Coming of AGE

Abstract

Live attenuated vaccine strains for bacterial pathogens are conventionally constructed by inactivating genes important for virulence or metabolism to render the organism less virulent. Ideally, these strains follow a natural route of infection, yet elicit an immune response without causing disease. In particular, live attenuated vaccines often have the advantage of generating cellular immune responses that killed organisms and purified antigens fail to stimulate. Importantly, a fine balance between attenuation and sufficient persistence in the host must be achieved in order to produce a protective and durable immune response. Unfortunately, the reality is that, in most cases, the complete repertoire of factors responsible for virulence is unknown or not well understood, hindering the creation of attenuated vaccine strains. Thus, the generation of live attenuated vaccines would be greatly facilitated by novel, portable approaches for attenuating Gram-negative pathogens.

Keywords: :

• AGE
• attenuated
• gene expression
• Salmonella
• vaccine

In this issue of Virulence, Yang et al.¹ used such an approach to generate an attenuated vaccine strain for Salmonella, a pathogen responsible for a large variety of foodborne and waterborne illnesses and a common vaccine vector (reviewed in refs. 2 and 3). The authors hypothesized that heterologous gene expression of antigens could render a wild-type pathogen more susceptible to host cell killing while maintaining immunogenicity, thus leading to protective immunity. They named their approach AGE, for Attenuating Gene Expression, and based it on a series of studies conducted by the senior author, David Pascual. Previously, Pascual’s group demonstrated that overexpression of enterotoxigenic E. coli (ETEC) fimbriae called colonization factor antigen I (CFA/I) in Salmonella resulted in an altered host immune response characterized by a Th2 response to the heterologously-expressed fimbriae while lacking stereotypical proinflammatory cytokine production by the infected macrophages.^4-8 Their studies suggested that the expression of CFA/I in Salmonella increased attachment and thus allowed for delivery of significantly more antigen to sites of the gut-associated and systemic lymphoid tissues. While others have used attenuated Salmonella strains to express foreign proteins and induce immunity (e.g., see ref. 9), Yang et al. have capitalized on these prior findings to create attenuated Salmonella strains by expressing the heterologous ETEC proteins.

Building on their initial findings¹⁰ through the generation of a series of optimized CFA expression constructs, Yang et al. were able to create promising live attenuated vaccine strains. Over a decade after their initial description of CFA/I expression in Salmonella, the authors of this study enhanced the expression of CFA/I proteins using an optimized tripartite promoter system. They found that overexpression of the ETEC fimbrial system proteins in Salmonella resulted in attenuation of the strain. They then proceeded to show that expression of the chaperone and usher proteins, CfaA and CfaB respectively, was essential for the observed attenuation. A comprehensive set of expression plasmids indicated that overexpression of CfaA, CfaC, and CfaE (minor fimbrial subunit) represented the best combination of CFA/I proteins to attenuate Salmonella. With these attenuated live vaccine strains, Yang et al. were then able to effectively orally immunize two different strains of mice, generating protective immunity with some constructs comparable to that achieved by the Salmonella aro negative attenuated control strain H647.

This study used a combination of in vitro macrophage infections and in vivo mouse infections to demonstrate the attenuation of Salmonella upon expression of CFA/I components. The CFA/I constructs induced a significant increases in serum IgG and fecal IgA specific for CFA/I and, most importantly, conferred protection after challenge with wild-type Salmonella. Furthermore, erythromycin sensitivity assays for the live attenuated vaccine strains have begun to shed light on one possible mechanism responsible for attenuation, with overexpression of CfaA and CfaC inducing permeability to erythromycin. The authors hypothesized that the observed in vivo attenuation is due to increased membrane permeability upon heterologous expression of CFA/I components.

This work presents important findings regarding a novel method for attenuation that may be applied to other Gram-negative pathogens. The proof-of-concept experiments set the framework for future applications of this method to other pathogens. A perhaps obvious next question is whether or not other heterologous fimbrial chaperone-usher pairs such as the E. coli PapCD or FimCD chaperone-usher pairs also attenuate heterologous hosts. The major potential for an AGE-based approach toward developing other vaccines lies in the portability of the system to other Salmonella or non-Salmonella strains as a method for general attenuation with concurrent antigen presentation. The CFA antigens have been used in Shigella to generate immunogenic live vaccine candidates; however, the host strain was already attenuated.¹¹ Thus, the extent to which AGE contributed to the attenuation of that live vaccine strain is not known.

Additional considerations come into the picture in examining AGE in the development of live, attenuated vaccines. First, conventional live attenuated vaccines produced by inactivation of metabolic genes are unable to replicate without an essential metabolite specifically garnered from the host recipient; however, AGE-attenuated strains may be competent for transmission outside the host. Additional safe guards may be necessary in AGE strains to limit their dissemination. Alternatively, after the validation of their safety, an AGE-based vaccine may have some advantage of producing herd immunity like many of the live attenuated viral vaccines.

A second consideration lies in the stability of the AGE-induced attenuation of the vaccine strain. Numerous mutations in the Cfa genes or promoters could result in a loss of attenuation and the emergence of a vaccine derivative with potentially enhanced virulence. Similarly, integration events in which the plasmid is incorporated into the genome may produce a loss of the Cfa portion of the plasmid while complementing the auxotrophy that is used to otherwise ensure plasmid maintenance. The authors have not reported the emergence of such strains in vivo, which may be due to a lack of specific assays to detect such emergent strains or, alternatively, a consequence of the relatively small number of animals in which the recombinant vaccine strains have been tested to date. Mutational events may be so rare that they would only be observed after administration of the vaccine to a large number of hosts. However, the transmissibility of the strains to other hosts would necessitate the development of sufficient genetic safety mechanisms to produce an abortive strain despite any of these speculative alterations.

One further consideration is the degree of attenuation produced by AGE. For Salmonella, maximal immune stimulation is generated by live attenuated vaccine strains that are able to effectively migrate to the regional lymph nodes, at which time they express vaccine antigens under environmentally sensitive promoters.¹² With the current engineering, the constitutive attenuation of the vaccine strain by AGE may limit the potential for generating complete immunity due to high expression of antigens from plasmid vectors, which is likely necessary to ensure sufficient attenuation. This potential limitation may be overcome through iterative engineering in which the expression of the AGE antigens is titrated through synthetic modification of the promoter or ribosome binding site to modify the level and timing of the vaccine antigen expression. Future studies optimizing current vaccine constructs will need to address these issues for the AGE vaccine strains to become therapeutically viable.

In summary, AGE is a novel approach for the generation of live attenuated vaccine strains that may find complementary utility to other emergent vaccine technologies. The flexibility and potential portability of the AGE system may allow for its adaptation in other Salmonella vectors as well as vaccine organisms derived from other bacterial genera or perhaps even eukaryotic organisms. While the applicability of AGE to other organisms remains to be tested, it is becoming increasingly evident that new vaccines and anti-infectives are drawing upon previous knowledge of bacterial components to use these against the same microorganisms. By engaging in this iterative process, a deeper understanding of bacterial regulation and how it affects immunogenicity in the host is being gained. Without a doubt, the initial fascinating observation made by Pascual and others nearly a decade ago has coalesced into a new twist on live attenuated vaccines: enhanced attachment may promote immunogenicity, but years later, we have learned that it is likely increased membrane permeability that is responsible for the impairment of an otherwise pathogenic strain.

Abbreviations:
AGE	=	attenuation through gene expression
ETEC	=	enterotoxigenic Escherichia coli
CFA	=	colonization factor antigen