Phase I clinical trials of aroA aroD and aroA aroD htrA attenuated S. typhi vaccines; effect of formulation on safety and immunogenicity.

PBCC211, an aroA aroD derivative of S. typhi strain CDC10-80, was tested in phase I trials as a single dose typhoid fever vaccine. Three different vaccine preparations, reconstituted lyophilized bacteria, freshly grown bacteria or lyophilized bacteria reconstituted from sachets, were orally administered to a total of 86 adult volunteers. An aroA aroD htrA strain, PBCC222, was also tested in 38 volunteers. Formulation impacted on the determination of a safe and immunogenic dose; reconstituted lyophilized cultures required higher doses than the broth cultures to stimulate seroconversion. In general, doses which seroconverted the majority of group members produced undesirable symptoms regardless of attenuation or formulation. The inability to separate the presence of symptoms from achieving significant immunogenicity in these aroA aroD or aroA aroD htrA strains precludes their use as single dose typhoid vaccines in the formulations tested. Multiple doses of these strains at a lower, safe level may be effective as vectors for foreign antigens.

Glycoprotein conjugate vaccines.

The polysaccharide capsule which surrounds bacterial species like Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, Salmonella typhi, is a potent virulence factor. It protects the bacterium from phagocytosis, but capsule specific antibodies plus complement binding to the capsule opsonise the organism for phagocytosis and elimination. Purified capsules elicit T-independent antibody responses without a memory function, and are often poorly immunogenic in infants where much of invasive H. influenzae type b (Hib) and pneumococcal infection is seen. Covalent linkage of the polysaccharide, or fractions thereof, to immunogenic carrier proteins creates glycoconjugates which are T-dependent antigens and which prime for boosting either with the glycoconjugate or the capsular polysaccharide; During the 1990s, four Hib glycoconjugate vaccines have been introduced and in countries that have vaccinated the majority of children, the success has been stunning. In countries with very high immunization coverage the disease has been virtually eliminated and, to a decline of over 95% in countries with slightly lower vaccine rates. Worldwide use of Hib glycoconjugate vaccines offers the possibility of elimination of invasive Hib disease. Pneumococcal (11 serotypes with coverage of approximately 85% of invasive disease) and meningococcal (A, C, W 135, Y but not B) glycoconjugates are in pre-registration phases and offer the prospect of being as successful as the Hib glycoconjugates.

Polyosides (encapsulated bacteria).

The polysaccharide capsule which surrounds bacterial species such as Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis and Salmonella typhi is a potent virulence factor by protecting the bacteria from phagocytosis. The host responds with antibody production and specific antibodies plus complement binding to the capsule facilitate opsonization of the micro-organism, which is phagocytized and eliminated. Purified capsular polysaccharides elicit T-independent antibody responses without a memory function, but are often poorly immunogenic in infants where much of the invasive H. influenzae type b (Hib) and pneumococcal infections is seen. Therefore purified polysaccharides have found limited use as vaccines. However, covalent linkage of the capsular polysaccharide, or fractions thereof, to immunogenic carrier proteins creates glycoconjugates which are T-dependent antigens and which elicit antibodies also in infants and which prime for boosting either with the glycoconjugate or the capsular polysaccharide. In the last decade Hib glycoconjugate vaccines have been successfully introduced and in countries with very high immunization coverage the disease has been virtually eliminated and a decline of over 95% has been seen in countries with slightly lower vaccine rates. World-wide use of Hib glycoconjugate vaccines offers the possibility of elimination of invasive Hib disease. Pneumococcal (11 serotypes with coverage of approximately 85% of invasive disease), meningococcal (A, C, W 135, Y but not B) and S. typhi glycoconjugates are in advanced development and offer the prospect of being as successful as the Hib glycoconjugates.

Vaccination against enteric pathogens: from science to vaccine trials.

Recent advances in scientific research and clinical trials have shown promise for vaccine development against enteric pathogens. Identification of new virulence factors, such as the two distinct shigella enterotoxins, has allowed the development of new immunogen or new attenuated strains. Improved knowledge facilitated the development of safer attenuated live microorganism and construction of multivalent vaccines. Finally, an important advantage is the use of nonreplicating plasmid DNA vectors to express protective antigens in the host.

Shigella flexneri type-specific antigen V: cloning, sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV.

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI-BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.

Molecular characterization of the genes involved in O-antigen modification, attachment, integration and excision in Shigella flexneri bacteriophage SfV.

Bacteriophage SfV is a temperate phage of Shigella flexneri responsible for converting serotype Y (3,4) to serotype 5a (V; 3,4) through its glucosyl transferase gene. The glucosyl transferase (gtr) gene of SfV has been cloned and shown to partially convert S. flexneri serotype Y to serotype 5a. In this study, we found that the serotype-converting region of SfV was approximately 2.5 kb in length containing three continuous ORFs. The recombinant strain carrying the three complete ORFs expressed the type V and group antigen 3,4, both indistinguishable from that of S. flexneri 5a wild-type strain. The interruption of orf5 or orf6 gave partial conversion in the S. flexneri recombinant strain indicated by the incomplete replacement of group antigen 3,4. The region adjacent to the serotype-conversion genes was found to be identical to the attP-int-xis region of phage P22. Altogether, an approximately 2.2-kb sequence covering a portion of the serotype-conversion (approximately 500 nt)-attP-int-xis regions of SfV was remarkably similar to that of P22.

Construction and characterization of genetically-marked bivalent anti-Shigella dysenteriae 1 and anti-Shigella flexneri Y live vaccine candidates.

Bivalent vaccine candidates were developed against Shigella dysenteriae 1 and Shigella flexneri, which are among the most frequent causative agents of shigellosis in developing countries. The rfp and rfb gene clusters, which code for S. dysenteriae serotype 1 O-antigen biosynthesis, were inserted into an arsenite resistance minitransposon and randomly integrated into the attenuated S. flexneri aroD serotype Y strain SFL124. Nine recombinant clones that efficiently expressed both homologous and heterologous O-antigens were obtained. Southern blot analysis showed that in one clone the S. dysenteriae 1 genes had integrated into the chromosome, whereas in all the others they had integrated into the virulence plasmid. All recombinant clones exhibited normal growth characteristics, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed efficiently the recombinant lipopolysaccharide within invaded cells. Immunization of mice with two of the recombinant clones resulted in the production of antibodies specific for both homologous and heterologous O-antigens. The recombinant clones constitute promising vaccine candidates which can readily be distinguished from endemic shigellae by their non-antibiotic resistance marker.

Construction and characterization of a live attenuated vaccine candidate against Shigella dysenteriae type 1.

Vaccine candidates against Shigella dysenteriae type 1, which is associated with the most severe cases of bacillary dysentery, were constructed. The rfp and rfb gene clusters, which code for S. dysenteriae 1 O antigen biosynthesis, were randomly integrated into either the chromosome or the virulence plasmid of the rough attenuated Shigella flexneri aroD strain SFL124-27 with a minitransposon carrying an arsenite resistance selection marker. The recombinant clones efficiently expressed the recombinant O antigen, exhibited a normal growth pattern, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed the recombinant antigen within invaded cells. A clone was selected as the vaccine candidate, which was demonstrated to be immunogenic and safe in animal models, leading to 47% full protection and 53% partial protection against challenge with the wild-type strain.

Quantitative assessment of IgG and IgA subclass producing cells in rectal mucosa during shigellosis.

AIMS: To assess quantitatively both the morphological changes in the rectal mucosa and the changes in the relative frequency of IgA and IgG subclass producing cells found in the rectal mucosa during the acute phase of shigellosis and at convalescence. METHODS: Rectal biopsies from 25 Shigella dysenteriae 1 infected patients, 10 Shigella flexneri infected patients, and 40 uninfected controls were studied. Morphological changes in the mucosa were graded. The frequency of IgA and IgG subclass producing cells was assessed. In addition, immunostaining for secretory component in epithelial cells was analysed. RESULTS: Using morphological grading, 20% of the 35 patients studied had advanced inflammation (grade 3) in the acute phase of the disease. At convalescence, grade 1 inflammation was seen in 37% of the patients and in 10% of the controls. In the acute phase, as well as at convalescence, the number of IgA1, IgA2, and IgG2 positive cells was significantly higher than in the controls. The results were related to the histopathological degree of inflammation. CONCLUSIONS: In shigellosis, there is evidence for a prolonged humoral response residing in the mucosa long after the clinical symptoms have resolved, suggesting that shigellosis induces persisting mucosal humoral immune and inflammatory responses, remaining at least until 30 days after the infection.

An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible.

In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis.

Shigella species cause bacillary dysentery in humans by invading epithelial cells of the colonic mucosa leading to colonic epithelial cell destruction and inflammation. For further analysis of local gut inflammation, morphological changes and the potential involvement of mediators in regulatory mechanisms of cell activation and cell proliferation were studied immunohistochemically in rectal mucosal biopsies taken from patients during the acute phase of shigellosis and at convalescence. Rectal biopsies from 25 Shigella dysenteriae-1 and 10 Shigella flexneri-infected patients and from 40 controls were studied. The frequencies of proliferative cells (Ki67-positive cells), p53-immunostaining cells, and cells coexpressing Ki67 with CD3 or with p53 were analyzed. Immunostaining for the inducible nitric oxide synthase (iNOS) and the endothelial NOS was assessed. In addition, the frequencies of apoptotic cells and CD68+ cells that engulf apoptotic cells were assessed. By morphological grading, 20% of the patients had advanced inflammation (grade 3) in the acute phase; mild inflammation (grade 1) was seen in 37% of the patients at convalescence as well as in 10% of the controls. The findings in the present study suggest that in the acute phase of shigellosis inflammation is characterized by increased cell turnover in the lamina propria (LP) and the epithelium, increased iNOS expression in the surface epithelium, and apoptosis, which seems to be associated with LP macrophages. The findings also suggest that neither p53 nor iNOS are important factors for the induction of apoptosis in shigellosis. Expression of p53 may be related to early cell activation in crypt epithelium. Moreover, there is an indication of an active, low-level inflammatory process at convalescence. The results thus indicate that Shigella-induced inflammation is associated with a complex series of cellular reactions in the rectal gut mucosa which persist long after clinical symptoms have resolved.

Monoclonal antibodies against Haemophilus influenzae lipopolysaccharides: clone MAHI 4 binding to a pentasaccharide containing terminal beta-Gal residues and clone MAHI 10 recognizing terminal phosphorylated saccharide residues.

Mouse monoclonal antibodies MAHI 4 and MAHI 10 reactive with Haemophilus influenzae lipopolysaccharide (LPS), were generated by fusing mouse myeloma cells with spleen cells of mice immunized with H. influenzae strain RM.7004-XP-1. The antibody MAHI 4 reacted in whole-cell enzyme immunoassay (EIA) and colony-dot-immunoblotting with 20 of 123 H. influenzae strains and to a few other human Haemophilus spp. and Neisseria spp., but not to any Bordetella pertussis, B. parapertussis, Aeromonas spp. or Moraxella catarrhalis strains tested. This suggests a specific epitope accessible to recognition in just a few strains. This conclusion was supported by the data on binding of MAHI 4 to only three of 18 H. influenzae LPSs tested, but not to any Haemophilus ducreyi or enterobacterial LPSs. The antibody MAHI 10 bound to 80 of 123 strains of H. influenzae and to a few strains of Neisseria spp. and M. catarrhalis as evaluated by EIA and colony-dot-immunoblotting, which suggests an epitope accessible to recognition in 65% of the H. influenzae strains tested. The antibody MAHI 10 reacted with 10 of 18 H. influenzae LPSs as determined by EIA. By using polysaccharides, obtained after both mild acidic hydrolysis, strong alkali treatment, and dephosphorylation, as inhibitors of the antibodies binding to H. influenzae LPS antigens it was shown that phosphate groups were essential for the binding of MAHI 10 to LPS but they did not affect antigenic recognition by MAHI 4. None of the monoclonal antibodies bound to isolated lipid A, but the aggregation caused by the fatty acids of lipid A was essential for optimum epitope recognition. Enzymatic treatment of homologous LPSs with galactose-oxidase led to products which were between 20 to 30 times less effective as inhibitors of the binding of the MAHI 4 than the native LPSs. Taken together the results indicate that MAHI 4 has the following pentasaccharide as the epitope Gal beta 1-->2 Hep alpha 1-->2Hep alpha 1-->3Hep alpha 1--> Kdo(P). These results emphasize the importance of the terminal beta-Gal residue in the definition of the MAHI 4 specificity, and of the terminal phosphorylated saccharide residues of some of the Haemophilus LPSs for the MAHI 10 specificity.

Persistence of nontypeable Haemophilus influenzae in adenoid macrophages: a putative colonization mechanism.

That nontypeable H. influenzae (NTHI) can reside intracellularly in human adenoid tissue has been suggested by use of in situ hybridization of a fluorescein labelled 16S rRNA-targeted oligonucleotide probe (FISH). Adenoid tissues from 43 children operated on in a clinically infection-free interval were investigated. FISH revealed H. influenzae in macrophage-like cells, located subepithelially in the crypts in all 43 adenoids. Furthermore, H. influenzae was detected in 22/22 adenoids using immunohistochemistry with the monoclonal antibody MAHI-3 recognizing a conserved H. influenzae LPS inner-core region. FISH and staining with monoclonal antibodies against immunophenotypic markers were performed simultaneously in order to characterize the cellular interrelations in this microenvironment. The findings of widespread presence of H. influenzae in cells of which some strongly expressed the CD14 marker of the monocyte/macrophage lineage may correspond to an important aspect of the colonization mechanisms whereby NTHI persists in the nasopharynx of children.

Semiquantitative estimation of Shigella antigen-specific antibodies: correlation with disease severity during shigellosis.

Systemic and mucosal antibody responses during shigellosis were analysed and correlated with clinical severity. Patients infected with Shigella dysenteriae type 1 (SDIP), and S. flexneri (SFIP) were grouped according to disease severity. The disease was more severe in SDIP than in SFIP. Higher levels of Shigella antigen-specific serum antibodies were associated with severe disease. In contrast, decreased serum levels of IgG, IgM and total protein were associated with severe disease. Faecal levels of total IgA, secretory IgA, albumin, and LPS specific s-IgA were increased in severe disease. The results of the study indicate that the level of humoral immune response correlates with the disease severity in shigellosis and that the local immune system in the mucosa is directly engaged in the response. This is also reflected at the systemic level by increased serum levels of specific antibodies and non-specific inflammatory parameters.

Immunomagnetic separation and PCR detection show Shigellae to be common faecal agents in children from urban marginal communities of Costa Rica.

Culture and immunomagnetic separation-polymerase chain reaction assays (IMS-PCR) were used to isolate and identify Shigella flexneri, S. dysenteriae type I and S. sonnei in faeces from 250 children up to 5 years and from their mothers (n = 143) selected at random from a large urban marginal community of Costa Rica. Children hospitalized because of severe diarrhoea (n = 110) were also studied. Only S. flexneri, mainly serotype 2a, and S. sonnei were found by culture. All specimens in which Shigella was cultured were also positive by the corresponding IMS-PCR. S. flexneri was isolated by culture from 1 (0.7%) mother and 4 (1.6%) community children. S. sonnei was found in 2 (0.8%) children. An additional 12 S. flexneri and four S. sonnei in the community children were found by IMS-PCR. In total, Shigella was cultured from 0.7% of mothers and 2.4% of children. By the IMS-PCR 2% of mothers and 8% of children were positive. S. flexneri was isolated by culture from 14 (12.7%) hospitalized children and S. sonnei from 1 (0.9%). An additional 11 S. flexneri and three S. sonnei were found by IMS-PCR. In total, Shigella was cultured from 13.6% of hospitalized children. By the IMS-PCR 26% of them were Shigella positive. Thus IMS-PCR was more than twice as effective in diagnosing shigellae as culture. Twelve (60%) Shigella positive community children were above 3-years-old and 25% of them were under one year. Seven (35%) of the Shigella positive children had dysenteric and 9 (45%) normal stools. Half of the Shigella infected community children had been weaned before the 3 months of age. By the age of 5 months, 90% of them were already weaned. Seventeen (59%) of the hospitalized Shigella positive children were under 1 year of age. The stools were watery or semiliquid in 13 (45%) and dysenteric in 12 (41%) of them. We conclude that shigellosis is common in Costa Rica and represents an important cause of severe infant diarrhoea requiring hospitalization.

Dissociation between cytokine mRNA expression and protein production in shigellosis.

In our study, infection with Shigella dysenteriae type 1 (n = 16) or Shigella flexneri in adults (n = 5) was associated with a gradual accumulation of mRNA for interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, transforming growth factor-beta, IL-10, IL-4, TNF-beta, interferon (IFN)-gamma and perforin in the rectal biopsy samples during the convalescent stage of the disease demonstrated by in situ hybridization. In contrast, immunohistochemical staining in rectal tissues of cytokine protein-producing cells at the single-cell level exhibited a steady-state expression during 2-36 days after the onset of the disease. The frequency of cytokine mRNA-expressing cells varied in the range of 3-100-fold higher than that of the corresponding protein-synthesizing cells. The accumulation of cytokine mRNA in vivo during shigellosis represented a long-lasting phenomenon throughout the disease course, and may be linked to its immunopathogenesis. The results also indicate that assessment of both protein and mRNA in vivo may provide complementary information. Stimulation in vitro of peripheral blood mononuclear cells from normal healthy donors with Shigella-derived lipopolysaccharide or shiga toxin was carried out to elucidate the role of Shigella antigens in the regulation of translation of cytokine-specific mRNA. The incidence of cytokine (IFN-gamma, IL-6 and TNF-alpha) mRNA- and cytokine protein-expressing cells was very similar and congruent after both these Shigella-derived stimuli. We could, thus, not find evidence for shiga toxin-induced down-regulation of cytokine mRNA translation as the explanation for the observed discrepancy between cytokine mRNA and protein levels in the tissue biopsies.

Changes in the peripheral blood T-Cell receptor V beta repertoire in vivo and in vitro during shigellosis.

A sequential activation of T cells in peripheral blood during shigello sis has been observer (D. Islam, P.K. Bradham, A. A. Lindberg, and B. Christensson, Infect. Immun 63:2941-2949, 1995). To further investigate the cellular response during the course of Shigella infection, changes in the T-cell receptor (TCR) repertoire in the subsets in blood in patients during shigellosis was that Shigella antigens may modulate the function of T cells carrying TCRs capable of recognizing Shigella-specific epitopes or superantigens. Such a selective preference for T cells expressing certain TCR Vbeta types could lead to the expansion or deletion of these T cells. In the present study of 27 adult male Bangladeshi patients with dysentery (14 cases caused by Shigella Dysenteriae 1 and 13 cases caused by Shigella flexneri), the changes in the TCR Vbeta repertoire of peripheral blood CD4+ and CD8+ T-cell subsets have been analyzed with a panel of nine anti-Vbeta monoclonal antibodies by flow cytometry. Twenty healthy males from Bangladesh and 20 healthy males from Sweden served as controls. Compared with the Bangladeshi controls, the patients had an increased frequency of CD4+T cells expression Vbeta2, Vbeta3, and Vbeta17, with a maximum at day 7 after the onset of disease. The frequency of CD4+T cells expressing Vbeta5.1 was increased only in patients with S. flexneri infection. Peripheral blood T cells from Shigella-infected patients also responded to in vitro stimulation in a TCR Vbeta-specific manner. Stimulation with heat-killed S. dysenteriae 1 and Shiga toxin enhanced the frequency of cells expressing Vbeta2, Vbeta3, Vbeta5.1, Vbeta13.6, and Vbeta17, especially in samples obtained at day 7. The enhanced frequency of cells expressing Vbeta2, Vbeta3, Vbeta5.1, and Vbeta17 found both in in vivo and in vitro could suggest that in shigellosis antigens or superantigens are presented to the immune system and preferentially activate certain TCR Vbeta types in T-cell subsets. The kinetics of the change in the TCR Vbeta repertoire in blood during shigellosis may indicate that following local activation, the antigen activated T cells can be retrieved in the blood and restimulated in vitro. If confirmed by parallel analysis of T cells in the gut and blood by TCR sequence analysis, the possibility suggested by our findings would facilitate further analysis of the role of cell-mediated immune responses in the pathogenesis of and protection against Shigella infection.

Local entrapment of interferon gamma in the recovery from Shigella dysenteriae type 1 infection.

In healthy controls (n = 8) living in shigella endemic areas, accumulation of interferon gamma (IFN gamma) in the epithelial lining was seen in the rectal tissues. At the single cell level, however, few or no IFN gamma protein producing cells or mRNA expressing cells were detected at that site indicating the involvement of the whole large intestine in the production of IFN gamma in controls. Persistent numbers of IFN gamma producing cells were detected in the rectum of patients with Shigella dysenteriae type 1 infection (n = 8) throughout the course of disease with a tendency to increase in the convalescent stage. A significantly increased extra cellular deposition of secreted IFN gamma in tissue was seen in convalescence when compared with the acute stage (p < 0.05). In addition, enzyme immunoassay showed increased stool concentration of IFN gamma in patients at the convalescent stage as well as in healthy controls. In situ hybridisation confirmed the results by showing increased frequency of IFN gamma mRNA containing cells at the late stage of the disease (p < 0.05). Extensive message for IFN gamma was evident in cells in the lamina propria with no detectable transcripts in the surface epithelium. A colocalisation of IFN gamma with the IFN gamma receptor expression, predominantly found in the epithelial lining was detected by immunohistochemistry. Semiquantitative evaluation by computerised image analysis showed a gradual increased expression of IFN gamma and its corresponding receptor in the convalescent stage of shigellosis. This suggested progressive entrapment and binding of IFN gamma to its specific receptor at the local site. The enhanced surface expression of IFN gamma receptor evident at the convalescent stage of shigellosis was comparable to the constitutive level of expression in the healthy subjects. Thus, immunity to shigellosis correlated to up-regulation of IFN gamma production and expression of IFN gamma receptor.