Japanese Encephalitis Vaccine

Japanese Encephalitis Vaccine
Dr Monjori Mitra Associate Professor Institute of Child Health Kolkata

Issues to Consider Epidemiological status Currently Available Vaccines
New Vaccination Modalities The Clinical Trial Currently Underway

Japanese Encephalitis Virus History
Minor epidemics of “summer encephalitis in Japan since at least 1870; large outbreak in 1924 causes 6,125 cases with 3,797 deaths Initially called Japanese type B encephalitis to differentiate from epidemic encephalitis lethargica, type A encephalitis Virus first isolated from the brain of a fatal case in 1935 Isolated from Culex tritaeneorhinchus in 1938 Now known to be the principal mosquito vector in most of the geographic distribution of the disease

Epidemiology Primarily a disease of rural Asia
Vector mosquitoes proliferate in close association with birds and pigs Birds and pigs are the major amplifying hosts Many other mammals and reptiles infected as well, long term viremia documented in bats, others Culex tritaeniorhynchus the principal vector but many other mosquitoes are competent and can transmit C. pipiens C. quinquefasciatus Species of Aedes, Anopheles Virus overwinters in mosquitoes as well as vertical transmission Traditional seasonal spread (spring/summer) heavily impacted by rice paddy flooding

Cattle May Serve to Modulate JE Activity
Photo by George Risi

Incidence and Prevalence
Commonest cause of encephalitis in Asia In hyperendemic areas half of all cases occur in children under 4 years of age, nearly all before age 10 Nearly 100% seroprevalence by adulthood in heavily infected areas Epidemic and endemic forms 20,000 cases and 6,000 deaths annually a gross underestimate Mathematical modeling predicts 175,000 annual cases, 43,750 fatalities, 78,750 with disability

Incidence and Prevalence
Ratio of apparent to inapparent infection ranges from 1:250 in susceptible Asians to 1:63 in adult US marines, 1:18 in Torres strait outbreak Ratio affected by age, virulence of the strain of virus, cross protective immunity from other flaviviruses (dengue) Risk to travelers 1 case per 50,000 months of exposure

Epidemiology Geographic range expanding; new areas infected by
Viremic migratory birds- Guam, Saipan Windblown mosquitoes- Torres strait of Australia

Epidemiology Uttar Pradesh Bihar July 2005 an outbreak began in northern India and Nepal; by November 10, 2005 Uttar Pradesh and Bihar had 6097 cases, 1400 deaths (23% mortality) Outbreaks clearly related to difficulties and expense of currently available vaccine

Epidemiology Japanese Encephalitis Virus is transmitted to Humans by the bite of infected Mosquito species. Different mosquitos genera and species of mosquito serve as intermediate host and transmit JE virus. Anopheles species: - hyrcanus, subpictus Culex species: - tritaeniorhynchus, vishnui Mansonia species:- annulifera, indiana Pigs & birds are primary reservoirs wherein the virus is maintained & amplified 30- 50,000 overt JE cases and 10,000 deaths reported annually worldwide (likely underreported). 30% of survivors suffer from lasting damage to central nervous system In India JE has shown increasing trend in occurrence and expansion of disease to non- endemic areas in India In JEV endemic areas, JE is primarily a pediatric disease

JEVirus –Transmission, Prevalence, Risk
Military deployed to endemic areas Expatriates in rural areas Travelers Key risk groups Residents of rural areas in endemic locations

JE Vaccination Program – Overview JE campaign States & districts
1 Andhra Pradesh 10 2 Kerala 3 Uttar Pradesh 34+1 4 Goa 5 Assam 11 6 Bihar 7 Haryana 8 Karnataka 7+1 9 Tamil Nadu 9+1 Maharashtra West Bengal 12 Manipur 13 Nagaland 14 Arunachal Pradesh 15 Uttarakhand Total 112 Map showing 112 JE vaccination campaign districts

The JE Mass vaccination Drives ( Campaigns ) Coverage
S.No. Year No. of States covered No. of Districts covered Target population years Total JE vaccination campaign coverage JE vaccination campaign coverage % 1 2006 4 11 88.39 2 2007 9 27 84.71 3 2008 22 84.24 2009 10 30 66.61 5 2010 8 19 81.7 6 2010*re- campaigns 97.85 Total 118 80.67 * Based on the emergence of new cases JE/ AES and low coverage as per CES report in 9 districts 2 states , it was decided to conduct re-campaigns

JE vaccination campaigns - Year coverage 2006 - 2011

JE Vaccination Campaign 2006

Japanese Encephalitis Disease
Incubation 6-16 days. Spectrum from mild febrile headache to severe encephalitis Headache, fever, nausea, vomiting, drowsiness. Abdominal pain and diarrhea common in children Progression over several days to severe disease Dull, mask-like facies Muscular rigidity Cranial nerve palsies Tremulous eye and extremity muscle movements Generalized and localized paresis, incoordination, pathologic reflexes Seizures frequent in children, <10% of adults Associated Press

Clinical Manifestations
Death in 5-40% Some deaths after acute fulminant course, others from cardiopulmonary complications with prolonged coma Children under 10 more likely to die or have residual neurological defects Poor prognosis associated with Respiratory dysfunction Babinsky’s sign Frequent or prolonged seizures Prolonged fever Albuminuria High viral replication in the brain Source: Reuters News Agency

Epidemiological Data

Type of vaccine:  1) Live attenuated vaccine (SA strain)  2) Inactivated, Vero cell-derived, alum-adjuvanted vaccine (SA strain)  3) Inactivated Vero cell-derived based vaccines (Beijing-1 strain) Schedule:  1) In China, the first dose of the live attenuated vaccine is given subcutaneously at age 8 months, followed by a booster dose at 2 years of age. In some areas, an additional booster is offered at 6–7 years of age. Protection for several years may be achieved also with a single dose of this vaccine.  2) Primary immunization of the inactivated, alum-adjuvanted vaccine consists of two intramuscular doses, 4 weeks apart  3) The inactivated (Bejjing-1-) vaccines: three doses at days 0, 7 and 28, or two doses given preferably 4 weeks apart (0.25 ml for children <3 years, 0.5 ml for all other ages).

Booster: The duration of immunity is not well established for the above vaccines. 1) the live attenuated vaccine, a booster dose is recommended in some countries. 2) the Japanese vaccines, a booster is recommended after year 1, and thereafter every 3 years. 3) the inactivated, alum-adjuvanted vaccine, one booster is recommended 12–14 months after completion of the primary immunization; the possible need for further boosters to be determined. Adverse reactions: Occasional mild local or systemic reactions Before departure: The immunization series should be completed at least 1 week before potential exposure to JEV.

Japanese Encephalitis Vaccine
A new live attenuated, JE–yellow fever chimeric vaccine has recently been licensed in Australia and Thailand , and will be commercialized from 2012. This vaccine requires a single dose for primary immunization; the possible need for booster doses remains to be determined.

Efficacy of the SA 14-14-2 Vaccine against Japanese Encephalitis.
Effectiveness of One Dose of SA Vaccine against Japanese Encephalitis N Engl J Med 2009; 360: April 2, 2009 Kumar R et al. N Engl J Med 2009;360:

Immunogenicity and efficacy of Live Attenuated SA 14-14-2
Several studies have demonstrated an excellent immune response after a single dose of SA vaccine, with neutralizing antibody responses produced in 85%-100% of non-immune children. Several field trials in China have yielded protective efficacy rates above 95%. One early case control study found 80% vaccine efficacy in children receiving one dose and 98% for two doses. A more recent study in an endemic area of Nepal reported 99.3% efficacy of a single dose. One year after immunization, a follow up study in the same region reported efficacy of 98.5%.

Global Advisory Committee on Vaccine Safety - The SA 14-14-2 live attenuated JE vaccine
GACVS has reviewed safety aspects of this vaccine at two of its meetings (twelfth, held on 9-10 June 2005, and fifteenth, held on November 2006). GACVS reviewed data related to the safety, immunogenicity and efficacy of the vaccine, and scrutinized data on co-administration with measles vaccine. GACVS concluded that the short-term safety profile of live JE vaccine appears satisfactory and that there appears to be a high level of vaccine efficacy after the administration of a single dose. In relation to serious adverse events reported after mass vaccination campaigns in India during 2006, no direct causality has been established between the reported illnesses and the SA JE vaccine. Nevertheless, GACVS recommended that in future, potential vaccine-related serious adverse events should be better investigated. Furthermore, more investigations are required to assess the possible risk of low frequency adverse events (especially neurological). Since live JE vaccine is currently used in “catch-up” campaigns on many millions of children in Asian countries, the opportunity should be taken to examine whether the vaccine safety profile remains valid in large study populations.

Development of Vero cell-derived inactivated JE vaccine

Other JE Vaccine Manufacturers
Name of the Vaccine Mfg. Strain Doses Schedule Route Presentation Inactivated Mouse Brain purified inactivated JE vaccine CRI Kasauli Nakayama 3 (>3yrs– 1ml and 1-3yrs – 0.5 ml) 0, 7 & 30 SC Liquid Mouse Brain purified inactivated JE vaccine (JENCEVAC) Green Cross – Shantha Biotech 3 (Adult – 1.0 mL & Children – 0.5 mL) Mouse Brain purified inactivated JE vaccine (JE-VAX) Sanofi Pasteur 3 [Adult – 1.0 mL & Children (1-3 Yr.)– 0.5 mL] Lyophilized Vero cell – Inactivated vaccine (IXIARO) Intercell SA 2 (only >17 Yr mL) 0 & 28 IM Liquid PFS Live Attenuated Live attenuated JE Vaccine China 1 (Adults & children mL) Live attenuated JE Vaccine (Chimerivax) Acambis 1 [Adult – 1.0 mL & Children (9-36 Months.)– 0.5 mL] Vero- derived Purified inactivated JE Vaccine (JENVAC®) BBIL Kolar 2 (Adults & children mL)

Comparison between different JE vaccines
(Mouse brain, Live (PHK) and Vero cell based ) Inactivated (Biken) Live attenuated (Chinese) Intercell and Bio E) IXIARO/ JEEV Strain Nakayama, Beijing-1 SA Substrate Mouse brain Primary hamster kidney (PHK) cells Vero Cells (Monkey Kidney cells Formulation Lyophilized Liquid Licensed 1954 – Japan 1993 – US 1988 – China Ixiaro-Licensed in USA, Australia, Canada & many other countries JEEV – Licensed in India Geographic use Worldwide: traveller vaccine SE Asia – childhood China, India Traveller vaccine Administration Subcutaneous IM Dosage 0.5 mL-children 1.0 mL-adults 0.25 mL-children 0.5 mL-adults Booster At one year & every 3 years At 6 years Studies on going Efficacy 91% – 2 dose 80% – 1 dose 97.5% – 2 dose 96 % – 2 dose in adult 95.7% -2 dose in children Protection Antibody levels > or = 1:10 Safety Rare cases of urticaria, angioedema, dyspnea, acute encaphalo-myelitis Serious adverse event reported lower rate of Adverse Events. *IXIARO Page 33

Collaboration with NIV & iOWH
For JE Vaccine Development has collaborated with Dr. Milind Gore National Institute of Virology, Pune, India, Dr. Richard Chin, Director Dr. Raj Shankar Ghosh, Regional Director, South Asia. (now PATH)

Global Scenario - JE Vaccine
First Generation Vaccines (Mouse Brain Derived): BIKEN- Japan has been the largest manufacturer and international distributor, but has ceased production. JENCEVAC- Manufactured by Green cross, South Korea. Other manufacturers are found in Taiwan, Thailand and Vietnam 35

Global Scenario - JE Vaccine
Second Generation Vaccines: Vero cell derived, Purified inactivated JE vaccine: IXIARO: Manufactured by Intercell AG, Austria. The vaccine was approved for adults. Phase III clinical trials completed in Indian children (BE collaboration). Manufactured by Bharat Biotech International Limited. The vaccine was approved for conducting Phase-II/III clinical trial and trials completed in adults & children. 36

Innovative aspects of BBIL JE Vaccine
Novel inactivation process - to keep the Antigenicity increase immunogenicity Increased stability & shelf-life of the vaccine Thermo- stable strain

NIV-History of JE virus seed
Obtained from : NIV, Pune, India Isolation : JE infected encephalitis patient Strain : Thermostable Kolar Strain (JEV XY) Passage history : 17 times in suckling mice Original Seed titer : LD50 per mL = 107 38 38

Product & Production profile
Purified, inactivated Japanese encephalitis protein Not Less Than 5.0µg/0.5mL (Single Human Dose) Robust manufacturing technology Production facility- Fully validated commercial scale Production capacity- 25 Million doses annually

Pre-clinical study

Type of Sample Injection Type of Sample Injection
Pre-clinical toxicity (BBIL) Systemic toxicity Group Type of Sample Injection Dose/Dosage (Intramuscular) No. of Rats per group Male Female Group I (Control) PBS buffer 4 doses (day 0, 7, 14 & 28)/0.5mL PBS buffer 10 Group II JENVAC® 4 doses (day 0, 7, 14 & 28)/0.5mL NLT 5µg Group III JENCEVAC 4 doses (day 0, 7, 14 & 28)/ 1mL Group Type of Sample Injection Dose/Dosage (Intramuscular) No. of Rabbits per group Male Female Group I (Control) PBS buffer 4 doses (day 0, 7, 14 & 28)/0.5mL PBS buffer 3 Group II JENVAC® 4 doses (day 0, 7, 14 & 28)/0.5mL NLT 5µg Group III JENCEVAC 4 doses (day 0, 7, 14 & 28)/ 1mL Pre-Clinical studies done as per Schedule-Y 41

Pre-clinical toxicity (BBIL) Systemic toxicity
Dose schedule of the vaccine is a maximum of 2SHDs, but in this study 4SHDs were given to the rats and rabbits and no impact was found on the animal safety. Blood samples for evaluation of serum chemistry and hematology were collected from all the animals on 0th day & 42nd day. A terminal body weight was obtained shortly prior to necropsy and a complete gross necropsy was conducted on all animals sacrificed during the study. There was no treatment related effects on mortality, clinical observations, body weight, food consumption, water consumption, coagulation, hematology or clinical chemistry analysis and histopathology in both rats & rabbits. Conclusion: Based on the study, Purified Inactivated Japanese Encephalitis Vaccine injection did not alter any of the above parameters in rats and rabbits in the systemic toxicity study conducted for a period of 42 days. 42

Animal Potency study

Animal Potency study (Thailand)
Project: Study on potency of inactivated Japanese encephalitis vaccines in adult mice Site of Study: Center for Vaccine Development, Mahidol University at Salaya (WHO approved center for JE vaccines) Animal: Female Swiss Albino Inbred strain SPF mice, age 4 weeks Immunization dose/schedule: Vaccine 1:10 dilution 1 2 3 4 5 6 7 Day 1st dose at Day 0, by I.P. route 2nd dose at Day 7, by I.P. route

Vaccines: Inactivated JE vaccine: Batch-88DP9001, Source: Bharat Biotech, India Inactivated JE vaccine: Batch-JJ , Source: GPO, Thailand Inactivated JE vaccine: Batch Source: Korean Green Cross, S. Korea (Nakayama), Serum collection: Collected on day 14 post dose 1 Serologic test: A validated Plaque Reduction Neutralization Test, 50% end point in continuous LLC- MK2 cells as per SOP using JE wild type Beijing strain as challenging virus, was used to evaluate all sera collected during the study period.

Result: To evaluate the magnitude of change in circulating neutralizing antibody titers after immunization, titers were measured in all 10 mice immunized. With 2 doses of the Bharat Biotech JE vaccine with GMT and 100% seroconversion rate. For GPO, GMT of PRNT was found to be Seroconversion rate of those 10 mice being used in the study revealed 90%. For KGC vaccine evaluation, GMT was fount to be and 80% seroconversion rate. Conclusion: Bharat Biotech JE Vaccine, like GPO JE Vaccine confers higher GMT than the Korean Green Cross JE Vaccine. For seroconversion rate, the Bharat JE Vaccine revealed 100% seroconversion rate after 2 doses, while the other 2 vaccines could not.

Phase I Clinical Trial

Phase I Clinical Trial Protocol Title:
A Phase I, Randomized, Double Blind, Placebo Controlled and Parallel Assignment Study to Evaluate the Safety, tolerability and immunogenicity of inactivated Japanese encephalitis Vaccine Produced by BBIL in healthy adult volunteers. Protocol Number: BBIL/JEV/I/2010 Study Investigator & Centre: Dr. Murali Mohan, MD-General Medicine, Professor, Dept of Medicine, Vydehi Institute of Medical Sciences, Bangalore.

Study Population 52 08 Number of subjects enrolled
A total of 60 healthy adult male subjects of age 18 to 50 years were participated in this study. Cohort 1: 25 vaccine and 5 placebo = 30 subjects (2 doses, day 0 & 28)* Cohort 2: 25 vaccine and 5 placebo = 30 subjects (3 doses, day 0, 7 & 28) * *Dose: As other commercially available vaccines are either 2 doses or 3 doses, hence BBIL has selected 2 & 3 dose schedule in Phase I Clinical Trial. Number of subjects enrolled Number of Subjects completed Number of subjects dropped out 60 52 08

Dose and Mode of administration
Subjects received either cell culture Inactivated Japanese encephalitis vaccine containing NLT 5µg protein or placebo by intramuscular route as per randomization. Liquid 0.5ml of vaccine/placebo is injected as two doses on day 0 and day 28+/-2 (Cohort-1) and three doses on Day 0, Day 7±1 and Day 28±2 (Cohort-2) by intramuscular route in to the deltoid region

Study Objectives Primary objective:
Evaluate the safety and tolerability in healthy volunteers of 18 to 50 years. Secondary objective: Immunogenicity in healthy volunteers of 18 to 50 years.

Study Procedure & Plan Safety Evaluation:
Adverse events, vital signs, Physical and clinical evaluation and laboratory tests. Lab investigations for safety evaluation done at baseline and 56±2 days following administration of either vaccine or placebo. Immunogenicity Evaluation: Immunological assessment at base line, 28±2 and 56±2 day for 50% plaque- reduction neutralization test (PRNT50) antibody titre increase against the JE virus

Adverse events observed
Results (Safety) Adverse events observed Test vaccine (121 doses)* Placebo (25 doses)** Fever 10 (8.26%) Headache 4 (3.35%) 1 (4%) Pain at Injection site Bodyache 3 (2.48%) Weakness 0 (0.00%) Swelling 1 (0.83%) Cold No AEs 98 (80.90%) 23 (92%)

Results (Safety) There was no clinically significant change in any of the vital parameters as well as haematological and other biochemical lab parameters after two and three doses of vaccine administration Subjects were also followed up till day 90 for safety, none of the enrolled subjects were withdrawn from study for vaccine related adverse reactions There was no significant difference between the vaccine and placebo groups for all the common adverse reactions (headache, weakness, swelling & cold) and there is a difference between the groups for fever, pain at injection site & body ache noted. Adverse Events observed in the study group are similar/less with other published clinical studies (Intercell and Sanofi Pasteur). (Ref: Assessment report for IXIARO - European Medicines Agency, 2009)

Geometric Mean Titre (n)
Results (Immunogenicity) The Antibody estimation was carried out by PRNT50 method for the Immunogenicity evaluation. JE vaccine strain (homologous virus) is used as a challenge virus. Comparison of GMT: Group Geometric Mean Titre (n) Day 0 Day 28 Day 56 Vaccine (2-Doses) 6.75 (25) 148.72 (21) 411.23 (20) Vaccine (3-Doses) 6.14 189.59 432.47 Placebo 7.16 (10) 8.08 8.53 (07) Note: No statistical difference between 2 and 3 dose group (p-value >0.05) on 28th and 56th day.

Results (Immunogenicity)
Comparison of % of Seroprotection Group % of Seroprotection (n) Day 0 Day 28 Day 56 Vaccine (2-Doses) 23.81 (25) 100 (21) (20) Vaccine (3-Doses) 16 Note: No statistical difference between 2 and 3 dose group (p-value >0.05) on 28th and 56th day.

Comparison of % of Seroconversion Group % of Seroconversion (n) Day 0 to 28 0 to 56 Vaccine (2-Doses) 90.48 (21) 100 (20) Vaccine (3-Doses) 96 (25) Placebo (10) (07) Note: No statistical difference between 2 and 3 dose group (p-value >0.05) on 28th and 56th day.

There is no statistically significant difference between the Geometric Mean Titres of the subjects given two and three doses of BBIL’s JE vaccine on day 28 & 56. The percentage of Seroprotection in subjects given two and three doses of BBIL Japanese encephalitis vaccine is 100% on day 28 or day 56 . The percentage of Seroconversion (≥4-Fold titer rise) with subjects given two and three doses of BBIL’s Japanese encephalitis vaccine on day 56 was 100%.

Analysis & Cross reactivity study - at NIV
Considering the expertise of NIV, Pune in the field of JE vaccine sera testing, representative set of blinded samples were sent to NIV, Pune for test validation and cross reactivity evaluation. Serum samples of the phase I clinical trial were tested for anti-JE neutralizing antibodies against homologous (821564) and internationally accepted heterologous (057434) JEV strains by Plaque Reduction Neutralization Test (PRNT) at the National Institute of Virology, Pune. NIV results BBIL results Parameter Heterologous virus Homologous virus Time period 0 Day 28 Day 56 Day GMT 6.5 14.8 18.5 8.9 23.0 27.9 30.4 51.5

Conclusion It can be concluded that BBIL’s Japanese encephalitis vaccine is safe, well tolerated and immunogenic against homologous (821564) and heterologous (057434) JE virus strains in healthy volunteers of age years. Hence as the immune response is adequate (100% seroconversion) with two dose vaccination, we wish to carry a large-scale multi-centre Phase III study in diverse population for evaluation of extended safety and immunogenicity of Inactivated Japanese encephalitis vaccine.

Phase II/III Clinical Trial

Phase II/III Clinical Trial
Protocol Title: A Phase II/III, Randomized, Single Blinded, Active Controlled Study to Evaluate the Immunogenicity and Safety of inactivated Japanese encephalitis Vaccine in healthy volunteers. Protocol Number: BBIL/JEV/II/III/2011 Study Centers: We have conducted the study in 9 centers in 4 different states stated below across India. 1. Andhra Pradesh 2. West Bengal 3. Karnataka 4. Rajasthan

Service providers Randomization, Labeling & Decoding
Asian Clinical Trials Statistical Analysis: Dr.G.S.R Murthy, Indian Statistical Institute, Hyderabad Sera Sample Analysis NIV – Pune BBIL - Hyderabad

Study Investigators and sites
Dr. J. Venkateswara Rao, Gandhi Medical College, Secunderabad Dr. G. Sampath, Institute of Preventive Medicine, Hyderabad. Dr. P. Venugopal, King George Hospital, Visakhapatnam. Dr. Mukesh Guptha, Saumya Child Clinic, Jaipur Dr. B. Krishnamurthy, Mysore Medical College, Mysore Dr. Monjori Mitra, Institute of Child Health, Kolkata Dr. Sudhakar, Priya Children’s Hospital, Vijayavada Dr. Sri Krishna, Mahavir Hospital, Hyderabad Dr. Bhuvaneswar Rao, Sri Sreenivasa Children’s Hospital, Vijayavada

Inclusion Criteria Healthy volunteers of 50 to 1 years.
Available for all study related visits and procedures for the entire duration of the study, without any known exposure to JE prior to the first screening visit based on previous clinical history. Willing to give signed written Informed Consent.

Exclusion Criteria Subjects with the age less than 1 and above 50 years. Fever of any origin of duration more than 3 days within one month prior to screening or on the day of screening. History of malaise, head ache, anorexia at the time of screening or during the administration of the vaccine under study. Past history of JE infection. Life threatening or serious cardiac (NYHA grades III-IV heart failure), respiratory gastrointestinal, Hepatic, renal, Endocrine, hematological or immune disorders. Past history of / current allergic diseases.

Exclusion Criteria Any confirmed or suspected immunosuppressive or immunodeficient condition Use of any marketed or investigational or herbal medicine or nonregistered drug or vaccine for JE or other vaccine in the past 2 months. Clinically relevant abnormal hematology or biochemistry values in the opinion of the investigator. Any criteria, which in the opinion of the investigator, suggests that the subject would not be compliant with the study protocol. Intention to travel out of the area prior to final or follow-up Visit on day 56±2. Previous history of hypersensitive reaction to vaccine or vaccine component.

Study Population The sample size has been calculated on the following assumptions: allocation ratio of 3:1 (test: reference), 90 % power, a non-inferiority margin of 15%, one sided alpha % CI. Based on the above inputs, a total of 600 evaluable subjects are needed (450 in test group and 150 in the Reference group). A total of 644 healthy subjects of age ≤50 to >1 year participated in this study across 9 centers in India Category Age in years Test group Reference group 1 ≤50 to >18 156 56 2 ≤18 to > 6 144 57 3 ≤6 to ≥1 178 53 Total 478 166 Number of subjects enrolled Number of Subjects completed Number of subjects dropped out 644 608 36

Study Endpoints Primary Endpoint:
Proportion of participants achieving 4-fold or greater neutralizing antibody titer in subjects seropositive at baseline (≥1:10) at day 28±2 after a single dose of vaccination. Proportion of participants that are seronegative at baseline (<1:10) will require a PRNT50 titer of ≥1:10 to meet the criteria for seroconversion at day 28±2 after a single dose of vaccination.

Study Endpoints Secondary Endpoint:
GMT in each group on day 0 and day 28±2. Occurrence of solicited and unsolicited local and systemic AEs within 28 days and 56 days of post vaccination on day 0, 28±2 and 56±2. Occurrence of vaccine-associated SAEs throughout the course of the study. Proportion of participants achieving 4-fold or greater neutralizing antibody titre at day 56 after two doses of vaccination.

Dose and mode of administration
Based on the results obtained from Phase I study we have selected the 2 dose schedule in Phase III study. As per the insert instruction, one dose of reference vaccine selected. Test vaccine: Liquid Purified, inactivated Japanese encephalitis protein NLT 5.0µg/0.5mL was injected as two doses on day 0 and day 28±2 by Intramuscular route. Reference vaccine: Lyophilized Reference Vaccine (Live attenuated, SA Chinese vaccine) 0.5mL was injected subcutaneously after reconstitution with the diluent supplied as one dose on day 0 and Placebo was administered as second dose on day 28±2.

Study Objectives The primary objective is to compare the immunogenicity of the Test vaccine with Reference vaccine in terms of seroconversion and Geometric Mean Titers of JEV neutralizing antibody four weeks after two doses. The Secondary objective is to assess and to evaluate the Safety of the cell cultured inactivated Japanese encephalitis Vaccine in healthy volunteers of 50 to 1 years.

Trial Profile

Reference Vaccine Group
Results (Safety) Distribution of Adverse Events in ≤50 to >1 year age group Adverse events observed Test Vaccine Group Reference Vaccine Group After 1st Dose (478 doses) After 2nd Dose (450 doses) (166 doses) After 2nd Dose (156 Placebo) General Adverse Events Fever 93 (19.5%) 6 (1.3%) 32 (19.3%) 2 (1.3%) Body ache 12 (2.5%) 0 (0%) 5 (3.0%) Vomiting 3 (0.6%) 2 (1.2%) Diarrhoea 1 (0.6%) Cold 2 (0.4%) Cough Myalgia 1 (0.2%) Headache 9 (1.9%) 3 (1.8%) Local Adverse Events Pain at Injection site 47 (9.8%) 18 (4%) 22 (13.2%) 6 (3.8%) Total AEs 172 (36%) 26 (5.8%) 69 (41.6%) 9 (5.8%) p-values are given in the next to the graphical presentation slide

Results (Safety) 58.4% 94.2% 94.2%

Results (Safety) There was no significant difference between the Test vaccine and Reference vaccine groups for adverse reactions noted after first dose of vaccination (p-value >0.05). Adverse Events were reported significantly lower after second dose, when compared to after first dose of test vaccination (p-value <0.001). The AEs reported after second dose in Test group were not significant with the AEs reported after Placebo administration as second dose in Reference group (p-value >0.05).

The Antibody estimation by PRNT50 method for the Immunogenicity. JE vaccine strain (homologous virus) is used as a challenge virus. Seroprotection: A PRNT50 antibody titre of more than 1:10 generally is accepted as evidence of protection. Seroconversion: % of subject’s ≥4-fold titer rise from pre to post vaccination titer called as % of Seroconversion (4-fold). Note: For subjects with a minimum dilution factor <10 (PRNT50), the titre is set to 5.

Comparison of Seropositive & Seronegative percentages between the vaccine groups (Age category ≤50 to≥1 year): Parameter Test Vaccine Reference Vaccine P-value % of subjects seronegative at base line (Day 0) 88.72 82.05 >0.05 % of subjects seropositive 11.28 17.95 after single dose (Day 28)* 98.50 72.66 <0.001 *Excluding subjects seropositive at base line

Seroprotection & Seroconversion of study Groups in ≤50 to ≥1 years Response Time period Test vaccine Reference vaccine p-value % of Seroprotection Day 0 11.28 17.95 >0.05 Day 28 98.67 77.56 <0.001 % of Seroconversion (4-fold) Day 0 to 28 93.14 57.69 All the subjects included

Seroprotection & Seroconversion of Test vaccine in different age groups Response Time period Age Group ≤50->18 years ≤18->6 years ≤6-≥1 years P-value % of Seroprotection Day 0 11.11 13.67 9.46 >0.05 Day 28 97.22 99.28 99.41 Day 56 99.31 100 % of Seroconversion (4-fold) Day 0 to 28 89.58 93.53 95.8 Day 0 to 56 93.06 98.56 98.82

GMT, Seroprotection & Seroconversion comparison in three different age groups Group Vaccine Group Geometric Mean Titre % of Sero prevalence protection conversion Day 0 Day 28 Day 0 to 28 ≤50->18 years Test Vaccine 6.02 105.6 11.11 97.22 89.58 Reference Vaccine 7.09 36.50 25.49 78.43 54.90 ≤18->6 years 6.61 134.71 13.67 99.28 93.52 6.46 29.75 14.81 79.63 59.26 ≤6-≥1 years 5.67 202.0 9.46 99.41 95.85 6.04 53.63 13.73 74.51 58.82

Comparison of GMT titers

Comparison of % of Seroprevalence & Seroprotection

Comparison of % of Seroconversion

Results (Immunogenicity) of Test Vaccine
1st dose 2nd dose 98.67% Seroprotection 93.14% Seroconversion 99.78% Seroprotection 96.90% Seroconversion Enrolled Completed Day 0 Day 28 Day 56 85 85

% of Seroconversion (Seronegative to Seropositive)
Results (Immunogenicity) Seroconversion comparison Day Group % of Seroconversion (4-Fold) % of Seroconversion (Seronegative to Seropositive) p-values Day 28 Test vaccine 93.14 96.46 >0.05 Reference vaccine 57.69 75 <0.05 Day 56 97.25 99.34 41 55.13 When two different seroconversion methods are compared, there is no significant difference in the test vaccine group, but there is a significant difference observed in the reference vaccine group. Since there was only one dose of vaccination in reference vaccine group, % of seroconversion decreased on day 56 by both the methods. Due to the second dose vaccine administration in test vaccine group, % of Seroconversion is increased slightly but not significantly on day 56 by both the methods.

Batch consistency Response Day Batch 88DP10001 (SD) 88DP10002 88DP10003 88DX10001 (MD) 88DX10002 88DX10003 GMT 6.6 6.2 5.7 5.0 5.6 28 96 183.7 165.8 184.4 102.0 233.6 56 308 547.5 531 387.4 441.2 958.7 Sero protection 15.8 13.2 8.1 0.0 8.7 98.5 97.1 100.0 99.3 4-Fold Rise 0 to 28 91.7 91.9 95.9 93.3 87.0 0 to 56 97.0 94.9 99.2 95.7 SD: Single Dose vial, MD: Multi Dose vial No significant difference between batches with respect to final Seroconversion (4-fold raise). Confidence Intervals of all these batches are within the interval (80-120).

Responses of two different vaccine vial presentations of Test Vaccine Response Presentation Overall Single Dose Multi Dose Number of Subjects 392 60 452 Geometric Mean Titre Day 0 6.2 5.2 6.1 Day 28 142.8 160.2 145.0 Day 56 446.0 567.7 460.5 % of Seroprotection 12.5 3.3 11.3 98.5 100.0 98.7 99.7 99.8 % of Seroconversion (4-Fold) Day 0 To 28 93.1 93.3 Day 0 To 56 96.9 96.7 Test vaccines in two different presentations i.e. single and multi-dose vaccine vials are bioequivalent with respect to the responses in seroprotection and seroconversion proportions. Confidence Intervals of two different presentations are within the interval (80-120).

Zone wise comparison of the results: A total of 608 subjects completed the study in 9 centers across India, 6 centers from Andhra Pradesh (Coastal zone: 3 & Hyderabad zone 3), 1 from Rajasthan, 1 from West Bengal and 1 from Karnataka. Centers were categorized according to the zones as below: Zone Area % of Seroprevalence AP 1 Coastal, Andhra Pradesh 14.18 AP 2 Hyderabad, Andhra Pradesh 11.11 B Jaipur, Rajasthan 15.38 C Kolkata, W. Bengal D Mysore, Karnataka 8.82

Interpretations: From the data it reveals that both one dose and two doses show the significant immunogenicity. There is no difference in % of subjects seronegative and seropositive at baseline, but there is significant difference in % of subjects seropositive after single dose on day 28 between Test & Reference vaccine groups. From the data of 2 dose study it shows that single dose of test vaccine is sufficient to elicit the immune response. As 28th day blood sample, subjects has received a single dose were 98.67% seroprotected and 93.14% seroconverted (4 fold) for ≤50- ≥1 years.

Seroconversion & Seroprotection percentages on 28th and 56th day between different age groups are statistically not significant (>0.05). After second dose of test vaccine GMT titre was increased exponentially from day 28 (145) to day 56 (460.5). Seroconversion & Seroprotection percentages on 28th day between Test and Reference vaccine groups are statistically significant (p-value <0.001). There is no significant difference among two presentations i.e. single & multi-dose vaccine vials and among different centers with respect to final Seroconversion (4-fold raise) and Seroprotection. Confidence Intervals are within the interval (80-120). Detailed statistics have done.

Discussion

Geometric Mean Titre (n) % of Seroconversion (n)
Comparison with other vaccines Bharat Biotech has compared the trial results to other commercially available vaccine data such as IXIARO from Intercell, JEEV from BE and JE-VAX from Sanofi Pasteur (EMEA, 2009). Vaccine Geometric Mean Titre (n) % of Seroconversion (n) Day 0 Day 56 Day 0 to 56 JENVAC® (Bharat Biotech) 6.06 (452) (452) 98.59 (452) IXIARO (Intercell) 5.0 (365) 243.6 (361) 96.4 (352) JEEV (Intercell- BE) 9.7 (304) (277) 92.42 (277) JE-VAX (Sanofi Pasteur) 5.0 (370) 102.0 (364) 93.8 (347) As from the comparative table, BBIL has higher GMTs and % of seroconversion than the other commercially available vaccines. It shows that BBIL's vaccine is not inferior to the other commercially available vaccines.

Other Clinical studies with SA 14-14-2
In a single dose of SA vaccine study done by NIV, Pune and seroconversion (>10.0.) was 74.28% after 30 days and 5% after 6 months against internationally accepted JE virus strain (057434). Titers are in the range of (Ref: National Institute of Virology annual report ) Another study in China tested between a two-dose one-month immunization schedule and three-month immunization schedule. After the first dose, seroconversion rates varied from 72% (n=53) to 100% (n=56). (Ref: Dr. Robert Siegel, HBIO 115B: The Vaccine Revolution, June 3, 2000) In one early case control study of SA vaccine it reported 80% vaccine efficacy in subjects receiving one dose and 98% for two doses (Ref: product insert).

Other Clinical studies with SA 14-14-2
Post Marketing surveillance studies carried out in India by ICMR show that the seroconversion is lower (ranging between 35% - 43%) than that reported in other countries. Independent evaluation of vaccine coverage shows that vaccine coverage in the programme were very low. UNICEF coverage report shows a big difference between reported and evaluated coverage figures e.g., In Dibrugarh it was 90.5% vs. 35.9% and Gorakhpur 97% vs % for reported and evaluated coverage respectively. (Ref: Minutes of the Expert Group meeting on JE Vaccine constituted by Sec. (DHR) and DG ICMR was held at ICMR Hqs. on 25th Jan.2010.) Whereas, results obtained for SA in our study was 77.56% of seroprotection on the 28th Day. For the test vaccine (BBIL’s vaccine) 98.67% of seroprotection was observed on the 28th day.

Summary Collaborated with NIV & Institute for OneWorld Health
Indian Thermo-stable strain Well characterised-MVB/WVB & MCB/WCB Well equipped Production Facility & Capacity Experience in QC testing (Rabies, Polio, H1N1& Rota) Vaccine Potency study at Thailand Pre-Clinical & Human Clinical studies Sera Testing at NIV, Pune Comparable with other vaccines

Conclusion

Overall Conclusion It can be concluded that the BBIL’s inactivated Japanese encephalitis vaccine is safe, well tolerated and immunogenic in healthy volunteers in the age group between ≤50 to ≥1, after one or two doses of vaccination. Hence, a single dose schedule can be used for the campaign immunization (~95% seroprotection and seroconversion after a single dose). Two dose schedule can be used for routine immunization (~97% seroprotection and seroconversion after two doses).

KOLKATA 40 yrs on, Japanese encephalitis claims life in Kolkata October 11, 2008 | Prithvijit Mitra & Saikat Ray , TNN KOLKATA: Japanese encephalitis - which kills one in every three persons it infects - is back in the city. On Thursday, 34-year-old Behala resident Pralay Choudhury died from the disease after being under treatment for over three months. This is the first death in Kolkata due to Japanese encephalitis in over four decades. It sparked panic in Pralay's locality in ward 126, with residents accusing KMC of doing nothing to stop mosquitoes from breeding. Pralay's neighbour, a... DELHI Two more in grip of Japanese Encephalitis November 1, 2011 | TNN NEW DELHI: Two more people have been confirmed positive for Japanese Encephalitis in the city, taking the total number of patients suffering from the mosquito-borne disease to 14. According to Dr V K Monga, the MCD health committee chairman, they have not found the source of the infection yet. "The blood samples collected from pigs in the affected areas and the ones being slaughtered have tested negative. Now, we are going to expand the screening... CHANDIGARH Haryana ready to combat vector-borne diseases October 14, 2003 | TNN CHANDIGARH: The Haryana health department has geared up to combat the spread of vector borne diseases such as dengue, malaria and Japanese Encephalitis in Faridabad, Sonepat, Karnal, Jhajjar, Rohtak, Ambala and Panchkula districts and is taking all necessary preventive and curative measures to control the spread of these diseases. Haryana director general of health services Dr B S Dahiya said that regular monitoring and evaluation was being carried out and the situation was well... INDIA Encephalitis claims 3 more in UP, 180 dead since Jan August 25, 2009 | PTI GORAKHPUR: Three more children have succumbed to Japanese Encephalitis here, taking the toll in the viral infection since January to 180. The children belonging to Kushinagar, Siddharthanagar and Mahrajganj districts died in the last two days at the BRD Medical College here, additional director health L P Rawat said here on Tuesday. Twenty people suspected to be suffering from the viral fever have been admitted to the hospital during the same period. ... SCIENCE Beware! New viruses are here October 29, 2004 | TNN NEW DELHI: Scientists are investigating the possibility of new and lesser known viruses emerging in the country even as a brain fever in Baghpat and Saharanpur in UP has claimed 60 lives. With new killer bugs having emerged in several parts of the country in the recent past, Japanese Encephalitis is not the only bug that is being tested. In Baghpat, at least, experts have ruled out Japanese Encephalitis as the cause of the death of 13 children. A lethal bug, spread... GUWAHATI Encephalitis claims 3 more in Dibrugarh, toll 32 August 3, 2012 | TNN Dibrugarh/GUWAHATI: Three more people have died of encephalitis in Dibrugarh district over the past two days. With this, 32 people have so far lost their lives after contracting the vector-borne disease in the district. Confirming the deaths, joint director of health services (Dibrugarh) DN Bangthai on Thursday said 11 deaths have occurred due to Japanese Encephalitis (JE), while 21 died of Acute Encephalitis Syndrome (AES). "As of now, we've 49 other people, who have been found JE... ALLAHABAD 79% kids administered many essential vaccines July 31, 2012 | TNN ALLAHABAD: When it comes to carrying out special immunization campaign in low performing areas including urban slums, migrant and mobile populations and marginalised population, the district health department on Monday claimed to have covered 79% children of set target who were administered with BCG, measles, DPT, Polio and other essential vaccines during four days special drive in the district. District immunization officer Dr Ashutosh Kumar told TOI that 3,705 children were administered... Spurt in encephalitis cases before onset of the season July 16, 2012 | Shailvee Sharda , TNN GORAKHPUR: A short spell of rain has provided a breeding ground for culex, the encephalitis causing mosquito, just outside a hospital ward. Water in choked drains along the passage leading to the epidemic ward too is home to mosquitoes. Add to these the poor management of hospital waste. These factors are accompanied by the sight of helpless parents who crowd the hospital corridors. Welcome to Baba Raghav Das (BRD) Medical College, Gorakhpur -- the infamous encephalitis capital of... NAGPUR Chandipura virus claims 14 children July 13, 2012 | TNN NAGPUREPUNE: Scanty rains have once again activated the sand fly, known to transmit the Chandipura virus. The virus has killed 14 children since June 15 in six districts of Nagpur circle. The figures are scary as the deaths have occurred in a short duration. The virus in monsoon is generally known to claim children over a period of 3-4 months. This year, the toll has already reached 14 in less than 30 days. The Pune-based National Institute of Virology (NIV) has... Centre’s largesse for UP government July 12, 2012 | Swati Mathur , TNN LUCKNOW: Call it prudent politics ahead of Lok Sabha elections 2014 or the fruits of persistent letter writing, either way it paid off for Uttar Pradesh on Tuesday, when the state walked away with a bag full of goodies from the Centre. In a three-and-a-half-hour meeting with PM Manmohan Singh and senior bureaucrats in New Delhi, UP chief secretary Jawed Usmani along with the state's senior bureaucrats managed to extract promises of funds for nearly all pending projects proposed by the state...

Vaccine strategy for disaster and outbreak situation
The growing need is being felt to stockpile of vaccines against certain diseases with potential to cause outbreaks such as Cholera, JE and H1N1 and other seasonal influenza. These vaccines are required for an affected target population and the quantity needed for stockpile should be assessed together with the National Disaster Management Agency (NDMA) • The manufacturers of these vaccines have to be communicated of the decision ahead of time for planning production and when the stock expires or is utilized. • Adequate budgetary provision for such stockpiles should be created and adequate cold chain equipment earmarked for storage. The NDMA also needs to be intimated about the locations of these stockpiles and effective communication maintained with the agency for delivery of these vaccines during an emergency situation

IAPCOI perspective Routine vaccinations to be recommended in high risk zone ???

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