Med Microbiol Immunol (2007) 196:227–231
Immunogenicity and protective eYcacy of the E. coli-expressed
domain III of Japanese encephalitis virus envelope protein in mice
Alka · Kaushik Bharati · Y. P. S. Malik ·
Received: 16 December 2006 / Published online: 22 March 2007
© Springer-Verlag 2007
Abstract Domain III of Japanese encephalitis virus
(JEV) envelope protein (E-DIII) was synthesized in E. coli
as a fusion protein containing maltose-binding protein
(MBP-E-DIII) or six contiguous histidine residues (His-E-
DIII) at its N-terminus. MBP-E-DIII was found both in the
soluble as well as the insoluble fraction of the bacterial
lysate, while His-E-DIII was found exclusively in the inclu-
sion bodies. These puriWed proteins were examined in mice
for their immunogenicity in presence of an aluminium
hydroxide based-adjuvant Alhydrogel and Freund’s adju-
vant. While both proteins generated anti-JEV antibodies
that neutralized JEV activity in vitro, His-E-DIII generated
higher antibody titers than MBP-E-DIII. Mice immunized
with His-E-DIII in presence of Alhydrogel generated anti-
body titers similar to those induced by the commercial vac-
cine and protected mice against lethal JEV challenge.
Keywords Neutralization · Enzyme-linked
immunosorbent assay · Adjuvant
Japanese encephalitis (JE), the most important cause of epi-
demic encephalitis is caused by Japanese encephalitis virus
(JEV), an arbovirus belonging to the family Flaviviridae
that includes several medically important pathogens such as
yellow fever, dengue and West Nile encephalitis viruses. JE
is a zoonotic disease of public health importance, largely
because of its epidemic potential and high case fatality rate.
The disease is endemic in the Indian subcontinent, virtually
all of Southeast Asia, China and parts of Oceania. It has
been estimated that approximately 35,000–50,000 cases of
JE occur annually, with 10,000–15,000 cases proving to be
fatal . The principal clinical manifestation of illness is
encephalitis. The disease is characterized by seizures,
poliomyelitis-like paralysis and Parkinsonian movement
disorders. A large proportion of survivors develop neuro-
psychiatric sequelae that include impaired cognition,
behavioral disturbances and convulsions . There is no
speciWc treatment for JE and hence vaccination of suscepti-
ble populations is the sole logical alternative. The only
World Health Organization (WHO)-approved vaccine that
is available, namely the mouse brain-derived formalin-inac-
tivated JEV vaccine, is inherent with certain drawbacks.
Besides being expensive and in short supply, it causes aller-
gic reactions in some recipients . Moreover, the immu-
nity conferred by the vaccine is of short-term duration .
A formalin-inactivated, primary hamster kidney cell cul-
tured vaccine based on the P3 strain of JEV is in use in
China since the late 1960s. Besides, a live, attenuated vac-
cine using the SA14-14-2 strain of JEV is in use in China.
However, both P3 and SA14-14-2 vaccines are not yet pro-
duced as per the internationally acceptable quality norms.
Hence, there is an urgent need to develop alternate vaccine
candidates that are eVective, safe as well as aVordable .
Alka and Kaushik Bharati contributed equally to this work.
Alka · K. Bharati · Y. P. S. Malik · S. Vrati (&)
National Institute of Immunology,
Aruna Asaf Ali Marg, JNU Complex,
New Delhi 110 067, India
Y. P. S. Malik
Jawaharlal Nehru Krishi Vishwa Vidyalaya,
Jabalpur, MP, India