Japanese Encephalitis Vaccine

Dr Monjori MitraAssociate ProfessorInstitute 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

Photo by George Risi

Cattle May Serve to Modulate JE Activity

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

• 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

Uttar Pradesh

Bihar

Epidemiology

Dr. Goetz Reiner 11

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

12

Military deployed to endemic areas

Expatriates in rural areas

Travelers

Key risk groups

Residents of rural areas in endemic locations

JEVirus –Transmission, Prevalence, Risk

JE campaign States & districts

1 Andhra Pradesh 10

2 Kerala 2

3 Uttar Pradesh 34+1

4 Goa 2

5 Assam 11

6 Bihar 6

7 Haryana 6

8 Karnataka 7+1

9 Tamil Nadu 9+1

10 Maharashtra 8

11 West Bengal 5

12 Manipur 5

13 Nagaland 2

14 Arunachal Pradesh 1

15 Uttarakhand 1

Total 112

JE Vaccination Program – Overview

RAJASTHAN

ORISSA

GUJARAT

MAHARASHTRA

MADHYA PRADESH

BIHAR

UTTAR PRADESH

KARNATAKA

ANDHRA PRADESH

JAMMU & KASHMIR

ASSAM

TAMIL NADU

CHHATTISGARH

PUNJAB

JHARKHANDWEST BENGAL

ARUNACHAL PR.

HARYANA

KERALA

UTTARANCHAL

HIMACHAL PRADESH

MANIPUR

MIZORAM

MEGHALAYANAGALAND

TRIPURA

SIKKIM

GOA

A&N ISLANDS

D&N HAVELI

PONDICHERRY

LAKSHADWEEP

Map showing 112 JE vaccination campaign districts

The JE Mass vaccination Drives ( Campaigns ) Coverage

* Based on the emergence of new cases JE/ AES and low coverage asper CES report in 9 districts 2 states , it was decided to conduct re-campaigns

S.No. YearNo. of States

covered No. of

Districts covered

Target population- 1-15 years

Total JE vaccination campaign coverage

JE vaccination campaign

coverage %

1 2006 4 11 10531554 9308688 88.39

2 2007 9 27 21758223 18431087 84.71

3 2008 9 22 20040262 16881941 84.24

4 2009 10 30 27170604 18097182 66.61

5 2010 8 19 9336609 7626354 81.7

6 2010*re- campaigns 2 9 7659936 7495380 97.85

Total 118 96497188 77840632 80.67

JE vaccination campaigns - Year coverage2006 - 2011

20062007

20082009

2010*

2010 reca

mpaigns0

20

40

60

80

10088% 84.7% 84.2%

66.6% 69.6%

97.9%

C

over

age%

Year of Campaign

JE vaccination campaigns - Year coverage2006 - 2011

Around 78 million children have been immunized in the JE vaccination campaigns from 2006 – 2011

Around 4 million children have been immunized with JE vaccine under RI

JE Vaccination Campaign 2006

JE Vaccination Campaign 2007

JE Vaccination Campaign 2008

JE Vaccination Campaign 2009

JE Vaccination Campaign 2010

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

24

Type of vaccine: 1) Live attenuated vaccine (SA 14-14-2 strain) 2) Inactivated, Vero cell-derived, alum-adjuvanted vaccine (SA 14-14-2 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.

Efficacy of the SA 14-14-2 Vaccine against Japanese Encephalitis.

Kumar R et al. N Engl J Med 2009;360:1465-1466.

Effectiveness of One Dose of SA 14-14-2 Vaccine against Japanese EncephalitisN Engl J Med 2009; 360:1465-1466April 2, 2009

Immunogenicity and efficacy of Live Attenuated SA 14-14-2

Several studies have demonstrated an excellent immune response after a single dose of SA 14-14-2 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 29-30 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 SA14-14-2 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

Vaccine Name of the Vaccine Mfg. Strain Doses Schedule Route Presentation

Inactivated

Mouse Brain purified inactivated JE vaccine

CRI Kasauli Nakayama3 (>3yrs– 1ml and

1-3yrs – 0.5 ml)0, 7 & 30 SC Liquid

Mouse Brain purified inactivated JE vaccine

(JENCEVAC)

Green Cross – Shantha

BiotechNakayama

3 (Adult – 1.0 mL & Children – 0.5 mL)

0, 7 & 30 SC Liquid

Mouse Brain purified inactivated JE vaccine

(JE-VAX)

Sanofi Pasteur

Nakayama3 [Adult – 1.0 mL & Children (1-3 Yr.)–

0.5 mL]0, 7 & 30 SC Lyophilized

Vero cell – Inactivated vaccine (IXIARO)

Intercell SA-14-14-22 (only >17 Yr. - 0.5

mL)0 & 28 IM Liquid PFS

Live Attenuated

Live attenuated JE Vaccine

China SA-14-14-21 (Adults & children -

0.5 mL)0 SC Lyophilized

Live attenuated JE Vaccine (Chimerivax)

Acambis SA-14-14-21 [Adult – 1.0 mL &

Children (9-36 Months.)– 0.5 mL]

0 SC Lyophilized

InactivatedVero- derived Purified inactivated JE Vaccine

(JENVAC®)BBIL Kolar

2 (Adults & children - 0.5 mL)

0 & 28 IM Liquid

Comparison between different JE vaccines (Mouse brain, Live (PHK) and Vero cell based )

 Inactivated

(Biken)Live attenuated

(Chinese)

InactivatedIntercell and Bio E)

IXIARO/ JEEV

Strain Nakayama, Beijing-1 SA14-14-2 SA14-14-2

SubstrateMouse brain Primary hamster kidney

(PHK) cellsVero Cells (Monkey Kidney cells

Formulation Lyophilized 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 vaccineSE Asia – childhood

China, India Traveller vaccine

Administration Subcutaneous Subcutaneous IM

Dosage 0.5 mL-children 1.0 mL-adults

0.5 mL-children 1.0 mL-adults

0.25 mL-children0.5 mL-adults

Booster At one year & every 3 years At 6 years Studies on going

Efficacy91% – 2 dose 80% – 1 dose

97.5% – 2 dose96 % – 2 dose in adult 95.7% -2 dose in children

Protection Antibody levels > or = 1:10 Antibody levels > or = 1:10 Antibody levels > or = 1:10

SafetyRare cases of urticaria, angioedema, dyspnea, acute encaphalo-myelitis

Serious adverse event reported

lower rate of Adverse Events.

PAGE 33*IXIARO

Collaboration with NIV & iOWH

Dr. Milind GoreNational Institute of Virology,Pune, India,

Dr. Richard Chin, DirectorDr. Raj Shankar Ghosh, Regional Director,South Asia. (now PATH)

For JE Vaccine Development

has collaborated with

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

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.

Innovative aspects of BBIL JE Vaccine

Novel inactivation process - to keep theAntigenicity 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 821564 XY)

Passage history : 17 times in suckling mice

Original Seed titer : LD50 per mL = 107

• 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

Product & Production profile

Pre-clinical study

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 10

Group II JENVAC® 4 doses (day 0, 7, 14 &

28)/0.5mL NLT 5µg 10 10

Group III JENCEVAC 4 doses (day 0, 7, 14 & 28)/ 1mL 10 10

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 3

Group II JENVAC® 4 doses (day 0, 7, 14 &

28)/0.5mL NLT 5µg 3 3

Group III JENCEVAC 4 doses (day 0, 7, 14 & 28)/ 1mL 3 3

Pre-Clinical studies done as per Schedule-Y

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.

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

1st dose at Day 0, by I.P. route

0 1 2 3 4 5 6 7Day

2nd dose at Day 7, by I.P. route

Animal Potency study (Thailand)

• Vaccines:

Inactivated JE vaccine: Batch-88DP9001, Source: Bharat Biotech, India

Inactivated JE vaccine: Batch-JJ 5210 3, Source: GPO, Thailand

Inactivated JE vaccine: Batch 00410002 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 153.55 and 100% seroconversion rate.

• For GPO, GMT of PRNT was found to be 187.22. Seroconversion rate of those 10 mice

being used in the study revealed 90%.

• For KGC vaccine evaluation, GMT was fount to be 46.21 and 80% seroconversion rate.

Animal Potency study (Thailand)

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.

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.

Study Population

Number of subjectsenrolled

Number of Subjects completed

Number of subjectsdropped 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

Results (Safety)

Adverse events observed

Test vaccine(121 doses)*

Placebo(25 doses)**

Fever 10 (8.26%) 0

Headache 4 (3.35%) 1 (4%)

Pain at Injection

site4 (3.35%) 0

Bodyache 3 (2.48%) 0

Weakness 0 (0.00%) 1 (4%)

Swelling 1 (0.83%) 0

Cold 1 (0.83%) 0

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)

Results (Immunogenicity)

The Antibody estimation was carried out by PRNT50 method for the Immunogenicity

evaluation. JE vaccine strain 821564 (homologous virus) is used as a challenge virus.

Comparison of GMT:

GroupGeometric 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(25)

189.59(25)

432.47(25)

Placebo7.16(10)

8.08(10)

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)

100(20)

Vaccine (3-Doses)

16(25)

100(25)

100(25)

Note: No statistical difference between 2 and 3 dose group (p-value >0.05) on 28th and 56th day.

Results (Immunogenicity)

Comparison of % of Seroconversion

Group

% of Seroconversion

(n)Day 0 to 28

Day 0 to 56

Vaccine (2-Doses)

90.48(21)

100(20)

Vaccine (3-Doses)

96(25)

100(25)

Placebo0

(10)0

(07)

Note: No statistical difference between 2 and 3 dose group (p-value >0.05) on 28th and 56th

day.

Results (Immunogenicity)

• 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 Homologous virus

Time period 0 Day 28 Day 56 Day 0 Day 28 Day 56 Day 0 Day 28 Day 56 Day

GMT 6.5 14.8 18.5 8.9 23.0 27.9 8.9 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 18-50 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

Randomization, Labeling & Decoding

Asian Clinical Trials

Statistical Analysis:

Dr.G.S.R Murthy, Indian Statistical Institute, Hyderabad

Sera Sample Analysis

NIV – Pune

BBIL - Hyderabad

Service providers

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.

• 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

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.

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 - 97.5% 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

Study Population

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 subjectsenrolled

Number of Subjects completed

Number of subjectsdropped 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.

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.

Study Endpoints

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 14-14-2

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

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)

After 1st Dose (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%) 0 (0%)

Vomiting 3 (0.6%) 0 (0%) 2 (1.2%) 0 (0%)

Diarrhoea 3 (0.6%) 0 (0%) 1 (0.6%) 1 (0.6%)

Cold 2 (0.4%) 0 (0%) 2 (1.2%) 0 (0%)

Cough 2 (0.4%) 0 (0%) 0 (0%) 0 (0%)

Myalgia 1 (0.2%) 0 (0%) 2 (1.2%) 0 (0%)

Headache 9 (1.9%) 2 (0.4%) 3 (1.8%) 0 (0%)

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 821564 (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).

Results (Immunogenicity)

Note: For subjects with a minimum dilution factor <10 (PRNT50), the titre is set to 5.

Results (Immunogenicity)

Comparison of Seropositive & Seronegative percentages between the vaccine groups (Age

category ≤50 to≥1 year):

*Excluding subjects seropositive at base line

ParameterTest

Vaccine

Reference

Vaccine

P-value

% of subjects seronegative at base line (Day 0)

88.72 82.05 >0.05

% of subjects seropositive at base line (Day 0)

11.28 17.95 >0.05

% of subjects seropositive after single dose (Day 28)*

98.50 72.66 <0.001

Results (Immunogenicity)

Seroprotection & Seroconversion of study Groups in ≤50 to ≥1 years

All the subjects included

ResponseTime

periodTest

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 <0.001

Results (Immunogenicity)

Seroprotection & Seroconversion of Test vaccine in different age groups

ResponseTime

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 >0.05

Day 56 99.31 100 100 >0.05

% of Seroconversion

(4-fold)

Day 0 to 28 89.58 93.53 95.8 >0.05

Day 0 to 56 93.06 98.56 98.82 >0.05

Results (Immunogenicity)

GMT, Seroprotection & Seroconversion comparison in three different age groups

Group Vaccine Group

Geometric Mean Titre

% of Seroprevalence

% of Seroprotection

% of Seroconversion

Day 0 Day 28 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

Test Vaccine 6.61 134.71 13.67 99.28 93.52

Reference Vaccine 6.46 29.75 14.81 79.63 59.26

≤6-≥1 years

Test Vaccine 5.67 202.0 9.46 99.41 95.85

Reference Vaccine 6.04 53.63 13.73 74.51 58.82

Results (Immunogenicity)

Comparison of GMT titers

Results (Immunogenicity)

Comparison of % of Seroprevalence & Seroprotection

Results (Immunogenicity)

Comparison of % of Seroconversion

Results (Immunogenicity) of Test Vaccine

1st dose 2nd dose

Day 0 Day 28 Day 56

Enrolled Completed

98.67% Seroprotection93.14% Seroconversion

99.78% Seroprotection96.90% Seroconversion

Results (Immunogenicity)

Day Group% of Seroconversion

(4-Fold)% of Seroconversion

(Seronegative to Seropositive)

p-values

Day 28Test vaccine 93.14 96.46 >0.05

Reference vaccine 57.69 75 <0.05

Day 56Test vaccine 97.25 99.34 >0.05

Reference vaccine 41 55.13 <0.05

Seroconversion comparison

• 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.

Results (Immunogenicity)

Batch consistency

Response Day

Batch

88DP10001(SD)

88DP10002(SD)

88DP10003(SD)

88DX10001(MD)

88DX10002(MD)

88DX10003(MD)

GMT

0 6.6 6.2 5.7 5.0 5.6 5.0

28 96 183.7 165.8 184.4 102.0 233.6

56 308 547.5 531 387.4 441.2 958.7

Seroprotection

0 15.8 13.2 8.1 0.0 8.7 0.0

28 98.5 97.1 100.0 100.0 100.0 100.0

56 100.0 99.3 100.0 100.0 100.0 100.0

4-Fold Rise0 to 28 91.7 91.9 95.9 93.3 87.0 100.0

0 to 56 97.0 94.9 99.2 93.3 95.7 100.0

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).

Results (Immunogenicity)

Responses of two different vaccine vial presentations of Test Vaccine

ResponsePresentation

OverallSingle Dose Multi Dose

Number of Subjects 392 60 452

GeometricMean 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

Day 0 12.5 3.3 11.3

Day 28 98.5 100.0 98.7

Day 56 99.7 100.0 99.8

% of Seroconversion

(4-Fold)

Day 0 To 28 93.1 93.3 93.1

Day 0 To 56 96.9 96.7 96.9

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).

Results (Immunogenicity)

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 15.38

D Mysore, Karnataka 8.82

Results (Immunogenicity)

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.

Results (Immunogenicity)

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

Comparison with other vaccines

VaccineGeometric Mean Titre (n) % of Seroconversion (n)

Day 0 Day 56 Day 0 to 56

JENVAC® (Bharat Biotech) 6.06 (452) 460.53 (452) 98.59 (452)

IXIARO (Intercell) 5.0 (365) 243.6 (361) 96.4 (352)

JEEV (Intercell- BE) 9.7 (304) 217.97 (277) 92.42 (277)

JE-VAX (Sanofi Pasteur) 5.0 (370) 102.0 (364) 93.8 (347)

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).

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 14-14-2 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 10.43- 133.03. (Ref:

National Institute of Virology annual report 2007-08)

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 14-14-2 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.

52.3 % 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 14-14-2 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

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).

Overall Conclusion

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Vaccine strategy for disaster and outbreak situation

The growing need is being felt to stockpile of vaccines against certaindiseases with potential to cause outbreaks such as Cholera, JE andH1N1 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 ofthe decision ahead of time for planning production and when thestock expires or is utilized.

• Adequate budgetary provision for such stockpiles should becreated and adequate cold chain equipment earmarked for storage.

• The NDMA also needs to be intimated about the locations of thesestockpiles and effective communication maintained with theagency for delivery of these vaccines during an emergencysituation

IAPCOI perspective

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