Hepatitis A Virus Cellular Receptor 1 Research Articles

Tim-1-mediated extracellular matrix promotes the development of hepatocellular carcinoma.

Tim-1 (T-cell immunoglobulin and mucin domain 1), also known as Kim-1 (kidney injury molecule 1) or hepatitis A virus cellular receptor 1 (HAVCR1), is a transmembrane protein expressed on various immune and epithelial cells. It plays a role in modulating inflammatory and immune responses. In this study, we find that Tim-1 is overexpressed in hepatocellular carcinoma (HCC) samples and that its expression is significantly correlated with postoperative survival. Bulk RNA sequencing reveals a general upregulation of extracellular matrix-related genes in HCC tissues with Tim-1 overexpression. The results of the cell and in vivo experiments reveal that Tim-1 in HCC not only affects biological processes such as the proliferation, migration, and invasion of HCC cells but also broadly promotes extracellular matrix processes by influencing cytokine secretion. Further studies demonstrate that Tim-1 mediates the activation of hepatic stellate cells and upregulates Th1 and Th2 cytokines, thereby promoting HCC progression. Thus, Tim-1 may represent a novel target for future interventions in HCC and liver fibrosis.

Abstract PO3-24-07: The role of Hepatitis A Virus Cellular Receptor 1 (HAVCR1) in breast cancer and the impact on penetration of blood brain barrier

Abstract Background: Hepatitis A Virus Cellular Receptor 1 (HAVCR1) is associated with the biological behaviour of several cancers, and has been shown to regulate junctional complexes. However, its role in breast cancer remains unexplored. This study aimed to investigate the effects of HAVCR1 on human breast cancer. Methods: We explored the UALCAN web portal, which is linked to the TCGA database, to determine the expression of HAVCR1 in breast cancer and the Kaplan-Meier plotter to evaluate the correlation with the survival of the patients. HAVCR1 siRNA was used to knock down the HAVCR1 expression in MCF-7 and MDA-MB-231 cell lines. In vitro cell growth, wounding healing, and electric cell impedance sensing (ECIS for assessing cellular proliferation, migration and adhesion) assays were used. Transepithelial resistance (TEER) and paracellular permeability (PCP) were assessed to determine changes in the barrier function of human breast cancer cells. The expression of HAVCR1 and prospective interactive binding molecules of HAVCR1 protein was assessed using quantitative polymerase chain reaction (qPCR). Cellular response to docetaxel was evaluated and shown (IC50). The invasiveness of breast cancer cells through the human cerebral microvessel endothelial cells (hCMEC/D3) cell layers was measured using an in vitro transwell cell invasion assay. Results: HAVCR1 gene expression levels were significantly lower (P< 0.001) in breast cancer tissue compared with normal tissues. Patients with high expression of HAVCR1 had a significantly better relapse-free survival than low expression (P< 0.001). There was no significant difference in overall survival (OS) between the HAVCR1 high and the low expression groups (P=0.22). However, in subgroup analysis, high HAVCR1 expression was associated with better OS in luminal A (P=0.04). Knockdown (KD) HAVCR1 in breast cancer cells influence cell function by enhanced adhesion, proliferation, and migration compared to wild type (WT) cell lines. It was noted that loss of HAVCR1 rendered both MCF-7 and MDA-MB-231 cells resistant to docetaxel (IC50 for WT and HAVCR1 KD respectively being 0.86nM vs 1.75nM for MCF-7 and 1.35nM vs 15.48nM for MDA-MB-231). HAVCR1 KD increase adhesion to hCMEC/D3 cells (53.14±8.23 vs ±99.37±8.97 for MCF-7, P< 0.001; 109.5±97.56 vs 147.1±96.63 for MDA-MB-231, P=0.228, WT and HAVCR1 KD). In the cerebral endothelial cell invasion assay, we found that HAVCR1 KD resulted in a significantly increased invasion of the breast cancer cells through the endothelium (19.27±8.74 vs 33.58±16.81 for MDA-MB-231, P=0.0114, WT and HAVCR1 KD). For the barrier function, the knockdown led to a decrease in TEER and an increase in PCP when compared with WT cells in both cell lines. HAVCR1 KD cells showed decreased barrier function, together with reduced expression of Claudin-1/8/10, occludin, MarvelD3, Tricellulin and ZO1. Conclusions: HAVCR1 is a potential biomarker for breast cancer prognosis. HAVCR1 influences the integrity of barrier function by influence the gene expression of tight junction in breast cancer, and influence the penetration to blood brain barrier. Together, it suggests that HAVCR1 may be a potential therapeutic target in breast cancer. Citation Format: Xiaoshan Cao, Binbin Cong, Wen Jiang, Tracey Martin. The role of Hepatitis A Virus Cellular Receptor 1 (HAVCR1) in breast cancer and the impact on penetration of blood brain barrier [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-07.

O123 Significance of hepatitis A virus cellular receptor-1 (HAVCR1) and its association with tumour immune microenvironment in breast cancer

Abstract Introduction Immunotherapy has an important role in the intervention of breast cancer, but single-agent efficacy of immunotherapy is poor, owing in part to influence of the surrounding tumour microenvironment. Hepatitis A virus cellular receptor-1 (HAVCR1), plays vital roles in immune microenvironment in pancreatic adenocarcinoma, but not clear in breast cancer. The present study aimed to characterize the role of HAVCR1 in immune microenvironment of breast cancer. Methods The Cancer Genome Atlas was used to evaluate functional role of HAVCR1. Kaplan-Meier plotter and Tumor Immune Estimation Resource were applied to illustrate correlation of HAVCR1 with the prognosis and immune infiltration of breast cancer. Results In Pietenpol subtype, high HAVCR1 levels indicated a favourable overall survival (OS) (HR=0.08, 95%CI:0.01-0.61, P=0.0024) in mesenchymal stem–like breast cancer. However, it was connected to worse OS (HR=2.29, 95%CI:1.04-5.04, P=0.035) in immunomodulatory subtype. Significant correlation was found between HAVCR1 expression level and immune infiltration in breast cancer, especially with B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and Dendritic cell (P<0.05), and other immune markers, such as PDCD1 and CTLA4 (P<0.05). Decreased of HAVCR1 in Mesenchymal stem cells, Natural killer T-cells and enriched of B cell, CD4+ memory T-cells, CD8+ T-cells, Type 1 T-helper cells, Type 2 T-helper cells significantly improved the prediction of OS (P<0.05). Decreased impression of HAVCR1 in B cells and CD8+ T-cells were correlated with worse OS (P<0.05). Conclusion HAVCR1 is closely correlated with immune microenvironment and may serve as a potential tumour immunotherapy target for breast cancer.

In-silico analysis of interacting pathways through KIM-1 protein interaction in diabetic nephropathy

BackgroundHuman Kidney Injury Molecule-1, also known as HAVCR-1 (Hepatitis A virus cellular receptor 1), belongs to the cell-surface protein of immunoglobulin superfamily involved in the phagocytosis by acting as scavenger receptor epithelial cells. The study focused on pinpointing the mechanisms and genes that interact with KIM-1.MethodsThis in-silico study was done from March 2019 to December 2019. The Enrichment and protein-protein interaction (PPI) network carefully choose proteins. In addition, the diagramed gene data sets were accomplished using FunRich version 3.1.3. It was done to unveil the proteins that may affect the regulation of HAVCR1 or may be regulated by this protein. These genes were then further considered in pathway analysis to discover the dysregulated pathways in diabetic nephropathy. The long list of differentially expressed genes is meaningless without pathway analysis.ResultsCritical pathways that are dysregulated in diabetic nephropathy patients have been identified. These include Immune System (Total = 237, P < 0.05), Innate Immune System (Total = 140, P < 0.05), Cytokine Signaling Immune system (Total = 116, P < 0.05), Adaptive Immune System (Total = 85) and Neutrophil degranulation (Total = 78).ConclusionThe top 5 genes that are interacting directly with HIVCR1 include CASP3, CCL2, SPP1, B2M, and TIMP1 with degrees 161, 144, 108, 107, and 105 respectively for Immune system pathways (Innate Immune System, Cytokine Signaling Immune system, Adaptive Immune System and Neutrophil degranulation).

Hepatitis A Virus Cellular Receptor 1 (HAVcr-1) Initiates Prostate Cancer Progression in Human Cells via Hepatocyte Growth Factor (HGF)-Induced Changes in Junctional Integrity.

Background: HAVcR-1 has been linked to cancer aetiology and may regulate junctional complexes, with its role in prostate cancer still unexplored. This study aims to investigate the expression of HAVcR-1 in prostate cancer samples and the exploration of the cellular/molecular impact of HAVcR-1. Methods: Levels of HAVcR-1 ectodomain in the serum of prostate cancer patients were compared to healthy controls, and assessed as the total protein and gene expression of HAVcR-1 and tissues sections. The manipulation of HAVcR-1 levels within prostate cancer cell lines determined changes in cell behaviour using in vitro cell models and barrier function assays. Protein/phosphoprotein levels were assessed using Western blotting. Results: Levels of HAVcR-1 ectodomain from serum were decreased in patients with prostate cancer. Ectodomain levels correlated with the Gleason score. Histologically, the total protein/gene expression of HAVcR-1 was overexpressed in prostate cancer. The overexpression of HAVcR-1 in prostate cancer cell lines resulted in key changes in cell behaviour and the phosphorylation of β-catenin with a concurrent decrease in membranous E-cadherin, increased nuclear β-catenin and increased cyclin D1 protein expression, which were associated with HGF-promoted changes in the barrier function. Conclusions: HAVcR-1 expression and ectodomain release coincides with the presence of prostate cancer; thus, indicating HAVcR-1 as a potential biomarker to aid in diagnostics, and implicating HAVcR-1 in the dysregulation of junctional complexes.

Novel insights into the disease transcriptome of human diabetic glomeruli and tubulointerstitium.

BackgroundDiabetic nephropathy (DN) is the most common cause of end-stage renal disease, affecting ∼30% of the rapidly growing diabetic population, and strongly associated with cardiovascular risk. Despite this, the molecular mechanisms of disease remain unknown.MethodsRNA sequencing (RNAseq) was performed on paired, micro-dissected glomerular and tubulointerstitial tissue from patients diagnosed with DN [n = 19, 15 males, median (range) age: 61 (30–85) years, chronic kidney disease stages 1–4] and living kidney donors [n = 20, 12 males, median (range) age: 56 (30–70) years].ResultsPrincipal component analysis showed a clear separation between glomeruli and tubulointerstitium transcriptomes. Differential expression analysis identified 1550 and 4530 differentially expressed genes, respectively (adjusted P < 0.01). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted activation of inflammation and extracellular matrix (ECM) organization pathways in glomeruli, and immune and apoptosis pathways in tubulointerstitium of DN patients. Specific gene modules were associated with renal function in weighted gene co-expression network analysis. Increased messengerRNA (mRNA) expression of renal damage markers lipocalin 2 (LCN) and hepatitis A virus cellular receptor1 (HAVCR1) in the tubulointerstitial fraction was observed alongside higher urinary concentrations of the corresponding proteins neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in DN patients.ConclusionsHere we present the first RNAseq experiment performed on paired glomerular and tubulointerstitial samples from DN patients. We show that prominent disease-specific changes occur in both compartments, including relevant cellular processes such as reorganization of ECM and inflammation (glomeruli) as well as apoptosis (tubulointerstitium). The results emphasize the potential of utilizing high-throughput transcriptomics to decipher disease pathways and treatment targets in this high-risk patient population.

Evaluation of cisplatin-induced injury in human kidney organoids.

Acute kidney injury (AKI) remains a major global healthcare problem, and there is a need to develop human-based models to study AKI in vitro. Toward this goal, we have characterized induced pluripotent stem cell-derived human kidney organoids and their response to cisplatin, a chemotherapeutic drug that induces AKI and preferentially damages the proximal tubule. We found that a single treatment with 50 µM cisplatin induces hepatitis A virus cellular receptor 1 (HAVCR1) and C-X-C motif chemokine ligand 8 (CXCL8) expression, DNA damage (γH2AX), and cell death in the organoids but greatly impairs organoid viability. DNA damage was not specific to the proximal tubule but also affected the distal tubule and interstitial cell populations. This lack of specificity correlated with low expression of proximal tubule-specific SLC22A2/organic cation transporter 2 (OCT2) for cisplatin. To improve viability, we developed a repeated low-dose regimen of 4 × 5 µM cisplatin over 7 days and found this caused less toxicity while still inducing a robust injury response that included secretion of known AKI biomarkers and inflammatory cytokines. This work validates the use of human kidney organoids to model aspects of cisplatin-induced injury, with the potential to identify new AKI biomarkers and develop better therapies.

HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

The hepatitis A virus cellular receptor 1 (HAVCR1) gene as a sensitive and specific biomarker has been reported in various diseases. Especially, HAVCR1 overexpression promotes the development and progression of several human cancers. Hence, we aimed to detect the effects of HAVCR1 on gastric adenocarcinoma (GAC). We first determined the expression of HAVCR1 in GAC tissues compared with normal gastric tissues based on the Cancer Genome Atlas (TCGA) database using bioinformatics analysis methods. Then, we assessed the biological function of HAVCR1 in GAC cells using quantitative real-time reverse transcription-PCR (qRT-PCR), western blot, cell counting kit-8- (CCK-) 8, colony formation assay, wound healing assay, and transwell assay. Our results showed that HAVCR1 expression was upregulated in GAC tissues and positively associated with poor survival. Loss-of-function analyses indicated that knockdown of HAVCR1 inhibited the proliferation, colony formation, migration, and invasion of GAC cells. Furthermore, reduction of HAVCR1 in GAC cells can decrease the expression of phosphorylated MEK/ERK. These findings suggested that HAVCR1 may represent a potential biomarker for GAC prognosis, as well as a novel therapeutic target for GAC treatment.

Canonical BMP signaling in tubular cells mediates recovery after acute kidney injury

Bone morphogenetic protein (BMP) signaling has been shown to modulate the development of renal fibrosis in animal models of kidney injury, but the downstream mediators are incompletely understood. In wild-type mice, canonical BMP signaling mediated by SMAD1/5/8 transcription factors was constitutively active in healthy renal tubules, transiently down-regulated after ischemia reperfusion injury (IRI), and reactivated during successful tubular regeneration. We then induced IRI in mice with a tubular-specific BMP receptor 1A (BMPR1A) deletion. These mice failed to reactivate SMAD1/5/8 signaling in the post-ischemic phase and developed renal fibrosis after injury. Using unbiased genomic analyses, we identified three genes encoding inhibitor of DNA-binding (ID) proteins (Id1, Id2, and Id4) as key targets of BMPR1A-SMAD1/5/8 signaling. BMPR1A-deficient mice failed to re-induce these targets following IRI. Instead, BMPR1A-deficiency resulted in activation of pro-fibrotic signaling proteins that are normally repressed by ID proteins, namely, p38 mitogen-activated protein kinase and cell cycle inhibitor p27. These data indicate that the post-ischemic activation of canonical BMP signaling acts endogenously to repress pro-fibrotic signaling in tubular cells and may help to prevent the progression of acute kidney injury to chronic kidney disease.

HAVCR1 expression might be a novel prognostic factor for gastric cancer.

Hepatitis A virus cellular receptor 1 (HAVCR1), which is also known as T-cell immunoglobulin and mucin domain 1 (TIM-1) is a TIM gene family member. In this study, we aimed to characterize the expression profile of HAVCR1 in GC, its prognostic value and the potential epigenetic mechanism leading to its dysregulation. Bioinformatic analysis was performed by using genomic, clinicopathological and survival data in the human protein atlas (HPA) and the Cancer Genome Atlas (TCGA). Results showed that HAVCR1 was significantly upregulated at the mRNA and protein level in GC tissues compared to the adjacent normal tissues. In addition, HAVCR1 upregulation was an independent indicator of shorter OS (HR: 1.698, 95%CI: 1.221–2.361, p = 0.002), after adjustment of older age, differentiation status, pathological stages and the presence of residual tumor and was also an independent indicator of shorter RFS (HR: 2.577, 95%CI: 1.583–4.197, p<0.001), after adjustment of gender and histological grade. The methylation level of two CpG sites (cg11188031 and cg07320595) was negatively correlated with HAVCR1 expression. However, only high methylation level of cg07320595 was associated with significantly longer OS (p = 0.018) and RFS (p = 0.021). Based on these findings, we infer that HAVCR1 upregulation might serve as a valuable prognostic marker in terms of OS and RFS in GC patients. Cg07320595 might be a critical CpG site influencing HAVCR1 expression.

Relationship of Severity of Hepatitis A with Polymorphisms in Hepatitis A Virus Cellular Receptor 1 (HAVCR1) Gene

Introduction and aim. HAVCR1 protein is the cellular receptor for hepatitis A virus (HAV). Genetic polymorphism in this gene may alter the outcome of HAV infection. In a previous study, a 6-amino acid insertion (157insMTTTVP) in HAVCR1 gene was associated with more severe disease. We decided to investigate this association further.Material and methods. We sequenced exon 4 of the HAVCR1 gene in patients with clinical hepatitis A attending our institution, and a group of healthy controls in a disease-endemic setting in India. Frequencies of different haplotypes of a genomic region with two overlapping insertion-deletion polymorphisms (indels; rs141023871 and rs139041445) were compared between patients and controls, as well as between patients with and without a severe form of disease (liver failure).Results. The gene had three haplotypes in the region of interest - a short form, an intermediate-form with a 5-amino acid 157insMTTVP insertion and a long-form with a 6-amino acid 157insMTTTVP insertion. The allele frequency (29/150 [19%] vs. 43/146 [29%]; p = ns) and haplotype frequency (29/75 [39%] vs. 39/73 [53%]; p = ns) of the 157insMTTTVP variant were similar in hepatitis A patients and healthy controls (30%). Further, the allele frequency (12/58 [21%] vs. 17/92 [18%]; p = ns) and haplotype frequency (12/29 [41%] vs.17/46 [37%]; p = ns) of the longest variant were also similar in patients with severe and mild disease.Discussion. In the study population, the 157insMTTTVP variant of HAVCR1 gene was not associated with more severe outcome of HAV infection. Further studies in other populations around the world are needed to assess the relation of this genetic variation with disease outcome.

HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection.

The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV.IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the mechanism by which the subsequent necroinflammatory process clears the infection remain a puzzle that most likely involves the HAV-HAVCR1 interaction. Based on negative data, a recent paper from the S. M. Lemon and W. Maury laboratories (A. Das, A. Hirai-Yuki, O. Gonzalez-Lopez, B. Rhein, S. Moller-Tank, R. Brouillette, L. Hensley, I. Misumi, W. Lovell, J. M. Cullen, J. K. Whitmire, W. Maury, and S. M. Lemon, mBio 8:e00969-17, 2017, https://doi.org/10.1128/mBio.00969-17) suggested that HAVCR1 is not a functional HAV receptor, nor it is it required for HAV infection. However, our data, based on regain of the HAV receptor function in HAVCR1 knockout cells transfected with HAVCR1 cDNA, disagree with their findings. Our positive data show conclusively that HAVCR1 is indeed a functional HAV receptor and lays the ground for the identification of alternative HAV receptors and how they interact with HAVCR1 in cell entry and the pathogenesis of HAV.

Association between the TIMD4-HAVCR1 variants and serum lipid levels, coronary heart disease and ischemic stroke risk and atorvastatin lipid-lowering efficacy.

Little is known about the association of the TIMD4 (T-cell immunoglobulin and mucin domain 4 gene)-HAVCR1 (hepatitis A virus cellular receptor 1) variants and lipid metabolism, the risk of coronary heart disease (CHD) and ischemic stroke (IS). The present study aimed to determine the TIMD4-HAVCR1 variants, their haplotypes and gene–environment interactions on serum lipid levels, the risk of CHD and IS, and the lipid-lowering efficacy of atorvastatin in a southern Chinese Han population. Genotypes of three variants in 622 controls, 579 CHD, and 546 IS patients were determined by the Snapshot technology. Atorvastatin calcium tablet (20 mg/day) was given in 724 hyperlipidemic patients for 8 weeks after genotyping. The rs12522248 genotypic and allelic frequencies were different between controls and patients, and were associated with the risk of CHD and IS. The rs1501908G-rs12522248T-rs2036402T haplotype was associated with an increased risk of CHD; the G-C-T haplotype was associated with lower risk of CHD; and the C-C-C haplotype was associated with an increased risk of IS. Variants and their haplotypes in controls were associated with triglyceride (rs1501908), low-density lipoprotein cholesterol (LDL-C, rs1501908, G-T-T), high-density lipoprotein cholesterol (HDL-C, rs12522248, C-C-C) and the ratio of total cholesterol (TC) to HDL-C (C-C-C). Interactions of rs1501908- and rs2036402-alcohol (HDL-C); rs1501908- and rs12522248-high body mass index (hBMI, ≥24 kg/m2; TC); and TIMD4-HAVCR1 variants-atorvastatin on several lipid parameters were detected. Interactions of rs12522248TC/CC-hBMI, G-T-T-, and C-C-C-smoking on the risk of CHD; and C-C-C-smoking, C-C-C-, and G-C-T-hBMI on the risk of IS were also observed. These findings suggest that the TIMD4-HAVCR1 variants may be the genetic risk factors for CHD and IS.

Structural pierce into molecular mechanism underlying Clostridium perfringens Epsilon toxin function

Epsilon toxin of the Clostridium perfringens garnered a lot of attention due to its potential for toxicity in humans, extreme potency for cytotoxicity in mice and lack of any approved therapeutics prescribed for human. However, the intricacies of the Epsilon toxin action mechanism are yet to be understood. In this regard, various in silico tools have been exploited to model and refine the 3D structure of the toxin and its two receptors. The receptor proteins were embedded into designed lipid membranes within an aqueous and ionized environment. Thereafter, the modeled structures subjected to series of consecutive molecular dynamics runs to achieve the most natural like coordination for each model. Ultimately, protein-protein interaction analyses were performed to understand the probable action mechanism. The obtained results successfully confirmed the accuracy of employed methods to achieve high quality models for the toxin and its receptors within their lipid bilayers. Molecular dynamics analyses lead the structures to a more native like coordination. Moreover, the results of previous empirical studies were confirmed, while new insights for action mechanisms including the detailed roles of Hepatitis A virus cellular receptor 1 (HAVCR1) and Myelin and lymphocyte protein (MAL) proteins were achieved. In light of previous and our observations, we suggested novel models which elucidated the existing interplay between potential players of Epsilon toxin action mechanism with detailed structural evidences. These models would pave the way to have more robust understanding of the Epsilon toxin biology, more precise vaccine construction and more successful drug (inhibitor) design.

Preclinical Efficacy of an Antibody‐Drug Conjugate Targeting TIM‐1 in Ovarian, Renal and Lung Tumor Models

T cell immunoglobulin and mucin domain 1 (TIM‐1) is a type I transmembrane glycoprotein, also described as kidney injury molecule 1 (KIM‐1) and as hepatitis A virus cellular receptor 1 (HAVCR1). TIM‐1 has been shown to have upregulated expression in several human cancers, but has restricted expression in healthy tissues, thus it represents a promising target for antibody mediated therapy. To this end we have developed a fully human monoclonal antibody specific for the extracellular domain of TIM‐1, covalently linked to the potent cytotoxin monomethyl auristatin E (MMAE). We have designated this antibody‐drug conjugate as CDX‐014. Three TIM‐1 expressing xenograft models (Caki‐1, a renal clear cell carcinoma; IGROV‐1, an ovarian adenocarcinoma, and A549, a lung carcinoma) have been established to demonstrate the efficacy of CDX‐014 in vivo against human tumors. In all models, cells were grown in culture and then implanted subcutaneously into immunocompromised mice. When tumors reached an average of approximately 0.3cm3, mice were split into equivalently sized control and treatment groups. Dose dependent efficacy of CDX‐014 was shown in all models by a decreased rate of tumor growth and extended group survival. These studies support the further development of CDX‐014, including manufacturing activities and IND‐enabling studies, in order to advance towards clinical studies in clear cell carcinomas and potentially other TIM‐1 expressing tumors.

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1.

HAVCR1 Gene Haplotypes and Infection by Different Viral Hepatitis C Virus Genotypes

The hepatitis A virus cellular receptor 1 (HAVCR1) gene is highly polymorphic, and several variants have been associated with susceptibility to allergic and autoimmune diseases. The HAVCR1 gene region was identified as a candidate for hepatitis C virus (HCV) natural clearance in a genotyping study of selected immune response genes in both European-American and African-American populations. The aim of the present study was to explore the influence of HAVCR1 in the outcome of HCV infection in the Spanish population. Three cohorts, consisting of 354 subjects with persistent HCV infection (285 with persistent HCV monoinfection and 69 with natural clearance), 182 coinfected HIV/HCV patients, and 320 controls, were included. Samples were genotyped in several polymorphic positions, insertion/deletion variants in exon 4 and tag single nucleotide polymorphisms (SNPs), in order to define previously described HAVCR1 haplotypes (haplotypes A to D). No statistically significant differences were observed with spontaneous resolution of infection or with viral clearance after treatment. Nevertheless, different rates of infection by viral genotypes (G's) were observed among the HAVCR1 haplotypes. Individuals bearing haplotype C had the highest viral G1 infection rate when compared to individuals bearing other haplotypes (75.82% versus 57.72%, respectively; corrected P value [P(c)], 3.2 × 10(-4); odds ratio [OR], 2.30; 95% confidence interval [CI], 1.51 to 3.47). Thus, HAVCR1 could be involved in susceptibility or resistance to infection by a particular HCV genotype.

The interaction of hepatitis A virus (HAV) with soluble forms of its cellular receptor 1 (HAVCR1) share the physiological requirements of infectivity in cell culture

BackgroundHepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, usurps the HAV cellular receptor 1 (HAVCR1) to infect cells. HAVCR1 is a class 1 integral membrane glycoprotein that contains two extracellular domains: a virus-binding immunoglobulin-like (IgV) domain and a mucin-like domain that extends the IgV from the cell membrane. Soluble forms of HAVCR1 bind, alter, and neutralize cell culture-adapted HAV, which is attenuated for humans. However, the requirements of the HAV-HAVCR1 interaction have not been fully characterized, and it has not been determined whether HAVCR1 also serves as a receptor for wild-type (wt) HAV. Here, we used HAV soluble receptor neutralization and alteration assays to study the requirements of the HAV-HAVCR1 interaction and to determine whether HAVCR1 is also a receptor for wt HAV.ResultsTreatment of HAV with a soluble form of HAVCR1 that contained the IgV and two-thirds of the mucin domain fused to the Fc fragment of human IgG1 (D1 muc-Fc), altered particles at 37°C but left a residual level of unaltered particles at 4°C. The kinetics of neutralization of HAV by D1 muc-Fc was faster at 37°C than at 4°C. Alteration of HAV particles by D1 muc-Fc required Ca, which could not be replaced by Li, Na, Mg, Mn, or Zn. Neutralization of HAV by D1 muc-Fc occurred at pH 5 to 8 but was more efficient at pH 6 to 7. D1 muc-Fc neutralized wt HAV as determined by a cell culture system that allows the growth of wt HAV.ConclusionThe interaction of HAV with soluble forms of HAVCR1 shares the temperature, Ca, and pH requirements for infectivity in cell culture and therefore mimics the cell entry process of HAV. Since soluble forms of HAVCR1 also neutralized wt HAV, this receptor may play a significant role in pathogenesis of HAV.

The human homolog of HAVcr-1 codes for a hepatitis A virus cellular receptor.

The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA was isolated from a cDNA expression library of African green monkey kidney (AGMK) cells by using protective monoclonal antibody (MAb) 190/4, which blocks the binding of hepatitis A virus (HAV) to AGMK cells. The HAVcr-1 cDNA codes for havcr-1, a 451-amino-acid class I integral-membrane mucin-like glycoprotein of unknown natural function. To determine the existence of a human homolog(s) of HAVcr-1 (huHAVcr-1), we used HAVcr-1-specific primers to amplify cDNAs from human liver and kidney mRNA by reverse transcription-PCR. Nucleotide sequence analysis revealed that the amplified liver and kidney huHAVcr-1 cDNAs were identical and that they coded for a 359-amino-acid glycoprotein, termed huhavcr-1, which was approximately 79% identical to havcr-1. The six Cys residues of the extracellular domain of havcr-1 and its first N-glycosylation site were conserved in huhavcr-1. However, the number of hexameric repeats of the mucin-like region was reduced from 27 in havcr-1 to 13 in huhavcr-1. In addition, 12 C-terminal amino acids in the cytoplasmic domain of huhavcr-1 were deleted. Northern blot analysis of poly(A) RNA showed that huhavcr-1 is expressed in every organ analyzed, including the liver, small intestine, colon, and spleen, and that it is expressed at higher levels in the kidney and testis. Although dog cells transfected with the huHAVcr-1 cDNA did not express the protective 190/4 epitope, they bound hepatitis A virus (HAV) and gained limited susceptibility to HAV infection. Treatment with MAb 190/4 did not protect AGMK cell transfectants expressing huhavcr-1 against HAV, suggesting that HAV infected these cells via the huhavcr-1 receptor and not the endogenously expressed havcr-1, which was blocked by MAb 190/4. Our data demonstrate that huhavcr-1 is a binding receptor for HAV and suggest that it is also a functional receptor for HAV.

The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4.

The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1-, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.