Deepshi Thakral

Hepatitis E virus enters liver cells through receptor-dependent clathrin-mediated endocytosis

Summary.  We investigated the virus–host interaction for hepatitis E virus (HEV) by performing competitive binding assays using in vitro assembled virus‐like particles (VLPs). We used Escherichia coli expressed native capsid protein (pORF2) and its mutants with an attached Gly(5)‐Ala (linker) reporter [enhanced green fluorescent protein (EGFP)/firefly luciferase (Fluc)]. Transmission electron microscopy and nanoparticle tracking showed near uniform particles of approximately 30–35 nm in diameter for pORF2 VLPs and 60–100 nm for reporter‐linked VLPs. Binding of reporter‐linked full‐length (1–660aa) and N‐terminal truncated (Δ1–112aa) pORF2 VLPs to Huh7 cell surfaces was found to be specific with 1.92 ± 0.065 × 105 sites per cell. Saturation binding indicated an equilibrium dissociation constant (Kd) of 121.1 ± 23.83 and 123.8 ± 16.15 nm for pORF2‐linker‐EGFP and pORF2‐linker‐Fluc VLPs respectively. A similar binding pattern was observed for Δ1–112aa pORF2‐linker‐EGFP and Δ1–112aa pORF2‐linker‐Fluc VLPs with Kd values of 123.6 ± 10.60 and 135.6 ± 16.19 nm respectively. The affinity (log Ki) of pORF2 binding on Huh7 cells in the presence of EGFP‐tagged and Fluc‐tagged pORF2 VLPs was found to be approximately 2.0. However, no VLP formation or binding was observed with refolded C‐terminal truncated (Δ458–660aa) pORF2. We investigated HEV internalization using fluorescent VLPs (EGFP‐VLPs), which showed vesicle‐mediated uptake starting at 5 min post‐incubation. The uptake of VLPs could be stopped by inhibitors for clathrin‐dependent endocytosis, but not by caveosome inhibitors. No binding and uptake of EGFP‐VLPs were observed on non‐hepatic cell lines (HeLa and SiHa). These findings suggest that HEV attaches to the host cell via a specific high affinity receptor and enters the cytoplasm by clathrin‐mediated endocytosis.

RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) and Hepatitis B Virus (HBV) Replicon Transfected Huh-7 Cells

Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV.

Differential expression of HLA-G and ILT-2 receptor in human tuberculosis: Localized versus disseminated disease.

Human leukocyte antigen-G (HLA-G) is an anti-inflammatory and immunosuppressive molecule that can modulate immune cell activation. The role of HLA-G in tuberculosis, an immune-mediated and chronic bacterial disease remains to be elucidated. We investigated the expression profile of soluble and membrane bound HLA-G in pulmonary TB (PTB), TB pleural effusion (TB-PE, localized disease) and Miliary TB (disseminated form). The expression of HLA-G receptor, ILT-2 was also determined on the immune cells. We observed that the plasma sHLA-G levels were significantly increased in Miliary TB than in TB-PE patients. In contrast, immunophenotyping revealed that the percent frequency of CD3(+) T cells expressing HLA-G was significantly reduced in Miliary TB as compared to TB-PE, whereas frequency of CD14(+) monocytes expressing HLA-G was significantly higher in TB-PE patients. Strikingly in the TB-PE cases, comparison of disease site, i.e. pleural effusion with peripheral blood showed increased expression of both soluble and surface HLA-G, whereas ILT-2 expressing cells were reduced at the local disease site. Furthermore, we demonstrated that in TB-PE cases, HLA-G expression on CD3(+) T cells was influenced by broad spectrum MMP inhibitor. Thus, differential expression of HLA-G could potentially be a useful biomarker to distinguish different states of TB disease.

Unusual Association of Hemophagocytic Lymphohistiocytosis in Systemic Lupus Erythematosus: Cases Reported at Tertiary Care Center

Case series Patients: Female, 10 • Female, 15 Final Diagnosis: Secondary hemophagocytic lymphohistiocytosis Symptoms: Arthralgia • CNS manifestations • fever • pancytopenia • rash Medication: — Clinical Procedure: — Specialty: Hematology Objective: Rare co-existence of disease or pathology Background: Hemophagocytic lymphohistiocytosis (HLH) in the background of systemic lupus erythematosus (SLE) is rare. Inability to discriminate between these two entities may be fatal for the patient. Here we report two cases of SLE with secondary HLH, one of which manifested HLH as the initial presentation, and the significance of HLH’s timely diagnosis. Case Report: We describe two cases of SLE secondarily affected by HLH, which were diagnosed by various laboratory parameters and detection of profoundly reduced NK cell activity by using flow cytometry. Both our cases on investigation showed hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, and marked reduction or complete absence of NK cell activity. Conclusions: Association of secondary HLH with SLE is rare, and when it occurs, differentiating it from lupus flare requires a high degree of suspicion and awareness of this association. Both have overlapping clinical features, but HLH is characterized by hyperferritinemia, hypofibrinogenemia, hypertriglyceridemia, and a decrease in erythrocyte sedimentation rate (ESR) and NK cell activity unlike SLE. Therefore, early diagnosis of HLH in the background of SLE facilitates timely selection of an appropriate treatment modality to prevent fatal complications.

Distinct clinico-immunological profile of patients infected with human papilloma virus genotypes 6 and 11

Anogenital warts are primarily caused by Human Papillomavirus (HPV) type 6 and 11, which belong to the taxonomic family Papillomaviridae, genus alpha-papillomavirus and species 10. The presentation of the warts is varied and most of the patients have high recurrence rate of wart lesions. Studies had shown that an effective cellular immune response is required for the control of HPV infection. Here, we report distinct clinico-immunological profile of two patients presenting with venereal warts caused by HPV genotypes 6 and 11. The Case 1 manifested greater number of verrucous warts and case 2 had fewer subtle lesions. Further, evaluation of HPV antigen-specific cellular immune response revealed a robust T cell response against HPV6 peptide and a weak response against HPV11 in case 1. Interestingly, HPV genotyping revealed type 6 in case 1 with greater severity of infection and robust immune response against HPV6 peptide. In contrast, case 2 presented with milder infection and weak immune response and was positive for genotype 11. More extensive study with larger cohorts will strengthen our observation and could be relevant for designing immunotherapeutic adjunct strategies along with the standard treatment for rapid clearance of HPV infections in these patients. This communication reports immune status of two patients with venereal warts and their correlation with clinical presentation and the genotyping.

Functional characterization of CD4 and CD8 T cell responses among human papillomavirus infected patients with ano-genital warts

Ano-genital warts are considered one of the commonest and highly infectious sexually transmitted infections. These warts are primarily caused by the human papillomavirus (HPV) of the family Papillomaviridae, genus alpha-papillomavirus, species 10 and types 6 and 11. However the high recurrence rate of warts is a matter of serious concern to the patients and a challenge for the treating physician. The conventional treatment options are targeted only to the local site of warts. There is no systemic treatment modality as there is limited understanding of the disease immune-pathogenesis. The role of cell-mediated immunity in combating HPV infection is not clearly defined. Hence the present study is aimed at investigating the CD4+ T helper (Th1 and Th2) and CD8+ T cell responses among wart patients. In this study, we compared HPV6 and HPV11 antigen-specific T cell responses among venereal wart patients relative to healthy controls. Significant decrease in percent frequencies of IFN-γ producing CD4+ and CD8+ T cells were observed in HPV infected wart patients. On the other hand, the frequency of CD4+ T cells expressing IL-4 was significantly increased in these patients as compared to healthy controls. The observed functional skewing of HPV specific T cells from Th1 to Th2 response in patients indicated suppressed immunity against the HPV. Moreover, decrease in CD8 T cell function correlated with poor wart clearance. Our findings open future avenues for exploring potential immunomodulation strategies as an adjunct to standard treatment for better management of these patients and prevention of recurrence.

Immune Response Profiling of Patients with Anogenital Warts

The incidence of viral sexually transmitted infections (STIs) has shown an upward trend worldwide.1 Condyloma acuminata (anogenital warts) are one of the commonest STIs caused by human papillomaviruses (HPVs),2 primarily by HPV-6 and HPV-11 (in 90% of cases)3-6 and occasionally by HPV-16 and HPV-18.7,8 Warts are generally papillomatous lesions occurring on the mucus membrane or skin of the external genitalia as well as in the perianal region. Patients are not generally satisfied with the standard treatment of warts because of high recurrence rate caused due to persistence of HPV in normal-presenting adjacent skin,9 adding to the difficulty of the physicians. For successful treatment outcome, a more comprehensive understanding of the host immune response and its interaction with HPV is necessary. Detailed understanding of the host immune mechanism will lead to better clinical management in the future. Few studies have reported suppression of local immune response mediated by dysfunctional natural killer (NK) cells, dysfunctional Langerhans cells, and resultant T-helper cells skewing toward Th2 phenotype at the local disease site.10-12 The Langerhans cells in HPV-infected lesions appeared in clumps with altered distribution and morphology13,14 and no surface expression of maturation marker CD83.15 It has been found that proportion of T-regulatory cell correlated with the size of the warts. Studies have also reported reduced interleukin (IL)-10 and transforming growth factor (TGF)β1 expression and increased interferon (IFN)-γ and IL-2 expression in small-sized warts.16 In addition, the serum levels of IL-6 were increased and IFN-γ were decreased in HPV-infected patients.17 All these findings suggested an immune imbalance in patients with HPV-infected genital warts. The aim of this study was to understand how HPV antigen (6 and 11)-specific immune responses in peripheral blood of patients with venereal warts are related. Here we tried to assess T-helper cell response in vitro on stimulation with HPV-6 and -11 antigens both in patients and in controls, as well as IFN-γ and IL-4 response to detect skewing of T-helper cell to decreased cellular immunity. We demonstrated functional skewing of HPV antigen-specific T-helper cell response in these patients that may have future implications in treatment strategies to boost protective immune response. 1Scientist D, 2Research Scientist II, 3Senior Consultant and Former Director, 4Advisor 5Professor and Head 1Department of Epidemiology and Communicable Diseases Indian Council of Medical Research, New Delhi, India 2,5Department of Transplant Immunology and Immunogenetics All India Institute of Medical Sciences, New Delhi, India 3Department of Dermatology, Max Superspeciality Hospital; Dr. Ram Manohar Lohia Hospital, New Delhi, India 4Amity Institute of Virology and Immunology, Amity University Noida, Uttar Pradesh, India