Dace Skrastina
Humoral and cellular immunogenicity of chimeric derivatives of
hepatitis B core particles.
University of Latvia, 1997
SUMMARY
Hepatitis B virus (HBV) nucleocapsids (HBcAg) possess unusually strong
immunogenic properties, although HBc particles are unable to induce
protection against HBV infection. They may be used as ideal "carriers" of
foreign epitopes. On the contrary, S and preS polypeptides located on the
surface of HBV virions within the so-called surface antigen (HBsAg) are more
significant immunologically, but their intrinsic immunogenicity is rather low.
In this study, the ability of immunodominant HBV preS and S regions to induce
immunological response after their insertion into HBc carrier particles has been
investigated. For this purpose we used structures, where preS and S
fragments of different length were inserted into HBc carrier molecule at
positions (i) 144, with retained C-terminal fragment 145-183 or without it
(so-called HBcΔ derivatives). (ii) 78, within the major immunodominant region
(MIR), as HBcΔ variant. Special attention was devoted to structures carrying
deletions of different length within the MIR with the insertion of preS1
31-DPAFR-35 epitope of minimal, 5 amino acid (act) residues, length,
so-called immunomarker. In all cases, intensity of immunological response
against HBc carrier and inserted protein fragments has been compared. In
experimental studies we employed immunoenzymatic methods to detect
specific antibodies in the sera of immunized mice, as well as helper T-cell (Th)
proliferation test for detection of stimulation of T-lymphocytes after
immunization of mice with chimeras and with HBc carrier as a control.
We found that both HBc carrier and inserted foreign epitopes within these
chimeric proteins stimulated specifically proliferation of T-lymphocytes.
C-terminal 39 aa region of HBc molecule after position 144 did not contain any
important Th- and B-cell epitopes and therefore it was not necessary for the
formation of immunoresponse in living organisms. Using synthetic peptides (6
to 23 aa in length), we detected the following Th-cell epitopes: preSI
(31-DPAFRA-36) and preS2 (133-DPRVRGL-139) in the constructions, where
these preS polypeptides were inserted into position 144 of the C-terminally
truncated HBcΔ and S epitope (313-CTKPSDGNCT-322) in the construction,
where an immunodominant HBs region, so-called domain"a" (dom"a"), was
inserted between positions 78 and 82 of C-terminally truncated HBcΔ Weak
Th-cell proliferation response against preSl(21-47) peptide was observed in
vitro, when preSI sequence 31-DPAFRA-36 was inserted into HBc? molecule
between positions 78 and 79, although high Th memory cell response was
found only for this construction. As to B-cell response, all above mentioned
constructions induced an efficient production of anti-HBc anti bodies, but
relatively weak synthesis of specific anti-preS and anti-S antibodies.
Constructions where press 31-DPAFR-35 epitope was inserted into the MIR of
HBcΔ carrier after its shortening, induced Th-cell proliferation in vitro against
HBcΔ but not against preS l (21-47) peptide. The highest level of the synthesis
of anti-preS1 antibodies occurred, when 10 aa deletion was achieved within
the MIR of HBcΔ molecule. In this case, the titer of anti-preSI enhanced
significantly, but the level of anti-HBc dropped markedly. Moreover, the titer of
anti-preSI antibodies grew in correspondence with the extension of MIR
deletion from 3 to 10 aa
All chimeric proteins with dissected or shortened MIR were able to induce
formation of specific antibodies, which was preserved during the long time, at
least 73 days after the first immunization,without any special restimulation of
animals. Chimeric constructions induced specific antibodies irrespective of
haplotype (H-2d and H-2k) of immunized mice and of injection strategy, with
adjuvants (CFA. IFA) or without them. In the experiments with special strains of
mice we found that chimeric proteins enabled antibody formation only
T-lymphocyte dependent mechanism of antibody synthesis, in contrary to HBc
carrier itself. which was able to use both T-cell dependent (TD) and T-cell
independent (TI) ways of antibody synthesis.
In general. the antibody response against preS and S epitopes showed
tendency to 10.- 50 fold enhance when these epitopes were inserted into MIR,
in comparison to constructions, where foreign sequences were added to the
C-terminally truncated HBcΔ carrier.
The obtained results are essential for the further investigation of
immunoresponse mechanisms in living organisms, for the development of
basic principles in the construction of chimeric proteins, opening therefore the
chance for the design of essentially new vaccines.
The present work is described in 5 papers and presented in 7 international and
5 Latvian conferences.
In 1996, this work was awarded by Academia Prize to young scientists in Baltic
states, sponsored by the Swedish institute and Soros International Science
Foundation.
I have been certificated in 1996 by Category "C" of the FELASA
recommendations to work with Laboratory Animals, in accordance with the
rules of Norvegian College of Veterinary medicine.
LIST OF ORIGINAL PAPERS
This thesis is based on the following papers, which are referred to by their
Roman numerals:
I Zakis, V.? Skrastina. D., Borisova, G. & Kuranova, 1. (1992).
Immunodominance of T-cell epitopes on foreign sequences of the hepatitis B
virus nucleocapsid fusion proteins. In Vaccines 92 (Brown, F., Chanock, R.M.,
Ginsberg, H.S. & Lemer, R.A., ea.) pp.341-347. Cold Spring Harbor Laboratory
Press, New York.
II Borisova, G., Arya, B., Dislers, A., Borschukova, O., Tsibinogin, V.,
Skrastina. D., Eldarov, M., Pumpens, P., Skryabin, K.G. & Grens, E. (1993).
Hybrid hepatitis B virus nucleocapsid bearing immunodominant region from
hepatitis B virus surface. J. Virol. 67, 3696-3701.
III Borisova, G., Borschukova Wanst, O., Mezule G., Skrastina. D., Petrovskis,
I., Dislers, A., Pumpens, P. & Grens, E. (1996). Spatial structure and insertion
capacity of immunodominant region of hepatitis B core antigen. Intervirolofy
39, 16-22.
IV Borschukova, O., Skrastina. D., Dislers, A., Petrovskis, I., Ose, V.,
Zamurujeva, I. & Borisova, G. (1997). Modified hepatitis B core particles as
possible vaccine carriers. In Vaccines 97 (Brown, F., Burton, D., Deherty, P.,
Mekalanos, J. & Norrby, E., ea.) pp.33-37. Cold Spring Harbor Laboratory
Press, New York.
V Borisova, G., Borschukova, O., Skrastina~ D., Mezule, G., Dislers, A.,
Petrovskis, I., Ose. V., Gusars, I., Pumpens, P.& Grens, E. (1997). Display
vectors. I. Hepatitis B core particle as a display moiety. Proc. Latv. Acad. Sci.
USA 51, 1-7.