Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein.

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.

Immunisation of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen.

Live vaccines based on BRD509, an attenuated S. typhimurium (aroA, aroD) strain, were constructed that directed the expression of hepatitis B core antigen particles (HBcAg) (BRD969) or HBcAg harbouring human papillomavirus type 16 E7 protein sequences (BRD974), under the control of the in vivo inducible nirB promoter. These strains were used to orally or intravenously immunise different inbred mouse strains and humoral, secretory and cellular anti-E7 and anti-HBcAg responses were monitored. Both BRD969 and BRD974 induced anti-HBcAg humoral IgG responses following oral or intravenous immunisation of B10 mice, although responses were higher in BRD969 immunised animals. IgG subclass analysis revealed a predominantly IgG2a response in these animals. BRD974, but not BRD969, induced anti-E7 humoral IgG responses. Anti-HBcAg (BRD969 and BRD974) and anti-E7 (BRD974) IgA responses were detected in the intestines of orally immunised mice. Anti-Salmonella but not anti-HBcAg or anti-E7 T helper cell responses were detected in mice immunised with BRD509, BRD969 and BRD974. Thus Salmonella vaccine strains can be used to efficiently deliver HBcAg and E7 epitopes to the mucosal and systemic immune systems.

Development of Attenuated Salmonella Strains that Express Heterologous Antigens.

The ability of attenuated strains of Salmonella to induce humoral, secretory, and cellular immune responses following oral ingestion has made them attractive as a system for delivering foreign antigens to the mammalian immune system. DNA capable of driving the expression of heterologous antigens can be introduced into Salmonella vaccine strains using a variety of approaches. In general, there are two common methods of expressing a foreign antigen in salmonellae: from plasmid vectors or from the bacterial chromosome. Since there are many similarities in the cellular and molecular biology of Escherichia coli and Salmonella, most of the genetic manipulations required to construct expression cassettes can be carried out in E. coli. The resulting constructs can then be introduced into the vaccine strains using simple transformatron or other similar techniques. However, the laboratory manipulation of Salmonella strains should be undertaken using techniques that do not lead to the accumulation of undefined genetic lesions, which may compromise the immunogenicity of Salmonella growing in vivo. With this in mind, we will describe appropriate techniques for manipulating Salmonella with the aim of constructing effective oral vaccines.

Isolation, characterization, and molecular cloning of a specific Mycobacterium tuberculosis antigen gene: identification of a species-specific sequence.

A rabbit polyclonal antiserum exhibiting a specific recognition pattern for Mycobacterium tuberculosis proteins was used to screen an M. tuberculosis genomic library constructed in the expression vector lambda gt11. One clone, denominated C1:10, expressed M. tuberculosis-specific determinants as part of a large fusion protein with beta-galactosidase. The gene for this protein has been sequenced, and it encodes a protein of 134 amino acids (13.8 kDa) which did not display significant homology with any of the previously reported proteins in the data bases. Hybridization studies with restriction fragments of the cloned sequence revealed that it was not present in the genomes of related mycobacteria, namely, M. bovis, M. bovis BCG, M. flavescens, M. fortuitum, M. phlei, and M. vaccae. These findings suggest that we have detected a gene, or a fragment therefrom, unique for M. tuberculosis whose nucleotide and amino acid sequences could be useful tools in the design of an improved vaccine or a diagnostic method of greater accuracy for tuberculosis.