Introduction
Sporozoites, the initial infecting form of the malaria
parasite, are a key target for vaccine development. A
successful vaccine would either cause destruction of the
parasite, or block migration to the liver by preventing
binding to hepatocytes. The main epitope responsible for
mediating initial recognition and attachment of the
malarial sporozoite to hepatocytes is believed to be the
short amino acid sequence, EWSPCSVTCGNGIQVRIK,
referred to as region II (RII) (Cerami
et al., 1992a). The
RII motif has been identified on the surface of two
groups of sporozoite proteins, the circumsporozoite
protein (CSP) and the sporozoite surface protein-
2/thrombospondin related anonymous protein (Robson
et al., 1988; Cerami et al., 1992a; Rogers et al., 1992).
Addition of a peptide containing this motif (Cerami
et al., 1992b; Chatterjee et al., 1995), or addition of
polyclonal antibodies against this peptide (Chatterjee
et al., 1995) blocked invasion of sporozoites to Hep-G2
cells in vitro.
The production of recombinant BCG (rBCG) provides
a very promising prospect for development of a live
malaria vaccine that could provide long term protec-
tion. BCG is a stimulator of cellular immune response
and a remarkably safe vaccine which is administered at
birth in many countries.
Various methods to insert foreign genes into BCG have
been described previously (Stover et al., 1991; Winter
et al., 1992; Dellagostin et al., 1993) and shown to
induce protective immune response in animal models of
disease (Stover et al., 1991; Stover et al., 1993). These
studies also showed that rBCG strains are stable and
continue to produce the foreign proteins in vivo . We
therefore chose a peptide fragment containing the RII-
CSP motif of P. falciparum to clone into BCG as the
initial step towards the development of a potential
malarial vaccine.
Materials and methods
Preparation of
M. smegmatis
and
M. bovis
BCG competent cells
M. smegmatis mc
2
155 (Snapper et al., 1990) was grown
with shaking at 37
°C in Nutrient Broth No. 2 (Oxoid)
supplemented with 0.05% v/v Tween 80 and 0.2% v/v
glycerol for 48 h. The cells were harvested by centrifu-
gation and the pellet resuspended and washed in 25 ml
pre-chilled 1 mM Hepes. After centrifugation, the pellet
was resuspended in 5 ml of pre-chilled 10% v/v glyc-
erol and centrifuged again. The cells were resuspended
in 2 ml 10% glycerol and 200
l aliquots frozen in an
ethanol:dry ice bath and stored at -80°C until used.
M. bovis BCG Pasteur was grown in 50 ml 7H9 media
(Difco) containing oleic acid/albumin/dextrose (OADC;
Difco) and incubated at 37°C for 4 to 5 days. Isoniazid
was added to a final concentration of 4 g/ml one day
prior to harvesting as above.
11111
2
3
4
5
6
7
8
9
10111
1
2
3
4
5
6
7
8
9
20111
1
2
3
4
5
6
7
8
9
30111
1
2
3
4
5
6
7
8
9
40111
1
2
3
4
5
6
7
8
9
50111
1
2
3111
Biotechnology Letters, Vol 19, No 11, November 1997, pp. 1135–1137
Biotechnology Letters · Vol 19 · No 11 · 1997
1135
Cloning and expression of a candidate
malarial epitope in bacille Calmette
Guerin
M.N. Norazmi
1
and J.W. Dale
2*
1
Department of Immunology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian,
Kelantan, Malaysia.
2
Molecular Microbiology Group, School of Biological Sciences, University of Surrey, Guildford
GU2 5XH, England, United Kingdom
The candidate malarial vaccine epitope, region II of
Plasmodium falciparum
circumsporozoite protein (RII-CSP), was
cloned into
Mycobacterium smegmatis
and
M. bovis
BCG via a mycobacterial replicative plasmid pUS1762. This
plasmid contained a gene for the
M. leprae
18 kDa protein to provide expression signals. Transformation was achieved
by electroporation and selection for kanamycin resistance and verified by Southern hybridisation and sequencing.
The transformation efficiency was in the order of 10
4
cfu/g DNA for
M. smegmatis
and 10
3
cfu/g DNA for
M. bovis
BCG. Western blotting using a polyclonal antibody specific for the RII-CSP and a monoclonal antibody specific for
the
M. leprae
protein showed the expected 25 kDa band of the 18 kDa-RII-CSP fusion protein.
© 1997 Chapman & Hall
*Correspondence: Fax : 44–1483–300–374;
E-mail address: j.dale@surrey.ac.uk.
J BL,1135-1137,929 Norazmi 20/10/97 8:05 am Page 1135