with HIV a subset of these antibodies may also inhibit bacterial killing [7,8]. Alternatively, antibodies specific to outer membrane proteins such as porins are bactericidal via a complement-mediated pathway and are protective against infection in a mouse model [8,9]. Finally, antibodies specific to the FliC flagellar protein increase phagocytosis and reduce viable intracellular bacterial load in vitro [10]. An effective vaccine against iNTS may thus require the induction of antibodies to multiple targets.

Infants in endemic regions develop NTS-specific CD4⁺ T cells prior to developing bactericidal antibodies, suggesting T helper cell responses are required to facilitate antibody production, class switching, affinity maturation and memory B cell development [7]. This is supported by mouse experiments showing that adoptive transfer of both antigen-specific T cells and antibodies are needed for protection from oral challenge [11]. Whilst humoral immunity may be particularly important in countering extracellular infection either in the gut or during bacteremia, NTS are facultatively intracellular pathogens requiring a cellular immune response for robust clearance. This is evidenced by the high rates of infection and recrudescence in HIV-infected people, whose T_H1 and T_H17 responses are impaired. LPS alone is a T-independent antigen, so in order to generate effective humoral and cell mediated responses, an iNTS vaccine is likely to also need protein antigen components to act as ‘carriers’, enabling linked recognition similar to that observed with polysaccharide-protein conjugate vaccines.

3. Outer membrane vesicle-based vaccines

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are attractive vaccine platforms as they present bacterial cell surface protein antigens and LPS in their natural conformation, eliciting an optimal immune response whilst avoiding the risks inherent with live attenuated vaccines. OMV-based vaccines are already licensed for use against Neisseria meningitidis group B infection (e.g. Bexsero).

Traditional OMV vaccines may be produced via detergent or mechanical extraction, but these methods lead to reduced immunogenicity or increased reactogenicity respectively. An alternative process derives OMVs from genetically modified bacteria which exhibit hyper-blebbing of the outer membranes, with the resultant vesicles referred to as Generalised Modules for Membrane Antigens (GMMA).

A quadrivalent Shigella GMMA-based vaccine, altSonflex1-2-3, is in development for protection against the four most prevalent Shigella serotypes (S. flexneri 1b, 2a and 3a and S. sonnei). GMMA technology is used for delivery of the LPS O-antigen and the vaccine is currently being tested in a phase 1/2 clinical trial (clinicaltrials.gov NCT05073003). A first generation vaccine, monovalent S. sonnei 1790-GMMA, has already been shown to be well tolerated and immunogenic in healthy African adults (18–45 years) [12].

4. The iNTS-GMMA vaccine

iNTS-GMMA is a bivalent GMMA-based vaccine in development aimed at protection from iNTS, and combines GMMA derived from modified S. Typhimurium and S. Enteritidis strains. Mutations in the tolR gene in these strains induce hyper-blebbing of OMVs, whilst modified msbB and pagP genes reduce acylation of the lipid A component of LPS, thereby reducing reactogenicity [13]. GMMA are purified from bacterial cultures and subsequently concentrated in a robust and scalable process. The vaccine consists of aluminum hydroxide-formulated GMMA from the two serovars in equal proportions and is administered intramuscularly.

Pre-clinical results for iNTS-GMMA are promising. Immunization of mice with a prototype bivalent iNTS-GMMA (tolR deletion only) induced high antibody responses to S. Typhimurium and S. Enteritidis O-antigens and strong in vitro bactericidal activity against the targeted strains. Additionally, in a comparison between GMMA and glycoconjugates as O-antigen delivery platforms, GMMA elicited higher serum bactericidal titers than did the glycoconjugate formulations [14]. The bacterial load in spleen, liver and blood following in vivo challenge with S. Typhimurium strain D23580 was lower in mice immunized with a monovalent S. Typhimurium aluminum hydroxide-formulated GMMA than in mice who received a saline placebo, and immunized mice also demonstrated evidence of B cell memory induction [15].

The iNTS-GMMA vaccine has now progressed to clinical trials, with phase 1 studies in healthy adults currently underway at the University of Oxford (trial registration ISRCTN51750695) and planned shortly in an endemic population at the Kenya Medical Research Institute. The primary immunogenicity endpoint of these studies is the induction of antibodies targeting serovar-specific O-antigen, but exploratory assays will also delineate the antibody response to other antigens as well as serum bactericidal activity, B cell memory induction, T cell responses, transcriptomic profile, mucosal antibody responses and the effect on host enteric microbiota composition. Not only will these data support the development of a much-needed vaccine against iNTS, they will also shed light on the diverse immunological response to NTS infection.

5. Expert opinion

Despite the burden and high mortality rate of iNTS in mostly sub-Saharan African countries, no vaccines to prevent this disease are yet available. Several vaccine candidates, including the iNTS-GMMA vaccine, use the LPS O-antigen from S. Typhimurium and S. Enteritidis as active moieties and have already transitioned from preclinical to clinical development. We postulate that the iNTS-GMMA vaccine has the potential to elicit robust T-dependent immune responses similar to traditional polysaccharide–protein conjugate vaccines which exploit the phenomenon of linked recognition, as LPS and proteins in GMMA particles will be taken up together by antigen presenting cells. This will be assessed by measuring antibody functionality and the development of B cell memory in clinical trial samples.

iNTS-GMMA is a bivalent vaccine developed against Salmonella serovars Typhimurium and Enteritidis. However, there is considerable geographical overlap between endemic areas of iNTS and S. Typhi, the causative agent of typhoid fever in Africa. Typhoid fever is also a disease of young children and typhoid Vi polysaccharide-conjugate vaccines (TCV) are licensed and already being introduced into childhood immunization schedules in countries with high endemicity. A future trivalent Salmonella vaccine is an attractive proposition both to provide broad protection to vulnerable populations, and to facilitate incorporation into already crowded immunization schedules. Further work is needed to elucidate both the cost-effectiveness and the optimal timing of administration of a potential trivalent vaccine in infants. Clinical trials are planned of a trivalent version of iNTS-GMMA with the addition of a TCV component, as with other candidates in the current iNTS vaccine pipeline.

Following on from clinical trials of safety, immunogenicity and age de-escalation, the roadmap to vaccine licensure will likely require proof of efficacy. Large scale efficacy field studies may be challenging to resource and conduct, and a controlled human infection model for non-typhoidal Salmonella is therefore currently under development which may help accelerate this pathway.

Correlates of immune protection against iNTS disease also need further interrogation. Whilst LPS-specific antibodies appear important, humoral responses to proteins and cellular immunity may also play a role. The ongoing studies of iNTS-GMMA in humans will allow elaboration of the immune response to iNTS, and facilitate comparisons between endemic and non-endemic populations, thus informing future vaccine development.

Declarations of interest

R Canals is employed by the GSK group of companies, holds GSK shares, and is the Project Leader at GSK Vaccines Institute for Global Health Srl for developing the iNTS-GMMA vaccine funded by a EU Horizon 2020 and an EDCTP grants. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. MN Ramasamy is Chief Investigator on a Phase 1 trial of the iNTS-GMMA vaccine sponsored by the University of Oxford and funded by a EU Horizon 2020 grant. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contribution statement

All authors have (1) substantially contributed to the conception and design of the review article and interpreting the relevant literature, and (2) been involved in writing the review article or revised it for intellectual content.

Additional information

Funding

This manuscript was not funded. The iNTS-GMMA vaccine program has received funding from the EU Horizon 2020 research and innovation programme under the project Vacc-iNTS (grant agreement number 815439), and from the EDCTP2 programme supported by the European Union under the project PEDVAC-iNTS (RIA2019AMR-2658).