Advances in the development of Salmonella-based vaccine strategies for protection against Salmonellosis in humans

Abstract

Salmonella spp. are important human pathogens globally causing millions of cases of typhoid fever and non-typhoidal salmonellosis annually. There are only a few vaccines licensed for use in humans which all target Salmonella enterica serovar Typhi. Vaccine development is hampered by antigenic diversity between the thousands of serovars capable of causing infection in humans. However, a number of attenuated candidate vaccine strains are currently being developed. As facultative intracellular pathogens with multiple systems for transporting effector proteins to host cells, attenuated Salmonella strains can also serve as ideal tools for the delivery of foreign antigens to create multivalent live carrier vaccines for simultaneous immunization against several unrelated pathogens. Further, the ease with which Salmonella can be genetically modified and the extensive knowledge of the virulence mechanisms of this pathogen means that this bacterium has often served as a model organism to test new approaches. In this review we focus on (1) recent advances in live attenuated Salmonella vaccine development, (2) improvements in expression of foreign antigens in carrier vaccines and (3) adaptation of attenuated strains as sources of purified antigens and vesicles that can be used for subunit and conjugate vaccines or together with attenuated vaccine strains in heterologous prime-boosting immunization strategies. These advances have led to the development of new vaccines against Salmonella which have or will soon be tested in clinical trials.

Figure 1

Endogenous and exogenous acid resistance pathways exploited in Salmonella vaccines. The diagram illustrates the two‐component amino acid decarboxylase‐antiporter resistance (AR) systems that have been utilized in candidate Salmonella vaccines to increase acid resistance. These systems are from left to right—the glutamic acid–dependent acid resistance system (GDAR, yellow) comprised of the GadC glutamate/γ‐aminobutyric acid (GABA) antiporter and glutamate decarboxylases, GadA/GadB; the arginine‐dependent acid resistance system (ADAR, purple) comprised of the AdiC arginine/agmatine antiporter (Agm) and the inducible arginine decarboxylase, AdiA; and the lysine‐dependent acid resistance system (LDAR, green) comprised of the CadB lysine/cadaverine antiporter (Cad) and the cytoplasmic inducible lysine decarboxylase, CadA. The ADAR and LDAR systems are native to Salmonella, while the GDAR system has been imported from Shigella into Salmonella strains to further enhance acid resistance. In low pH conditions (deep pink colour extracellular to the bacteria, large H⁺) and in the presence of their cognate amino acid, the decarboxylase raises intracellular pH (indicated by the light‐coloured cytoplasm) by decarboxylating the amino acid substrate (indicated by the orange square, purple triangle and green rectangle) in a proton‐dependent manner releasing CO₂ in the process. The product binds the substrate‐specific antiporter in the inner membrane and exchanges the decarboxylated substrate with a new amino acid. IM, inner membrane; OM, outer membrane. Figure created with BioRender.com.

Figure 2

Reagent strains for the production of core and O‐polysaccharide (COPS) and flagellin. Live oral vaccine strains are highly attenuated and can be used to purify antigens safely and economically. This figure shows the deleted genes of reagent strains used for NTS COPS:FliC conjugate vaccines and the impact on phenotype and virulence particularly with respect to OPS (indicated by the green chains on the outer surface of the bacteria) length, flagellar (indicated as lavender hair‐like projections) expression, and flagellin protein FliC secretion (indicated as lavender circles). (a) Deletion of guaBA results in guanine auxotrophy and increases the LD₅₀ by 5 log₁₀. (b) The clpPX mutations results in hyperflagellated mutants. (c) Deletion of fliD eliminates the flagellar cap protein which results in monomers of the flagellin protein FliC being secreted into the media while deletion of fljB from Salmonella Typhimurium results in loss of phase 2 flagellin. (d) Expression of the protein that regulates the number of OPS repeats, wzzB, results in expression of long chain LPS and hence a more uniform product. Figure created with BioRender.com

Figure 3

Production of outer membrane vesicles (OMVs) and lipid A modifications. OMVs are spherical vesicles enriched with proteins. Their production can be manipulated to express antigens from unrelated pathogens as shown in this figure. Black dashed arrows indicate the movement of the antigen from cytoplasm to outer membrane (OM). (1) An unfolded heterologous antigen (HA) in the periplasm (yellow line with an N‐terminal secretion signal in red and a C‐terminal β‐signal in blue) is translocated from the cytoplasm across the inner membrane (IM) via the Sec translocase and the chaperone protein SurA directs HA to the BAM complex. (2) The BAM complex facilitates proper folding of HA and its insertion into the OM. (3) Vesicles subsequently bud from the OM and contain OM proteins, LPS, periplasmic proteins, and the foreign antigen. (4) The inset shows lipid A modifications used to reduce the reactogenicity of OMVs. Black solid arrows indicate the position at which the acyl chain (zigzag lines) modifications are made. These include deletion of HtrB which blocks the addition of a 12‐carbon secondary chain to the 2′ position; deletion of MsbB which blocks the addition of a 14‐carbon secondary chain to the 3′ position; deletion of PagP which blocks the addition of a 16‐carbon secondary chain to the existing acyl chain at the 2 position. PagL is a deacylase that removes the β‐hydroxymyristoyl chain at the carbon‐3 position and LpxE dephosphorylates lipid A at the carbon 1 position. COPS, core and O polysaccharide; IM, inner membrane. Figure created with BioRender.com.