FUNDED BY: National Health Mission (NHM), Health & Family Welfare Department, Government of West Bengal

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1 Characterizing circulating immune complexes complement consumption Chemokine and cytokine responses for the early diagnosis and follow up of arthralgia severity in Chikungunya patients FUNDED BY: National Health Mission (NHM), Health & Family Welfare Department, Government of West Bengal STUDY PERIOD: February 2012 to February 2014 STUDY POPULATION: One Thousand Two hundred and Twenty Principal Investigator: Dr. Bibhuti Saha, Head of the Department, Department of Tropical Medicine, Calcutta School of Tropical Medicine

2 INTRODUCTION: The Chikungunya virus (CHIKV) is an arthropod-borne virus which possesses a single stranded positive RNA as its genetic material. This virus belongs to Alphavirus genus in the Togaviridae family. CHIKV has been shown to be transmitted by Ades aegyptii and Ades. albopictus mosquitoes. It was identified in East Africa in the early 1950s and since then has caused epidemics in continental Africa, the Indian Ocean region, and countries of Southeast Asia such as India 1-4, where since 2006 suspected cases have been estimated to be 1.39 million. The virus is currently causing one of the largest reported outbreaks of CHIK fever in the last 40 years. Since January 2005, countries in the Indian Ocean are facing an unparalleled outbreak (More than million cases) caused by CHIKV. According to NVBDCP during a total chikungunya suspect cases were recorded from 27 states of India 5. Acute onset of fever and polyarthralgia are the primary clinical findings. Some patients might have relapse of rheumatologic symptoms (e.g. polyarthralgia, polyarthritis, tenosynovitis) in the months following acute illness. Studies report variable proportions of patients with persistent joint pains for months to years. Currently treatment for CHIKV induced polyarthralgia is mainly symptomatic. During the course of any infection, antibodies are formed in the circulation. These antibodies normally react with specific antigens to form antigen antibody complexes. In Hepatitis C Virus (HCV) infection the appearance of articular symptoms in the form of either arthralgias or frank arthritis in a patient with chronic HCV infection often puzzles the caring physician. Joint manifestations in patients with chronic HCV infection can be encountered in four different forms: a coexistent arthropathy (not related to HCV), arthritis in the setting of mixed cryoglobulinemia, arthritis directly related to HCV (HCV associated arthritis), and arthritis induced by antiviral therapy (interferon-alpha [IFN-α]) 6. Parvovirus B19 is a member of the family of small, non enveloped, singlestranded DNA viruses Parvoviridae and is the cause of the common childhood illness erythema infectiosum as well as of transient aplastic crisis, hydrops fetalis, and bone marrow suppression in immune compromised patients 7 The soluble form of these immune complexes (Circulating Immune Complexes) may play a pathogenic role in immune injury by depositing activating immune complement in tissues and releasing vasoactive substances and mediate the inflammations. Though Chikungunya infection is not fatal but severe manifestation mainly polyarthralgia will persist for few weeks. Currently there is no specific relevant drugs for treatment for CHIKV induced polyarthralgia 8,9. Cytokines are thought to play an important role in viral immunopathalogy. Both TH1 and TH2 regulated cytokine could serve as important marker of arthralgia severity. It has been suggested that IL-1ß, IL-6, and RANTES were associated with disease

3 severity in acute infected patients 10. Identification of biomarkers for severe polyarthralgia may be useful in identifying subjects at risk. Globally Chikungunya virus (CHIKV) induced arthralgia has resulted in a huge work hour loss and productivity. Urgent need therefore exists for the identification of unique arthralgia severity biomarkers, which could be useful for patient care. The objective of this research scheme was thus to identify novel molecules which could serve as diagnostic and prognostic biomarkers of chikungunya induced arthralgia. The identification and validation of such early biomarkers could thus help in the proper patient management. So, we investigated the various cytokine and chemokine parameter along with characterization of circulating immune complex and HLA B27 status of patients with chikungunya fever at onset and follow up. METHODS: Selection of study subjects: Fever cases (duration </=5 days) of all age groups and either sex attending STM, Kolkata were selected as per WHO criteria (An acute febrile illness, rash, joint pain). Exclusion criteria: patients with underlying medical disorders e.g. Haematological malignancies, Bleeding disorders, Chronic liver disease, Diabetes mellitus, Severe cardiorespiratory disease, Renal diseases, preexisting arthritis. STUDY POPULATION: The study population consisted of total1220 Chikungunya suspect patients enrolled in the outdoor department and Virology department of School of Tropical Medicine during the study period, May 2013 to February 2015 These patients were referred to Dept. of Virology of School of Tropical Medicine for Chikungunya diagnosis by IgM ELISA and Dept of Biotechnology for confirmation by RT- PCR. Among these 7 patients were confirmed by chikungunya IgM ELISA and 4 were confirmed by RT- PCR. However, only five chikungunya patients could be followed up. Table 1, below shows the month wise break up of enrolled patients. Table 1: Month wise break up of enrolled patients Month Virology Department Fever clinic and O.P.D. 16 th May 15 th August th August- 31 st January st February-30 th August st September-14 th February 676 The above table shows that the maximum cases of chikungunya suspect patients were in the month July.

4 Fig- 1. Showing symptomatic Chikungunya infected patients Study design- descriptive, cross sectional observational study It includes: Informed consent of the patient, Detailed medical history, Physical examinations, Investigation: Diagnosis of Chikungunya: Testing of Chikungunya suspect patient serum samples was done according to manufacturer s instructions (procedure as per National Institute of Virology, Pune guidelines). Among the Suspect population 11patients were positive by IgM ELISA and RT PCR. Estimation of LFT profile: LFT profiles of patients were investigated using standard biochemical assays. Identification and characterization of novel chikungunya biomarker: Protein profiling of patients Circulating Immune Complex was carried out by using standard Gel electrophorosis technique. Immune complex measurement (IC): Serum IC was quantified by standard PEG precipitation and measured spectrophotometrically.

5 Measurements of Anti C3( complement component) : This was measured using standard kits. Measurements of Cytokine, Chemokine and anti CCP: These were quantitated by standard kit Study of HLA B27 association with chikungunya infection. The status of HLA B27 was estimated using standard ELISA based assay. s no: 1 M / 18 yrs no: 2 F / 51yrs no: 3 M / 30 yrs No:4 F/28 yrs No:5 M/26 yrs No:6 M/38 yrs No:7 F/ 23 yrs No:8 F/ 19 yrs No:9 F/13 yrs No:10 F/21 yrs No:11 M/28 yrs Collection time 2 days (July 2013) 3 month (July 2013) 7 days (Aug 2013) 5 days (Aug 2013) 5 days (Sep 2013) 2 days (Nov 2013) 5 days (Nov 2013) 10 days( Aug2014) 14 days (Sep 2014) 15 days (Oct 2014) 7Days 2014) Table no. 2. Details of confirmed Chikungunya patients: Clinical Chik Chik SGOT BILI (T) Comments symptoms at RT IgM SGPT BILI(D) presentation PCR ELISA Joint pain, +VE -VE NA NA *Six months Myalgia, No Follow up rash collected Joint pain, -ve +ve OT-34 BI (T)- 0.9 *Nine Myalgia, Rash PT- 39 BI (D)- 0.4 months Follow up collected Joint pain, -ve +ve OT-216 BI (T)-0.8 Stays at Myalgia, Rash PT-66 BI (D)-0.3 Bangalore (Nov Joint pain, Myalgia, No rash Joint pain, Myalgia, No rash Joint pain, Myalgia, No rash Joint pain, Myalgia, No rash Joint pain, Myalgia, rash Joint pain, Myalgia, rash Joint Myalgia pain, Joint pain, Myalgia, rash +ve -ve OT-154 PT-72 +ve -ve OT-35 PT-32 +ve -ve OT-34 PT-39 -ve +ve OT-36 PT-39 BI (T)-0.8 BI (D)-0.4 BI (T)-0.7 BI (D)-0.3 BI (T) -0.7 BI (D)-0.4 BILI (T)-0.8 BILI(D)-0.4 Lost follow up Lost follow up Lost follow up Lost follow up -ve +ve OT-25 BILI (T)- 1.0 Five months PT-40 BILI(D)-0.4 Follow up collected -ve +ve OT-352 BILI (T)- 1.2 Four months PT-302 BILI(D)-0.5 Follow up collected -ve +ve OT-100 BILI (T)- 0.3 Three PT-122 BILI(D)-0.2 months Follow up collected -ve +ve NA NA Lost to follow up to to to to

6 Table no. 3. Details of Follow up Chikungunya patients: s no: 1 Follow up no: 2 Follow up no: 3 Follow up no: 4 Follow up no: 5 Follow up Collection time 180 days (January 2014) 270 days (January 2014) 90 days ( January 2015) 120 days( January 2015) 160 days ( January 2015) Clinical symptoms at time of collection No Joint pain, no Myalgia, No rash Joint Myalgia, pain, No Joint pain, no Myalgia, No rash No Joint pain, no Myalgia, No rash No Joint pain, no Myalgia, No rash Chik PCR RT Chik IgM ELISA SGOT SGPT -ve -ve NA NA -ve -ve NA NA -ve -ve OT-30 PT-26 -ve -ve OT-20.7 PT ve -ve OT-18.5 PT-26.4 BILI (T) BILI(D) BILI (T)- 0.6 BILI(D)-0.3 BILI (T)- 0.5 BILI(D)-0.2 BILI (T)- 0.6 BILI(D)-0.2 Analysis of Circulating Immune complexes (CIC): To analyze the inflammatory responses (CIC) were measured by PEG precipitation and the corresponding PEG index was calculated. Fig 2: Comparative status of CIC in Chikungunya, Dengue and Healthy donors Observation: fold increased CIC were obtained in Chikungunya patients as compared to HD, demonstrating increased inflammatory response during the infection Identification of circulating immune complexes by SDS PAGE analysis Method: Circulating immune complexes were isolated by centrifuging the precipitates with 8% PEG at rpm for 20 minutes at 4 C. The supernatants were removed, and the precipitates were washed and re-suspended in 3% PEG in PBS (ph 7.2). This material then was centrifuged at rpm for 20mins. The supernatants were then removed and the precipitates were dissolved in equal amounts of PBS and incubated at 37 C for 1 hour. Following the incubation period, the complexes were dissociated and analyzed by 7.5 % SDS-PAGE. The isolated patient CIC was subjected to 70V constant voltage for 3 hrs. The Gel was stained with silver stain and photographed. The Relative mobility of the protein

7 standards were calculated and standard graph generated. The Mol wt of the unknown patient proteins CIC were derived from the standard graph generated. RESULTS: According to our previous report, The CIC of chikungunya patients were composed of different polypeptides, including albumin, immunoglobulin, complement, and acute phase reactants. In this report we have further analyzed CIC of Chikungunya patients (at presentation and follow up samples) with a more sensitive silver stain. Figures below show the representative profile of patient A B C D Figure 3: Silver stained SDS-PAGE gel (7.5%) of immune complexes isolated from the serum of patients subjects ( 1). (LaneA: Chikungunya (2 days) Lane B: Rheumatoid Arthritis. Lane C: Dengue Lane D: Chikungunya (180 days) [follow up] Observation: As observed above from CIC isolated from Chikungunya confirmed (patient no :1) there was overexpression of one protein band with molecular weight of 39kDa. Further, this molecule was completely absent in the CIC of patients with Dengue and Rheumatoid Arthritis. Additionally, 39kDa protein was absent in the patient no 1 after 6 months follow up. Records show, this patient didn t have arthralgia. Similarly, CIC analysis of two more chikungunya patients were carried out. Figure below demonstrates the results.

8 A B C D Figure 4: Silver stained SDS-PAGE gel (7.5%) of immune complexes isolated from the serum of patients. (Lane A: Standard Protein marker; Lane B: Chikungunya (patient no 5; 2 days); Lane C: Chikungunya (patient no 6; 5 days) ; Lane D: Healthy control. Observation: As observed above in the CIC of both the chikungunya patients there was overexpression of one protein band with molecular weight of 39kDa. This molecule was completely absent in the CIC of healthy donors. In continuation with our investigation of the CIC protein profile we further analyzed two more chikungunya patients. The gel picture below reveals the protein pattern obtained. A B C D Figure 5: Silver stained SDS-PAGE gel (7.5%) of immune complexes isolated from the serum of patients. (Lane A: Standard Protein marker; Lane B: Chikungunya (patient no. 3; 5 days); Lane C: Chikungunya (patient no:7, 7 days); Lane D: Healthy control.

9 Observation: As observed above in the CIC of both the chikungunya patients there was overexpression of 39kDa protein band. This molecule was completely absent in the CIC of healthy donors. To investigate the status of 39kDa in follow up cases we next analysed the CIC of a chikungunya patient at presentation (at 3 months of illness) as well as 9 months after the onset. The patient was suffering with polyarthralgia. The gel picture of the analysis is shown below. A B C Figure 6: Silver stained SDS-PAGE gel (7.5%) of immune complexes isolated from the serum of patient no.2. (Lane A: Chikungunya (90 days); Lane B: Chikungunya (270 days) [follow up] Lane C: Healthy control. Observation: As observed above there was overexpression of protein band with molecular weight of 39kDa protein present in 90 and 270 days samples in patient with polyarthralgia which was absent in healthy person (fig-6).

10 A B C D E Figure 7: Silver stained SDS-PAGE gel (7.5%) of immune complexes isolated from the serum of patients. (Lane A: Standard Protein marker; Lane B: Chikungunya follow up (90 days); Lane C: Chikungunya follow up (120days); Lane D: Chikungunya follow up (160days) Lane E: Healthy Donor. Observation: As observed in the CIC of both the chikungunya follow up patients there was no overexpression of 39kDa protein band and records show that they were not suffering from arthralgia. This molecule was completely absent in the CIC of healthy donors y = x R² = Series1 Linear (Series1) Fig 8: Standard curve of 7.5% SDS-PAGE Molecular wt markers

11 Cytokine analysis: Analysis of IL-6: To analyse the TH2 mediated inflammatory responses by measuring IL6 titers Fig 9:- Serum IL-6 profile in Chikungunya,,Chikungunya follow-up patients and Control population. Observation: fold increased IL6 titers were obtained in Chikungunya patients as compared to HD demonstrating increased inflammatory response during the infection. IL-6, a pleiotropic cytokine, has a destructive role in Rheumatoid Arthritis, contributing to joint inflammation as well as joint pain this study demonstrated that the IL6 titers were even higher than Rheumatoid Arthritis. However, this parameter was not an early marker for prediction of persisting arthralgia in the chronic chikungunya patient. Analysis of HLA-B27 Tissue haplotype HLA-B27, has been suggested to have a possible genetic association with prolonged joint symptoms. Analysis of HLA B27 GENOTYPE of the 11 CHIK patients revealed that 6 had HLA B27 genotype, among them 1 patient with persistent CHIKV arthralgia too had HLA B27 Genotype. Thus, a genetic predisposition for Chik V induced arthralgia is possible, but this can be validated only with a sufficient number of patients. Analysis of Anti CCP: Anti CCP (anti-cyclic citrullinated protein) antibodies are known to be upregulated during inflammation and serve as diagnostic markers of Rheumatoid Arthritis. Analysis of anti CCP titers

12 revealed that among the 11 CHIK confirmed patients, 5 had high anti CCP titers at onset and when they were followed up only one patient with persisting arthralgia had positive anti CCP titers.thus, anti CCP can serve as a good predictor of arthralgia both in Rheumatoid Arthritis and Chikungunya induced arthralgia patients.however, this marker is not specific only for Chikungunya induced arthralgia. Analysis of IL12: To analyse the TH1 inducing responses in Chikungunya patients IL12 titers were also measured. Fig 10:-Serum IL-12 profile in Chikungunya (CHIK), Chikungunya follow-up patients and Control population. Observation: 2.31 fold increased IL12 were obtained in Chikungunya patients as compared to HD.Interestingly, the patient with persistent arthralgia had very low titers of IL12, indicating that there might be a downregulation in IL12 expression with persistent arthralgia manifestation.

13 Analysis of IL-7 IL-7 is known to have an important role in the development of rheumatoid arthritis, thus their status in Chikungunya patients remains unknown. Fig 11:-Serum IL-7 profile in Chikungunya (CHIK), Chikungunya follow-up patients and Control population Observation: fold increased IL7 were obtained in Chikungunya patients as compared to HD. The titers were even higher than Rheumatoid Arthritis patients, demonstrating profound T lymphocyte cell destruction. Interestingly, the patient with persistent arthralgia had two fold increased IL7 production at follow-up as compared to that detected at onset, indicating that there might be profuse cell destruction accompanied with inflammation. However, this parameter IL7 was not an early marker for prediction of persisting arthralgia in the chronic chikungunya patient.

14 Analysis of CXCL-9 CXCL9 is a chemokine induced by IFN-γ and have been demonstrated to be high in acute infection, hence their levels were measured. Fig 12:-Serum CXCL9 profile in Chikungunya (CHIK), Chikungunya follow-up patients and Control population Observation: In the present study, 3.60 fold increased CXCL9 were obtained in Chikungunya patients as compared to HD. The levels of CXCL9 were higher in chikungunya patients as compared to Dengue patients.the CXCL9 titers were very high inthe patient with persistent arthralgia both at onset and followup. Thus, CXCL9 appears to be a good predictor of Chikugunya induced arthralgia.

15 Analysis of CXCL-10 CXCL10 is a chemokine induced by IFN-γ and have been demonstrated to be high in acute infection, hence their levels were also measured. Fig 13:-Serum CXCL-10 profile in Chikungunya ( CHIK), Chikungunya follow-up patients and Control population Observation: In the present study, fold increased CXCL10 were obtained in Chikungunya patients as compared to HD. Interestingly, the levels of CXCL10 were much higher in Dengue patients as compared to Chikungunya patients. Analysis of RANTES: RANTES levels have previously been reported to be suppressed in severe CHIKF patients, hence their levels were also measured in acute and follow up patients in the present study. Fig 14:-Serum RANTES profile in Chikungunya(CHIK), Chikungunya follow-up patients and Control population

16 Observation: Interestingly, as compared to Dengue, the levels of RANTES were lower in Chik patients at onset and at follow up. Interestingly, the patient with persistent arthralgia had almost 2 fold increased RANTES titers at follow up demonstrating a high persisting inflammatory response. Analysis of C3 complement: Heightened Inflammatory responses are characterised with increased complement protein consumption, thus anti C3 complement component was measued at onset and follow up. Fig 15:-Serum complement C3 profile in Chikungunya (CHIK), Chikungunya follow-up patients and Control population Observation: 8.93 fold increased Complement C3 was obtained in Chikungunya patients as compared to HD. The levels further increased in Chikungunya Followup patients demonstrating induction of post inflammatory response.

17 CONCLUSIONS: High levels of CICs, IL6, IL12, CXCL9, IL7 titers were obtained, demonstrating a mixed TH1/TH2 cytokine and chemokine response in acute Chikungunya patients. However, the TH2 response outweighs the TH1 response. Interestingly, RANTES, CXCL10 titers were lower in acute Chikungnya patients as compared to Dengue fever patients, demonstrating that these two parameters can serve as differential biomarkers of these otherwise similar diseases. High levels of anti CCP antibodies were also obtained in acute Chikungunya patients, which persisted in patients with arthralgia, demonstrating that this parameter can serve as a novel arthralgia marker both in Chikungunya and RA In the present study, % of Chikgunya patients had HLA B27 genotype demonstrating that this is a predisposition factor to chikungunya infection. Complement activation was high in acute and follow up chikungunya patients demonstrating significant tissue injury. Protein profiling of CIC from Chik patients demonstrated the presence of a unique 39Kda protein band, which was absent in normal and dengue patients. The arthralgia severity marker remained in the follow up patient with persisting chikungunya induced arthralgia.

18 REFERENCES: Aikat BK, Konar NR, Banerjee G. Haemorrhagic fever in Calcutta area. Indian J Med Res 1964; 52: Halstead SB, Udomsakdi S, Scanlon JE, Rohitayodhin S. Dengue and chikungunya virus infection in man in Thailand, V. Epidemiologic observations outside Bangkok. Am J Trop Med Hyg 1969; 18(6): Lahariya C, Pradhan SK. Emergence of chikungunya virus in Indian subcontinent after 32 years: A review. J Vector Borne Dis 2006; 43(4): Mason PJ, Haddow AJ. An epidemic of virus disease in Southern Province, Tanganyika Territory, in ; an additional note on Chikungunya virus isolations and sertrop Med Hyg 1957; 51(3): National Vector borne disease control Programme (NVBDCP), Chikungunya cases, 2013, Vassilopoulos D, Calabrese LH: Rheumatic manifestations ofhepatitis C infection.curr Rheumatol Rep 2003, 5: Young NS, Brown KE: Parvovirus B19. N Engl J Med 2004, 350: Deep AB, Parashar D. Chikungunya: an overview. J Biosci 2008; 33(4): Lebrun G, Chadda K, Reboux AH, Martinet O, Gaüzère BA.Guillain-Barre syndrome after chikungunya infection. Emerg Infect Dis 2009; 15(3): Chirathaworn C, Poovorawan Y, Lertmaharit S, Wuttirattanakowit N, Cytokine levels in patients with chikungunya virus infection, Asian Pacific Journal of Tropical Medicine, (2013)

19 PROJECT REPORT SUBMITTED BY: Prof. Bibhuti Saha (Principal Investigator),HOD, Dept. of Tropical Medicine Dr. Sumi Mukhopadhyay, Dept of Laboratory Medicine, CSTM (Co-PI) Dr. Bhaswati Bandyopadhyay, Dept of Virology, CSTM (CoI) Dr. Sudesna Mallik, Associate Professor, Dept. of Tropical Medicine, CSTM (CoI) Dr. Manab Ghosh, Assistant Professor, Dept. of Tropical Medicine, CSTM (CoI) Dr. Ranajoy Shankar Bhattacharya, Assistant Professor, Dept. of Tropical Medicine, CSTM(CoI) Dr. Soumen Halder, Assistant Professor, Dept. of Tropical Medicine, CSTM (CoI) Dr. Yogiraj Ray, RMO cum clinical tutor, Dept. of Tropical Medicine, CSTM (CoI) Dr. Arindam Naskar, RMO cum clinical tutor, Dept. of Tropical Medicine, CSTM (CoI) Dr. Anusri Tripathi, Dept of Biochemistry and Medical Biotechnology, CSTM (CoI) Mr. Goutam Patra, Research Assistant