to leu-108 of HA-1 which is common to at least 12 H3N2 influenza virus strains (Arnon &. Shapira, 1984) was tested for immunogenicity. According to Wilson et ...
J. gen. Virol. (1987), 68, 2249-2252. Printed in Great Britain
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Key words: influenza A virus~syntheticpeptide/immunogenicity
lmmunogenicity of a Synthetic Peptide Corresponding to a Portion of the Heavy Chain of H3N2 Influenza Virus Haemagglutinin By E. H A M S I K O V A , 1 H. Z A V A D O V A , 1 M. Z A O R A L , 2 J. J E Z E K , 2 K. B L A H A 2 AND V. V O N K A 1 1Department of Experimental Virology, Institute of Sera and Vaccines, Prague and 2Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, Czechoslovakia (Accepted 21 April 1987)
SUMMARY
Rabbits were immunized with a synthetic octadecapeptide corresponding to the sequence ser-91 to leu-108 of the haemagglutinin heavy chain of H3N2 influenza A viruses. They developed antibodies reactive in solid-phase radioimmunoassay (SPRIA) with the peptide and with haemagglutinins of various H3N2 viruses but not of heterotypic H1N1 and H2N2 viruses. The antibodies were also non-reactive in the haemagglutination-inhibition or neutralization test. Influenza H3N2 virus replicated in the lungs of mice immunized with the peptide to the same extent as in the control mice. Of 27 human sera possessing anti-H3N2 activity or seven sera from rabbits immunized with either virions or haemagglutinins of various influenza A viruses, none was reactive with the peptide in SPRIA.
Genetically engineered and synthetic vaccines and diagnostics are being developed in many laboratories. Special attention has been paid to the influenza viruses, because detailed information on the structure, function and antigenic properties of the two influenza virus surface glycoproteins, haemagglutinin (H) and neuraminidase (N), is available (Ward, 1981; Wilson et al., 1981 ; Colman et al., 1983). In the present series of experiments the sequence ser-91 to leu-108 of HA-1 which is common to at least 12 H3N2 influenza virus strains (Arnon & Shapira, 1984) was tested for immunogenicity. According to Wilson et al. (1981) this region contains a folded corner in the three-dimensional structure of H and it is adjacent to antigenic site D. The same sequence was selected for similar experiments by Mfiller et al. (1982) and was also investigated by Wilson et al. (1984). Two batches of the peptide were prepared. To prevent the dimerization of the peptide through S-S bonding, cysteine at position 97 was substituted by either methionine (batch denoted OP-1) or alanine (OP-2). Both peptides were synthesized by the standard solid-phase methodology (Barany & Merrifield, 1980). The crude peptides were purified by reversed-phase HPLC. For immunization, peptides were coupled to purified tetanus toxoid (TT) (Sevac Prague) by means of either 1-ethyl-3-(3'-dimethyl-aminopropyl)-carbodiimide hydrochloride or glutaraldehyde. For serological reactions, peptides were coupled to bovine serum albumin (BSA) using glutaraldehyde. Rabbits were immunized by subcutaneous injections of 1 mg of conjugate dissolved in 0.5 ml of phosphate-buffered saline and mixed with an equal volume of Freund's complete adjuvant for the first dose and incomplete adjuvant for the subsequent five doses administered at 2 to 3 week intervals. The immunoglobulin fraction was isolated from each serum specimen using caprylic acid precipitation (Steinbuch & Audran, 1969). The antibody response in these rabbits was monitored by solid-phase radioimmunoassay (SPRIA), haemagglutination inhibition and a neutralization test. SPRIA was performed using the procedure described by Such/mkovfi et al. (1984). The following preparations were used as 0000-7581 © 1987 SGM
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Table 1. Reactivity of sera from peptide-immunized rabbits with synthetic peptides and homotypic
haemagglutinin by SPRIA
Rabbit no.
Immunogen*
1
OP-1 (HPLC) + TT-CDI OP-I (HPLC) + TT-GA OP-2 +TT-GA OP-2 +TT-GA OP-2 (HPLC) +TT-GA OP-2 (HPLC) +TT-GA OP-2 (HPLC) +TT-CDI OP-2 (HPLC) + TT-CDI TT TT
2 3 4 5 6 7 8 9 (control)
Test antigenl OP-1 H OP-I H OP-2 H OP-2 H OP-2 H OP-2 H OP-2 H OP-2 H OP-2 H
+ BSA + BSA + BSA + BSA + BSA + BSA + BSA + BSA + BSA
r 1 8-____22 0-6 5-4 0.9 2.1 0.9 5_.___44 1.4 12.8 1.2 0.5 0.5 2.'/ 0.7 14-0 2-_._44 1-0 1-1
Reactivity index$ after indicated number of doses ~ 2 3 4 5 13-0 0-6 __7"9 0-9 3.9 1.4 5.__66 1-5 16.4 1.9 4.3 0.6 --14.3 2.4 0.9 1.0
13-7 1-6 4-1 0.5 6-9 1.2 5-5 1.9 17.6 2._._66 10-7 2.7 13.9 0-8 15-5 1.5 1.3 0-7
12-7 1.7 3-_.__22 1-0 -1-3 0.9 15-3 2.4 10.1 1-7 15.9 1.1 21.4 1.8 1-1 1.0
12.4 4.._.0 3-8 2.1 -1-2 -1-4 30.2 2-j 15.3 2-._O0 14-8 1.3 29.0 ._2.__66 0.8 0.8
6 12.3 3-4 6.0 2.__0 -1.1 -1-0 34.6 3.__.66 15.0 2._._33 17.4 1.5 29.4 1.6 1.4 0.9
* (HPLC), purified by HPLC; TT, conjugated to tetanus toxoid; CDI, coupled using carbodiimide, GA, coupled using glutaraldehyde. t BSA, conjugated to bovine serum albumin; H, haemagglutinin derived from A/Philippines or A/Victoria (H3N2) influenza virus. Concentration of immunoglobulins was approximately 0.3 mg/ml. $ Ratio of c.p.m, of test serum to c.p.m, of preimmune serum, values equal to or exceeding 2.0 are underlined.
antigens: (i) 2 gg per well of HPLC-purified peptide coupled to BSA; (ii) 2 ~tg per well of haemagglutinins of homotypic H 3 N 2 A/Philippines/85 and A/Victoria/75 and heterotypic [A/swine/31 (H1N1), A/PRS/34 (H1N1), A/Ostrava/80 (H1N1)] viruses, prepared from p u r i f e d virions by bromelain cleavage (Brand & Skehel, 1972), and (iii) 0.5 ~tg per well of virions purified according to Laver (1964). Various dilutions (starting at 1 : 10) of the Ig preparations were tested. The reaction was considered positive if the ratio of c.p.m, of test serum to c.p.m, of preimmune serum was 2.0 or more. The immunization procedure and the development of antibodies reactive with the synthetic peptides (OP-1 or OP-2) and H of H 3 N 2 virus are shown in Table t. It can be seen that antibodies reactive with either OP or with both OP and H developed in all rabbits. Two of the three rabbits which did not develop anti-H reactivity had been immunized with a preparation not purified by H L P C ; this suggests a lower immunogenicity of this preparation. N o m a r k e d differences in the immunogenic activity o f OP-1 and OP-2 were encountered and no m a r k e d influence on the immune response was produced by the coupling procedure used. The antipeptide antibodies were nearly always detected earlier and attained higher levels than those reactive with H. This might be associated with the higher number of antibody-binding sites in the wells coated with the peptide than those coated with H. Alternatively the differential reactivity may have been due to a heterogeneity of the OP preparation which might consist of molecules possessing the conformation of the native protein as well as molecules with other conformations and antigenicities; thus antibodies capable of reacting with antigens present in OP preparations but not in H might have developed. The reactivity in S P R I A of selected sera of OP-immunized rabbits with purified H and complete virions of homotypic and heterotypic influenza viruses are shown in Fig. 1 and 2. The antibody was reactive with H preparations from H 3 N 2 viruses but not with those derived from H1N1 and H 2 N 2 viruses (Fig. 1). These results, demonstrating specificity of the reaction of OPinduced antibodies, were in sharp contrast to the cross-reactivity observed when whole virions
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