Influence of Promoter, Gene Copy Number, and Preexisting Immunity on Humoral and Cellular Responses to a Vectored Antigen Delivered by a Salmonella enterica Vaccine

Abstract

Attenuated Salmonella strains are currently in production as vaccines for protection of animals against salmonellosis. Such commercial strains offer the potential to deliver heterologous antigen to protect animals against other diseases. One vaccine strain, attenuated Salmonella enterica serovar Typhimurium (STM-1), was tested for the ability to deliver ovalbumin and to induce immune responses in mice. Two vaccine trials were performed testing the influence of promoter choice, the location of the encoding DNA (plasmid or chromosome), and the effect of preexisting homologous or heterologous immunity. The results demonstrated that humoral and T-cell responses were induced from either of two promoters, from either the plasmid or the chromosome, and that preexposure to the empty homologous vector, STM-1, or the heterologous vector, S. enterica serovar Enteritidis, had no detrimental effect on subsequent antigen-specific responses. In the case of homologous preexposure, responses were generally greater, and this was correlated with an increased uptake of Salmonella by macrophages in vitro after opsonization with immune sera.Salmonella enterica strains have been well characterized for the delivery of heterologous vaccine antigens in several animal models (4,5,8, 11, 22,39,43,50). The potential advantages of live attenuated vaccines, such as the simple mode of inoculation and generation of strong immune responses, make them ideal candidates for the delivery of antigens (38-40, 57). Infection by Salmonella induces both humoral and cell-mediated responses, not only against homologous antigens, but also against the heterologous antigen for which they act as a carrier (2, 16, 32,36,41,43).STM-1 is an S. enterica serovar Typhimurium mutant developed by RMIT and harbors a mutation in the aroA gene region that renders it attenuated (M. Saxena, P. Smooker, and P. Coloe, submitted for publication). It is a commercial vaccine strain currently in use to protect livestock against Salmonella infection (1). It is delivered to the immune system of chickens by spraying or administration via drinking water and therefore enters via mucosal tissues. It has been previously demonstrated that STM-1 is capable of eliciting immune responses in mice to model plasmid-borne antigens (8) and from inserts integrated at a chromosomal location (55). We have extended these studies to further analyze the capacity of STM-1 in three ways: first, a comparison between plasmid-and chromosome-borne delivery routes using two different promoters; second, an analysis of the T-cell responses induced in mice, in addition to humoral responses; and last, an experiment designed to investigate the influence of preexisting immunity to the vaccine vector. Emphasis was given to an analysis of expression from the chromosomal location, since in order to progress to any commercial application, the use of plasmid-borne antigens should be eliminated. There are two main reasons for this: (i) to ensure the stability of antigen expression in the absence of plasmid selection an...

Pre-existing immunity against vaccine vectors – friend or foe?

“…The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al , 2009) (depicted in Fig. 2).…”

“…In these subjects, humoral responses were moderately elevated in pre-exposed individuals. Similarly, Saxena et al (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses.…”

“…The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al , 2001; Kohler et al , 2000a, b; Metzger et al , 2004; Saxena et al , 2009; Sevil Domènech et al , 2008; Whittle & Verma, 1997), with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al , 1997; Gahan et al , 2008; Roberts et al , 1999; Sevil Domènech et al , 2007; Vindurampulle & Attridge, 2003a, b). …”

“…Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al , 2007, 2008). Saxena and colleagues also reported that antigen-specific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al , 2009). …”

Attenuating gene expression (AGE) for vaccine development

“…The attenuated Salmonella strains by Hoiseth and Stocker 4 has led to the extensive development of vaccines for typhoid fever in humans 5 , 6 and salmonellosis in calves, 7 - 9 pigs, 10 - 13 and chickens 14 - 16 . Moreover, such live vaccines also allow for the expression of heterologous genes from other pathogenic organisms that can stimulate both humoral and cell-mediated arms of immunity 17 - 19 . When these are attenuated, one unique feature of live vector vaccines is their ability to stimulate both the mucosal and systemic immune compartments of the host while having limited capacity for growth and pathogenicity.…”