Elsevier

Vaccine

Volume 35, Issue 3, 11 January 2017, Pages 419-426
Vaccine

Characterization of O-antigen delivered by Generalized Modules for Membrane Antigens (GMMA) vaccine candidates against nontyphoidal Salmonella

https://doi.org/10.1016/j.vaccine.2016.11.089Get rights and content

Abstract

Invasive nontyphoidal Salmonella disease (iNTS) is a leading cause of death and morbidity in Africa. The most common pathogens are Salmonella enterica serovars Typhimurium and Enteritidis. The O-antigen portion of their lipopolysaccharide is a target of protective immunity and vaccines targeting O-antigen are currently in development. Here we investigate the use of Generalized Modules for Membrane Antigens (GMMA) as delivery system for S. Typhimurium and S. Enteritidis O-antigen. Gram-negative bacteria naturally shed outer membrane in a blebbing process. By deletion of the tolR gene, the level of shedding was greatly enhanced. Further genetic modifications were introduced into the GMMA-producing strains in order to reduce reactogenicity, by detoxifying the lipid A moiety of lipopolysaccharide. We found that genetic mutations can impact on expression of O-antigen chains. All S. Enteritidis GMMA characterized had an O-antigen to protein w/w ratio higher than 0.6, while the ratio was 0.7 for S. Typhimurium ΔtolR GMMA, but decreased to less than 0.1 when further mutations for lipid A detoxification were introduced. Changes were also observed in O-antigen chain length and level and/or position of O-acetylation. When tested in mice, the GMMA induced high levels of anti-O-antigen-specific IgG functional antibodies, despite variation in density and O-antigen structural modifications.

In conclusion, simplicity of manufacturing process and low costs of production, coupled with encouraging immunogenicity data, make GMMA an attractive strategy to further investigate for the development of a vaccine against iNTS.

Introduction

Salmonella enterica Typhimurium and Enteritidis are the most common serovars responsible for invasive nontyphoidal Salmonella disease (iNTS) in Africa [1], [2], [3], [4], resulting in case-fatality rates of around 20% [5]. iNTS is closely associated with malaria and malnutrition among African infants and children, and with HIV infection in all age groups [6]. Antibiotics are not always available in rural African settings, and increasing levels of multidrug-resistance are limiting their effectiveness [7], [8], [9], making this disease a high priority for vaccine development. Currently, there are no licensed vaccines against iNTS and efforts are ongoing to identify protective antigens and best strategies for vaccine development [10], [11].

Antibodies directed against the O-antigen (OAg) portion of Salmonella lipopolysaccharide (LPS) have been shown to be able to mediate killing [12], [13], [14], [15], [16] and protect against infection in animal models [14], [15], [17]. S. Typhimurium and S. Enteritidis OAg share a common backbone consisting of galactose (Gal), rhamnose (Rha), and mannose (Man), which serologically constitutes epitope O:12 [18]. A different 3,6-dideoxy-hexose residue is linked to Man in these two serovars: abequose (Abe), conferring O:4 specificity to S. Typhimurium, or tyvelose (Tyv), conferring O:9 specificity to S. Enteritidis (Fig. 1) [19], [20]. Salmonella OAg can demonstrate high levels of heterogeneity in terms of chain length and variation in O-acetylation and glucosylation of the repeating units [14], [21], [22], [23], [24]. For S. Typhimurium, Abe may be O-acetylated at position C-2, which adds the O:5 specificity [25]. The additional presence of O-acetyl groups at C-2 and C-3 of Rha has also been reported [22], [26]. OAg chains can also be variably glucosylated, with glucose (Glc) linked at C-4 (O:122 specificity) or C-6 (O:1 specificity) to Gal [21], [27]. Studies in mice indicated that all these structural modifications can impact the immunogenicity of the corresponding glycoconjugate vaccines [14], [23].

We are investigating a Generalized Modules for Membrane Antigens (GMMA) [28] approach to the development of a bivalent vaccine against S. Typhimurium and S. Enteritidis [29]. Gram-negative bacteria naturally shed outer membrane as blebs [30], [31]. The release of blebs can be greatly increased by genetic manipulation of the bacteria resulting in GMMA. In Salmonella, deletion of the tolR gene affects the stability of the linkage between the inner and the outer membrane, and results in an enhanced shedding process [28], [29], [32], [33]. GMMA derived from the surface of Gram-negative bacteria contain potent immunostimulatory components, such as lipoproteins and LPS, which can contribute to their immunogenicity, but also to reactogenicity [30], [31]. In GMMA vaccine development, removal or modification of such components may alter the balance between reactogenicity and immunogenicity. One way to reduce the reactogenicity of LPS is to modify its acylation pathway, for example by deletion of msbB, pagP and htrB genes [29].

In this study, we report that mutations introduced to increase GMMA release and decrease reactogenicity are associated with changes in the structure of surface OAg. We investigate the impact of these changes on the antibody response to GMMA in mice and serum bactericidal activity of these antibodies in vitro.

Section snippets

Strains

Salmonella enterica serovar Typhimurium isolate SGSC1418 (STm 1418) (LT-2 collection [34], University of Calgary) and Enteritidis SA618 (SEn 618) (CEESA EASSA II collection [35] of Quotient Bioresearch Limited), both isolated from animals, were chosen as parent strains [23]. Mutants were generated as previously described [29] (Scheme 1).

GMMA production and characterization

GMMA were produced and purified as described [29]. Total protein content was estimated by Lowry assay [36], [37]. OAg sugar content was quantified by

Results

As part of our previous work, several different S. Typhimurium and S. Enteritidis isolates were screened as sources of OAg for use in a bivalent glycoconjugate vaccine against iNTS [23]. Based on the results obtained, SEn 618 and STm 1418 strains were also selected for use as GMMA-producing strains. They were genetically modified through deletion of tolR gene for GMMA overproduction and of further genes (ΔmsbB, ΔhtrB and ΔpagP) to reduce reactogenicity [29]. IL-6 release was used as an

Discussion

GMMA are nano-sized particles, displaying high-densities of antigens and containing bacterial pathogen associated molecular patterns (PAMPs), with the potential to trigger strong immune responses [30], [31]. Furthermore, GMMA can be produced efficiently, economically and rapidly [28], [32], [33], making them an attractive vaccine candidate, particularly for low and middle income countries.

A comprehensive panel of analytical methods has been assembled for GMMA characterization with particular

Declaration of interests

RA, MC, LL, FN, AS, CAM, SR and FM were permanent employees of Novartis Vaccines Institute for Global Health (NVGH) at the time of the study. Following the acquisition of NVGH by the GSK group of companies in March 2015, RA, LL, FN, AS, SR and FM are now permanent employees of GSK Vaccines Institute for Global Health (GVGH), part of the GSK group of companies.

Contributorship

GDB, RA, PC, MC, AS, CAM, SR, FM were involved in the conception and design of the study. GDB, RA, MC, LL, FN, SR, FM acquired the data. GDB, RA, PC, MC, LL, FN, AS, CAM, SR, FM analyzed and interpreted the results. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors had full access to the data and approved the manuscript before it was submitted by the corresponding author.

Acknowledgments

This study was sponsored by Novartis Vaccines Institute for Global Health. The Institute has now become GSK Vaccines Institute for Global Health, part of the GSK group of companies. We also wish to thank Aurel Negrea for strains generation and Omar Rossi for SDS-PAGE analysis of GMMA from mutated strains. P. Cescutti and G. De Benedetto (Dipartimento di Scienze della Vita, Università di Trieste) thank the “Beneficentia Stiftung” (Vaduz, Lichtenstein) for funding the acquisition of the Bioline

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