Fritz Grunert

CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes

Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase‐variable opacity‐associated (Opa) outer membrane proteins mediate the non‐opsonized binding and internalization of N.gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52‐expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion‐associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52‐mediated interactions could be inhibited by polyclonal anti‐CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52‐expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N.gonorrhoeae.

Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae : identification of Opa adhesiotopes on the N-domain of CD66 molecules

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity‐associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N‐terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1‐ (CD66d) and NCA (CD66c)‐expressing cells. Because the C751 Opa proteins make up distinct structures via the surface‐exposed hypervariable domains (HV‐1 and HV‐2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To define the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N‐domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N‐domain that we constructed show that the binding of all Opa proteins tested occurs at the non‐glycosylated (CFG) face of the molecule and, in general, appears to require Tyr‐34 and Ile‐91. Further, efficient interaction of distinct Opa proteins depends on different non‐adjacent amino acids. In the three‐dimensional model, these residues lie in close proximity to Tyr‐34 and Ile‐91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N‐domain. These studies define the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.

Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–Claudin-1 associations†

The tight junction protein claudin‐1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry—the first step of viral infection. Due to the lack of neutralizing anti‐CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface–expressed CLDN1 specifically inhibit HCV infection in a dose‐dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti‐CLDN1 and anti‐CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti‐CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1‐E2 interaction. Using fluorescent‐labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti‐CLDN1 antibodies inhibit CD81‐CLDN1 association. In contrast, CLDN1‐CLDN1 and CD81‐CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81‐CLDN1 interactions. Conclusion: These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2‐CD81‐CLDN1 interactions. (HEPATOLOGY 2010.)

Carcinoembryonic Antigen Family Members CEACAM6 and CEACAM7 Are Differentially Expressed in Normal Tissues and Oppositely Deregulated in Hyperplastic Colorectal Polyps and Early Adenomas

Four members of the carcinoembryonic antigen (CEA) family, CEA, CEACAM1 (BGP), CEACAM6 (NCA-50/90), and CEACAM7 (CGM2), are coexpressed in normal colorectal epithelia but are deregulated in colorectal cancers, where they could play a role in tumorigenesis. As a basis for functional studies, their expression patterns in normal tissues first need to be clarified. This is well documented for CEACAM1 and CEA but not for CEACAM6 or CEACAM7. We have now carried out immunohistochemical expression studies on 35 different organs, using CEACAM6-specific (9A6) and CEACAM7-specific (BAC2) monoclonal antibodies. CEACAM7 was only found on the apical surface of highly differentiated epithelial cells in the colorectal mucosa and on isolated ductal epithelial cells within the pancreas. CEACAM6 was expressed in granulocytes and epithelia from various organs. CEACAM6 and CEACAM7 expression correlated with apoptosis at the table region of the normal colon, and both were absent from highly proliferating cells at the base of colonic crypts. CEACAM6 revealed a broader expression zone in proliferating cells in hyperplastic polyps and adenomas compared with normal mucosa, whereas CEACAM7 was completely absent. Down-regulation of CEACAM7 and up-regulation of CEACAM6 expression in hyperplastic polyps and early adenomas represent some of the earliest observable molecular events leading to colorectal tumors.

Monoclonal Anti-Claudin 1 Antibodies Prevent Hepatitis C Virus Infection of Primary Human Hepatocytes

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. The tight junction protein claudin-1 (CLDN1) has been shown to be required for entry of HCV into the cell. METHODS Using genetic immunization, we produced 6 monoclonal antibodies against the host entry factor CLDN1. The effects of antibodies on HCV infection were analyzed in human cell lines and primary human hepatocytes. RESULTS Competition and binding studies demonstrated that antibodies interacted with conformational epitopes of the first extracellular loop of CLDN1; binding of these antibodies required the motif W(30)-GLW(51)-C(54)-C(64) and residues in the N-terminal third of CLDN1. The monoclonal antibodies against CLDN1 efficiently inhibited infection by HCV of all major genotypes as well as highly variable HCV quasispecies isolated from individual patients. Furthermore, antibodies efficiently blocked cell entry of highly infectious escape variants of HCV that were resistant to neutralizing antibodies. CONCLUSIONS Monoclonal antibodies against the HCV entry factor CLDN1 might be used to prevent HCV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Expression of CEACAM6 in Resectable Colorectal Cancer: A Factor of Independent Prognostic Significance

PURPOSE CEACAM6, CEACAM1, and human carcinoembryonic antigen (CEA) are coexpressed in normal colorectal epithelia, but show deregulated expression in colorectal cancers (CRC). Upregulation of CEACAM6 expression in hyperplastic polyps and early adenomas represents one of the earliest observable molecular events leading to colorectal tumors. The aim of our study was to evaluate the prognostic relevance of CEACAM6, CEACAM1, and CEA tissue expression in patients with CRC. PATIENTS AND METHODS Immunohistochemical analysis was carried out on tissue microarrays from 243 paraffin-embedded biopsies from a randomized controlled clinical trial (Swiss Group for Clinical Cancer Research [SAKK] 40/81) of adjuvant fluorouracil-based chemotherapy with CEACAM-specific monoclonal antibodies. The median follow-up was 8 years. Overall survival (OS) and disease-free survival (DFS) were calculated using Kaplan-Meier estimates and hazard ratios (HRs) estimated using Cox proportional hazards models. RESULTS Tissue expression of CEACAM6, CEACAM1, and CEA was enhanced in 55%, 58%, and 94% of patients, respectively. Multivariate Cox analysis including sex, age, tumor site, stage, differentiation grade, treatment, and nodal status as covariates showed that CEACAM6 overexpression independently predicted poor OS (HR, 1.86; P =.0100) and DFS (HR, 2.00; P =.0028), whereas CEACAM1 or CEA were not significantly related to these outcomes. The data did not provide evidence for or against the hypothesis that the CEACAM6 effect on survival differs according to treatment. CONCLUSION Expression of the cell adhesion molecule CEACAM6 in CRC is an independent prognostic factor allowing subdivision of patients into low- and high-risk groups. Whether CEACAM6 or CEA and CEACAM1 might be useful as predictive markers of chemotherapy benefit remains unclear.

Biliary glycoprotein (BGP) expression on T cells and on a natural-killer-cell sub-population

Human T and natural‐killer (NK) cells, that are thought to be the major cytotoxic effector‐cell populations in the defence against neoplastic cells, were isolated from blood and decidua in order to analyze their expression of carcinoembronic‐antigen‐(CEA)‐family‐member proteins. Biliary glycoprotein (BGP, CD66a) was the only member of the carcinoembryonic antigen family detected. While freshly isolated T‐cells expressed low amounts of BGP, freshly isolated NK cells were negative. After in vitro stimulation for 3 days,T cells up‐regulated their BGP expression and a sub‐group of NK cells (CD16− CD56+), known to predominate in decidua, revealed de novo expression of BGP. In contrast, stimulated CD16+ CD56+ NK cells, which occur exclusively in the blood, remained negative. The expression of BGP could be shown on the protein level by using a panel of 12 well‐defined MAbs and on the transcription level in rt‐PCR and subsequent oligonucleotide hybridization. Interestingly, rIL‐2‐stimulated T cells expressed 3‐fold higher levels of BGP compared with those seen after stimulation with phytohemaglutinine (PHA). PHA, on the other hand, induced a higher expression of HLA‐DR, an activation marker of T cells. The differential regulation implies a distinct function of BGP and HLA‐DR.

The postbinding activity of scavenger receptor class B type I mediates initiation of hepatitis C virus infection and viral dissemination.

Scavenger receptor class B type I (SR‐BI) is a high‐density lipoprotein (HDL) receptor highly expressed in the liver and modulating HDL metabolism. Hepatitis C virus (HCV) is able to directly interact with SR‐BI and requires this receptor to efficiently enter into hepatocytes to establish productive infection. A complex interplay between lipoproteins, SR‐BI and HCV envelope glycoproteins has been reported to take place during this process. SR‐BI has been demonstrated to act during binding and postbinding steps of HCV entry. Although the SR‐BI determinants involved in HCV binding have been partially characterized, the postbinding function of SR‐BI remains largely unknown. To uncover the mechanistic role of SR‐BI in viral initiation and dissemination, we generated a novel class of anti–SR‐BI monoclonal antibodies that interfere with postbinding steps during the HCV entry process without interfering with HCV particle binding to the target cell surface. Using the novel class of antibodies and cell lines expressing murine and human SR‐BI, we demonstrate that the postbinding function of SR‐BI is of key impact for both initiation of HCV infection and viral dissemination. Interestingly, this postbinding function of SR‐BI appears to be unrelated to HDL interaction but to be directly linked to its lipid transfer function. Conclusion: Taken together, our results uncover a crucial role of the SR‐BI postbinding function for initiation and maintenance of viral HCV infection that does not require receptor‐E2/HDL interactions. The dissection of the molecular mechanisms of SR‐BI–mediated HCV entry opens a novel perspective for the design of entry inhibitors interfering specifically with the proviral function of SR‐BI. (HEPATOLOGY 2013)

Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding

The carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEACAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection‐based infection system, we showed that binding of Opa52 involves residues 27–42, which are predicted to form β‐strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino‐terminal domain. The replacement of residues 27–29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEACAM6 receptors that are bound by CEACAM1/CEACAM5‐specific Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC′ face of the N‐terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.

Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response

CD66a is an adhesion molecule member of the carcinoembryonic antigen immunoglobulin‐like family present on the surface of epithelial cells, granulocytes and IL‐2 activated T cells. We studied whether CD66a is expressed in vivo by T lymphocytes and whether it affects TCR‐mediated activation. CD66a was detected by histochemistry, flow cytometry analysis, reverse transcription PCR and Western blot on fresh colon biopsies and T cell clones. A fraction of T cells in the lamina propria express CD66a, which is induced by IL‐7 and IL‐15 cytokines. T cells express four different CD66a splice variants and at least two forms of the protein are glycosylated in a cell type‐specific manner. Triggering of CD66a on T cells with physiological ligands or with specific mAb increases TCR‐mediated lymphokine release, in an antigen dose‐independent manner. This effect requires the presence of the CD66a intracytoplasmic domain, which contains two immunoglobulin receptor family tyrosine‐based inhibitory motif‐like domains, as shown by stimulation of Jurkat cells transfected with different CD66a isoforms and is associated with increased induction of AP1 and NFκB transcription factors. These data indicate that CD66a amplifies T cell activation and thus could facilitate crosstalk between epithelial cells and T lymphocytes in intestinal immune response.