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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~ Acknowledgment
 ~  References
 ~  Article Figures

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BRIEF COMMUNICATION
Year : 2013  |  Volume : 31  |  Issue : 1  |  Page : 64-68
 

Evaluation of antigenicity and cell mediated immunity of Hepatitis E virus patients: Using non radioactive MTT assay


1 Department of Virology, PGIMER, Chandigarh, India
2 Department of Hepatology, PGIMER, Chandigarh, India

Date of Submission03-Aug-2012
Date of Acceptance06-Jan-2013
Date of Web Publication15-Mar-2013

Correspondence Address:
R Ratho
Department of Virology, PGIMER, Chandigarh
India
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Source of Support: Indian Council of Medical Research, Ansari Nagar, New Delhi, India, Conflict of Interest: None


DOI: 10.4103/0255-0857.108725

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 ~ Abstract 

Hepatitis E virus (HEV) is an important cause of hepatitis in developing nations. Disease spans from asymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). Cell-mediated immunity (CMI) is less studied. Studies document CMI in HEV patients using [ 3 H]-thymidine incorporation (radioactive in nature). The aim of this study was to evaluate the antigenicity of recombinant HEV ORF 2 peptide (452-617 a.a) (pORF2) by non-radioactive MTT assay and detecting the proliferation indices of primary PBMC culture. A total of 27 laboratory confirmed HEV patients (16 AVH and 11 ALF) and 20 apparently healthy individuals (HC) were included. PBMCs were isolated, plated and stimulated with pORF2. After an incubation of 4 days, cells were looked for blastogenic transformation and subjected to MTT assay. PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively. PI of AVH Vs HC, ALF Vs HC and AVH Vs ALF were found to be significantly higher ( P < 0.0001). This study demonstrates MTT to be an adaptable technique to evaluate CMI in HEV patients. Recombinant pORF2 was found to be antigenic in nature and PBMCs from AVH patients were immunologically more reactive than ALF patients.


Keywords: Acute liver failure, cell mediated immunity, Hepatitis E virus, MTT assay


How to cite this article:
Majumdar M, Ratho R, Chawla Y, Singh M P. Evaluation of antigenicity and cell mediated immunity of Hepatitis E virus patients: Using non radioactive MTT assay. Indian J Med Microbiol 2013;31:64-8

How to cite this URL:
Majumdar M, Ratho R, Chawla Y, Singh M P. Evaluation of antigenicity and cell mediated immunity of Hepatitis E virus patients: Using non radioactive MTT assay. Indian J Med Microbiol [serial online] 2013 [cited 2019 May 21];31:64-8. Available from: http://www.ijmm.org/text.asp?2013/31/1/64/108725



 ~ Introduction Top


Hepatitis E virus (HEV) is the causative agent for Hepatitis E, a water borne disease, occurring as sporadic cases or focused outbreaks. About 2 billion people are estimated to live in areas endemic for HEV infection. [1] The clinical presentation varies from the stage of asymptomatic infection to self-limiting acute viral hepatitis (AVH) and acute liver failure (ALF). Recently the chronic sequelae was reported in transplant patients under immunosuppression. [2]

The HEV virions are small, non-enveloped, 32-34 nm in diameter with icosahedral symmetry. The viral genome is approximately 7.2 kb long, single stranded, positive sense, polyadenylated RNA that contains three open reading frames (ORF). [3] ORF1 encodes the non-structural proteins responsible for the replication of viral genome and polyprotein processing, whereas the major capsid protein is being encoded by ORF2. ORF3 encodes a phosphoprotein that associates with the cytoskeleton having regulatory functions. [4]

Humoral immune response as is evident by the presence of anti-HEV IgM is used for the diagnosis of clinically suspected HEV cases. [5] In contrast, limited data are available on the cellular immune responses during HEV infection. Information about lymphoproliferative response is important to study the cell mediated immune response (CMI). Furthermore the immunogenic epitope can prove to be a potential vaccine candidate.

Most of the studies related to CMI of HEV have relied on the conventional [ 3 H]-thymidine incorporation assay for the assessment of cell growth. [6],[7] However, the assay is associated with several limitations and its use has been restricted in recent years because it is a) radioactive in nature thereby posing hazard to personnel and the environment, b) requires a separate facility and special equipment and c) expensive. Moreover, radioactive substances can be handled only in laboratories having a radioactive clearance certification from the authorised agencies further limiting the usage and practical application of such protocols.

A non-radioisotopic, colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) has been tried as an alternative because its rapid, cost efficient and safe. [8],[9],[10] MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent and the released formazan reagent is measured spectrophotometrically. Thus, it is a colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase. A comparative evaluation between the MTT assay and [ 3 H]-thymidine incorporation assay for lymphocyte proliferation [11],[12] has shown a close correlation.

This study aimed to investigate the immunogenic nature of HEV ORF 2 peptide (452-617 a.a) by its ability to promote blastogenic transformation in primary PBMC cultures from HEV infected (AVH and ALF) patients, and quantitate the proliferation indices using non-radioactive MTT assay.


 ~ Materials and Methods Top


A total of 27 patients (AVH; n = 16 and ALF; n = 11) with clinically suspected viral hepatitis, confirmed by a positive anti-HEV IgM and/or HEV RNA were included in this study. AVH was defined as patients presenting with at least five-fold rises in serum aspartate aminotransferase levels or clinical jaundice or both. ALF was indicated when the patient after having a typical acute hepatitis develops encephalopathy and/or coagulopathy within 2 to 8 weeks of onset of jaundice resulting in severe impairment of hepatic function, without any history of pre-existing liver disease. [13] Patients with history of hepatotropic drug use, chronic alcoholics, co-infection with HAV/HBV/HCV as determined by ELISA, and hepatitis due to non-infectious or known bacterial causes were excluded from the study proper. The study included ( n = 20) age and sex matched healthy volunteers as the control group (HC). The study protocol was approved by the Institutional ethics committee as per national guidelines. Written informed consent was obtained from the subjects before being sampled.

Approximately 10 ml of venous blood was collected aseptically by trained health care personnel. Three ml was utilised for serum separation and the rest 7 mL of anti-coagulated whole blood was processed for isolation of PBMC's. Serum samples were subjected to the markers of acute viral hepatitis, that is, anti-HAV IgM (Orgenics, Israel), anti-HEV IgM (ImmunoVision, USA), Hepatitis B virus surface antigen (HBsAg) (J Mitra, New Delhi), and hepatitis C virus (HCV) by anti-HCV third generation ELISA (J Mitra, New Delhi) using the commercially available kits as per the manufacturer's instruction. Healthy controls were tested for anti-HAV IgM, anti-HEV IgM, HBsAg, anti-HCV and also anti-HEV IgG (ImmunoVision, USA).

RNA was extracted from 27 serum samples of acute viral hepatitis using Qiagen RNA easy mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. RNA was reverse transcribed to cDNA using Moloney strain of Murine Leukemia Virus Reverse Transcriptase enzyme and random hexameric primers (Fermentas, USA). PCR amplification was carried out using specific primers against the ORF 1 gene by a nested PCR protocol. [14] Briefly a nested PCR was carried out targeting ORF1 gene using 1 st set of primers; HEV1 (4957-4928): 5′- CCA CAC ACA TCT GAG CTA CAT TCG TGA GCT-3′, HEV2 (4382-4407): 5′-AGG CAT CCA TGG TGT TTG AGA ATG AC-3′ and PCR master mix containing 1x Taq buffer (Fermentas, USA), 2.5 mM MgCl2 (Fermentas, USA), 0.2 mM dNTP (Fermentas, USA) and 1 U Taq polymerase (Fermentas, USA). Five micro-litre of 1 st round of PCR product acted as template for the second round of PCR amplification with constituents of master mix remaining same but amplified with different primers; HEV3 (4420-4439): 5′-CGA CTC CAC CCA GAA TAA CTT-3′, HEV4 (4747-4727): 5′-CAC AGC CGG CGA TCA GGA CAG-3′. The thermal profile for both the runs are same with denaturation at 94°C for 1 min, Annealing at 50°C for 1 min and extension at 72°C for 2 min, 25 cycles followed by final extension at 72°C for 7 min. The amplicon of 343 bp was visualised by 2% agarose gel electrophoresis following ethidium bromide staining. Representative strains were sequenced and phylogenetic analysis was performed to determine the circulating strains in this geographical region.

Seven ml of anti-coagulated blood (Acid citrate dextrose) was double diluted and then over layered on the ficoll-paque (Hi-sep, Himedia, India, specific gravity: 1.0770) in the ratio of 3:1, followed by centrifugation at 1600 rpm for 20-30 mins. The PBMC's were carefully taken out from the plasma-ficoll interface, washed three times with RPMI-1640 at 1600 rpm for 7-10 min each. Cell viability was checked by trypan blue (Gibco, Auckland, New Zealand) staining and cells were counted using haemocytometer as per standard procedure. Cells were finally suspended in RPMI-1640 supplemented with 10% fetal calf serum (FCS) (Gibco, Auckland, New Zealand). Counted cells were plated in flat bottomed 96 well tissue culture plates (BD Falcon, New Jersey, USA). Approximately 1 × 10 3 to 2 × 10 5 cells/well were plated to check for the presence of a linear relationship between cell number and the absorbance to allow accuracy in the quantification of cell proliferation. This standardisation is important as the assays gives best result when the control cell set up give a signal of 0.2 to 0.4 O.D at 570 nm according to the kit literature. MTT assay was performed using commercially available MTT Cell Growth Assay Kit (Millipore, Massachusetts, USA) according to the manufacturer's instructions. A cell count of 2 × 10 4 was found optimum to perform the MTT assay [Figure 1]. Hence, forth the cells were plated in duplicate with a count of approximately 2 × 10 4 PBMCs/well. One set was stimulated with 2 μg/well of recombinant HEV pORF2 (452-617 a.a) protein (Prospec, USA) and another set was mock treated with phosphate buffered saline pH 7.2. Similarly the PBMC's from healthy controls (HC) were stimulated with recombinant HEV pORF-2 to check for the specific antigenic reactivity of the epitope. Blanking was done by putting RPMI 1640 in the well. Thus, MTT tests were carried out in 4 groups a) HEV patients with and without stimulation and b) HC with and without stimulation. The cells were then incubated in the presence of 5% CO 2 at 37°C for 4 days. After 4 days the cells were first microscopically examined for the presence of blastogenic transformation followed by MTT assay. MTT solution measuring 10 μL of (5 mg/mL) was added to each well, incubated at 37°C for 4 h, 100 μL of isopropanol with 0.04 N HCl was added and mixed thoroughly by repeated pipetting for colour development. Within an hour the absorbance was measured on monochromator-based ELISA plate reader (Biotek, Highland Park, USA, Model: PowerWave XS) at a wavelength of 570 nm. The proliferation index (PI) was calculated by OD 570 stimulated sample-OD 570 unstimulated sample/OD 570 unstimulated sample. [15],[16]
Figure 1: Absorbance at 570 nm of PBMCs plated at a cell gradient of 1×103-2×106 cells/well. Y-axis – O.D values and X-axis – Number of cells plated were plotted. Cell concentration for plating is optimised to 2×104 as the next higher concentration of 1×105 showed considerably higher difference in O.D

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 ~ Results Top


All the patients included were positive for anti-HEV IgM and/or HEV RNA and were negative for other viral hepatitis markers tested. All the healthy controls were negative for the viral hepatitis markers and also for anti-HEV IgG. Out of 27 HEV patients included in this study, 16 presented with AVH and 11 with ALF. The mean age of the study population was 30.15 ± 13.70 years, male:female ratio is 1.9:1. Anti-HEV IgM was positive in 26 (96.29%) and RNA in 11 (40.74%) patients. The biochemical investigations of the patients revealed the mean bilurubin levels to be 13.49 ± 8.4 mg/dL, alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphates (ALP) were found to be raised in the patient group and ranged between 867.4 ± 568.4 IU/mL, 511 ± 318.6 IU/mL and 296.5 ± 201.5 IU/mL, respectively. On phylogenetic analysis of the representative strains, they found to belong to HEV Genotype I.

PBMCs from HEV patients on stimulation with HEV pORF-2 (452-617 a.a) showed the presence of blastogenic transformation when observed under 40×. The same was not appreciable in the mock treated PBMCs of the patient group as well as HC group [Figure 2].
Figure 2: Recombinant ORF2 antigen (452-617 a.a) stimulation in HEV AVH patient group observed under microscope (×20) and Blast cell formation (×40)

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On quantitating the lymphocyte proliferation by non-radioactive MTT assay, PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively [Figure 3]. Significantly higher antigenic reactivity expressed in PIs were observed between the PI of AVH Vs HC and ALF Vs HC ( P < 0.0001). When the PI of AVH patients were compared with ALF patients, AVH patients showed significantly more lymphocyte proliferation as compared to ALF patients ( P < 0.0001). Hence, the experiments indicate the antigenic nature of recombinant pORF2 (452-617). The peptide is also specific as it brings about blastogenic transformation of HEV infected patients PBMCs that were not detected in naive healthy controls.
Figure 3: The proliferation indices of HC, AVH and ALF group

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 ~ Discussion Top


Lymphocyte proliferation assay has been widely used for studying the pathogenesis of autoimmune and allergic diseases. [17],[18] Recently researchers have started exploring the utility of the same for understanding the immune mechanisms of infectious diseases. [15],[16],[19] Cellular immune response plays an important role against viral infections. We observed a pronounced lymphocyte proliferation of hepatitis E infected patients in the AVH group as compared to the ALF group suggesting; PBMCs of ALF patients are immunologically less reactive as compared to AVH patients. Pal et al., [6] while evaluating the Th1 and Th2 responses in HEV infected pregnant females observed PBMCs from them had lower lymphocyte proliferation response to phytohemagglutinin than those of control pregnant and non-pregnant females, suggesting generalised inability to mount a lymphocyte proliferation response. Srivastava et al. [20] have also documented that on stimulation with HEV ORF 3 peptide, T cells from fulminant Hepatitis E patients had less marked expansion of HEV specific IFN-γ and TNF-α secreting CD4+T cells than uncomplicated hepatitis E patients. Studies on cytokine profile of HEV patients revealed a Th2 shift in ALF patients, which was evident by an elevation in IL-12 levels in AVH while significant low levels of IFN-γ and IL-2 ALF cases. [21]


 ~ Conclusion Top


The inability to mount a robust lymphocyte proliferation on stimulation with specific HEV ORF 2 peptide (452-617 a.a) by ALF patients in our study demonstrates a state of T cell immunosuppression and points towards a Th2 bias which might play an important role in the disease severity and mortality. Further link between lymphocyte proliferation, cytokine profiles and liver injury following HEV induced hepatitis needs to be looked in, for understanding the immunopathogenesis of the disease.


 ~ Acknowledgment Top


Manasi Majumdar is a recipient of senior research fellowship from the Council of Scientific and Industrial Research, Govt. of India. We are thankful to Mrs. Jyoti Malik, Educationist-cum-Lecturer in English, Medical education and research cell PGIMER, Chandigarh for contributing towards the corrections in English text of the manuscript.

 
 ~ References Top

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  [Figure 1], [Figure 2], [Figure 3]

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