Identification and Quantitation of the

Transcription

1 APPLIED MICROBIOLOGY, My 1972, p Copyright i 1972 Americn Society for Microbiology Vol. 23, No. 5 Printed in USA. Identifiction nd Quntittion of the Components of Polyvlent Inctivted Influenz Virus Vccines by Immunodiffusion MARTIN G. MYERS AND NICOLA M. TAURASO Lbortory of Virology nd Rickettsiology, Division of Biologics Stndrds, Ntionl Institutes of Helth, Bethesd, Mrylnd Received for publiction 27 Jnury 1972 One of the bsic problems in the stndrdiztion of inctivted polyvlent influenz virus vccines hs been the determintion of the reltive potency of the individul strin components. The chicken cell gglutintion test mesures relibly the totl hemgglutinin content of these vccines. With immunodiffusion techniques, it is now possible to quntitte ech strin component of polyvlent vccines. Routine ppliction of these techniques would serve s n interim procedure to ssess ntigenic potency of individul strin components of commercil vccines until improved tests re developed. Reserch in the 1940's nd 1950's culminted in the development of n inctivted influenz virus vccine. However, it hs only been since the emergence of the Hong Kong vrint of A2 influenz in 1968 tht relible lbortory methods for mesuring the ntigenicity of these vccines hve been vilble (11). Since then, the stndrdized chicken cell gglutintion (CCA) test, employing reference vccine, hs llowed reproducible nlysis of the totl vccine hemgglutinin content. Although the CCA test hs llowed stndrdiztion of totl hemgglutinin content, it hs not been possible to mesure the reltive content of the severl ntigenic components in polyvlent vccine preprtion. Immunodiffusion (ID) techniques hve been shown to be suitble both for the study of influenz virus subunits nd the detection of homologous ntibody responses in vrious niml species including mn (1-3, 6-10). This report describes the ppliction of ID techniques to nlyze the ntigenic composition of Formlin-inctivted polyvlent influenz virus vccines. content. These ltter vccines were mde by dilution with 0.01 M phosphte-buffered sline (PBS), ph 7.2, (or, in the cse of zonl-purified concentrtes, with 0.85% sline contining 0.2% geltin) nd the following Formlin-inctivted influenz virus concentrtes: AO/PR/8/34, Al/AA/1/57, nd A2/Jpn/170/62 monovlent Shrples concentrtes; A2/Aichi/2/68 nd B/Mss/3/66 monovlent zonl-purified concentrtes. Immune ser. Immune chicken ser were used in these studies. Ser prepred for hemgglutintioninhibition (HI) ginst AO/PR/8/34, A2/Aichi/2/68, nd B/Mss/3/66 were obtined from the Center for Disese Control, Atlnt, G. Ser ginst A1/AA/1/57 were the 17-dy-convlescent ser of White Rock roosters inoculted intrvenously with 1.0 ml of experimentl vccine G, contining 1,000 CCA units of A1/AA/1/57 ntigen per ml. Nonimmune ser were obtined from uninoculted White Rock roosters. ID. Double diffusion ws performed in either 0.75% Iongr no. 2 (Colbs) or 0.75% grose (L'Industrie Biologique Frncise) prepred in disstilled wter contining vrious concentrtions of NCl nd 0.1% sodium zide. The medium ws utoclved t bout 120 C for 7 min, cooled t 60 C, nd then 2.5-ml mounts were dispensed into 35-mmdimeter plstic petri dishes (Flcon) which were stored in MATERIALS AND METHODS plstic bgs t 4 C until use. Six wells, 4 mm in dimeter, evenly positioned Vccines. The following inctivted vccines round nd 9 mm (center to center) from centrl 4- were employed in this study: (i) Ntionl Institutes mm-dimeter well, were cut with templte. Seril of Helth (NIH) 1967 civilin polyvlent (67CP) nd twofold dilutions of the rectnts were mde in the 1971 civilin bivlent (71C) reference vccines; (ii) PBS diluent mentioned bove. Antigen dilutions lots of commericlly prepred influenz vccines; were dded in duplicte to the outer wells. Precipitin bnds were recorded either by visul exmin- nd (iii) number of experimentl monovlent nd polyvlent vccines of known composition nd CCA tion with indirect light or by photogrphy employing 946

2 VOL. 23, 1972 COMPONENTS OF INFLUENZA VIRUS VACCINE 947 Polroid CU 5 Lnd cmer. ID endpoints were expressed s the reciprocl of the highest ntigen dilution giving precipitin rection on the seventh dy. The ID endpoints of the 71C reference vccine components were converted into CCA units (one ID unit being equivlent to CCA units of A2/Aichi/2/68 nd CCA units of B/Mss/3/66). The reltive contribution of ech of these ntigens to the totl CCA content of the vrious vccines could then be compred. Other ssy procedures. The CCA test, used to quntitte the hemgglutinting ntigen content of the vccines, ws performed by the method of Miller nd Stnley (4) s described in n NIH memorndum [Titrtion of chicken red cell gglutintion vlue (NIH memorndum of 16 September 1946, vilble from the Division of Biologics Stndrds, Ntionl Institutes of Helth, Bethesd, Md )]. The semiutomtic micro-rn test (5) ws employed to mesure the ntibody content of the ser. When ser were treted with receptor-destroying enzyme (RDE) previously reported methods were used resulting in 1: 4 dilution of ser (12). RESULTS Effect of different gr preprtions nd slt concentrtion upon precipitin rections. An exmple of typicl ID rection employing whole virus vccine is shown in Fig. 1. Initilly, 0.75% Iongr no. 2 contining 7.5% NCl ws used s the ID medium. Clerer precipitin rections, however, were obtined employing grose contining 5.0% NCl; s FIG. 1. Duplicte seril twofold dilutions of vccine ntigen A (Monovlent A2/Aichi/2/68) in outer wells: wells A nd B = undiluted; C nd D = 1:2 dilution; E nd F = 1:4 dilution. Homologous immune chicken serum (1:16 dilution) in centrl well. Photogrph is 2x enlrgement. the sensitivities of these two medi were found to be identicl, the ltter ws ultimtely dopted. A very indistinct rection ws obtined with Noble gr (Difco), nd slt concentrtions less thn 5.0% or greter thn 10.0% were found to be unstisfctory with these ser. Optiml serum dilution for ID precipittion nd specificity of rections. Precipitin rections could be obtined with undiluted to 1:64 to 1: 128 diluted ser; however, by block titrtion ginst the homologous vccine ntigens 1: 8 to 1: 16 serum dilutions were found to be the most sensitive. These dilutions were used subsequently throughout the study. The specificity of the untreted immune chicken ser used for ID ws shown by HI tests s outlined in Tble 1. There were no significnt differences between untreted nd RDEtreted ser when tested by ID (Tble 2). Consequently, untreted ser were generlly employed for ID. Quntittion of ntigen content by ID. Tble 3 records the quntittive immunoprecipitin rections s relted to the reltive CCA ntigen content of NIH reference vccines, lots of commercil vccines (1971 civilin formultion), nd experimentl vccines. There were no rections between ny of the test ntigens nd either untreted or RDE-treted norml chicken serum. Vccines contining A2/Aichi/2/68 nd A2/Jp/170/62 were indistinguishble by these mens. A vccine contining similr quntity of B/Md/1/59 ntigen ws only modertely rective ginst the B/Mss/3/66 immune serum; none of the other ntigens rected with heterologous ser. The commericl vccine lot 4, contining the A2/Aichi/2/68/X- 31 strin, did not cross-rect with AO/PR/8/34 serum. TABLE 1. Hemgglutintion-inhibition of immune chicken ser used for immunodiffusion Immune chicken Hemgglutintion inhibition for influenz virus ntigens ser AO/PR/ A1/AA/ A2/Aichi/ B/Mss/ 8/34 1/57 2/68 3/66 Norml AO/PR/8/ A1/AA/1/ A2/Aichi/2/ b 0 B/Mss/3/ Men reciprocls of highest dilution of five tests. Zero represents less thn 1:4. b Heterologous rection with A2/Jpn/170/62 ntigen ws 1:38.

3 948 MYERS AND TAURASO APPL. MICROBIOL. TABLE 2. Effect of RDE tretment of ser on immunodiffusion Hemgglutinin composition" Immunodiffusionc with: Experimentl Untreted ser RDE-treted ser vccines AO Al A2 B A2 B NC A2 B NC A B C D E F 1, G 0 1, Key to bbrevitions: AO = AO/PR/8/34; Al = Al/AA/l/57; A2 = A2/Aichi/2/68; B = B/Mss/3/66; NC - negtive control; RDE = receptor-destroying enzyme. b Chicken cell gglutintion (CCA) units per milliliter s determined from CCA vlues of virl concentrtes from which these experimentl vccines were prepred. c Expressed s the men (four tests) of reciprocl of highest ntigen dilution giving precipitin rection. Bsed on the results of the 71C reference vccine, the reltive proportions of A2/Aichi/2/68 nd B/Mss/3/66 ntigens in the bivlent vccines were determined. The correltion of the theoreticl content to the ctul determintions of the experimentl nd commericl vccines ws extremely high. On the bsis of these results, commericl vccine lot no. 4 contined less A2 nd more B ntigen thn the 71C reference nd commericl vccine lots no. 1 to 3. Commercil vccine lot no. 5 ws included in the study becuse it filed to meet the CCA requirements of the Division of Biologics Stndrds (DBS). Anlysis of these results reveled tht it ws pprently deficient predominntly in the B components. The sensitivity of the immunoprecipitin test s performed here is in the rnge of 100 to 150 CCA units per ml. DISCUSSION It is generlly believed tht dequte quntities of the pproprite inctivted influenz virus ntigens fford t lest some mesure of protection ginst influenz. However, one of the bsic problems tht hs been encountered in the stndrdiztion nd, therefore, the evlution of influenz virus vccines is how to determine the potency of inctivted ntigens. The stndrdized CCA test is both simple nd relible technique mesuring the totl hemgglutinin content of these vccines. However, it previously hs not been possible to mesure the reltive contribution to the totl immunizing mss of the severl ntigenic components of polyvlent vccines. In previous studies (3, 7-10) ID techniques hve been pplied to the study of disrupted influenz A virus subunits. These sme workers showed the soluble ntigens to be similr for the vrious influenz A subtypes. In turkeys, ntibody mesured by ID with S ntigen correlted with the complement-fixtion test but not with the HI test (6). Employing Formlin-inctivted ntigens, Berd hs shown correltion between the detection of type-specific influenz A ntibodies by similr ID technique nd complement fixtion in mn nd horses (2) nd the homologous HI tests in birds (1). Although the immune ser employed in this study undoubtedly contin ntibody to number of surfce ntigens in ddition to the hemgglutinin, the use of reference vccine of known strin nd hemgglutinin composition hs llowed the differentition in semiquntittive mnner between the influenz subtypes. In this study, lipid solvent split hemgglutinin vccines were not included s they required different templte nd ssy system possibly becuse of difference in the ntigen prticle size. A commercil vccine which filed to meet DBS stndrds ws exmined by these methods nd ws found to be deficient primrily in the B ntigen component. It is probble, therefore, tht n error ws mde during the preprtion of the bulk vccine lot. Conceivbly, vccine could lso fil the CCA test becuse of dilutionl error which would lso be pprent by the ID test. It is hoped tht by routine ppliction of these techniques, influenz vccines relesed for commericl distribution will be ssured not only of dequte totl hemgglutinin content but lso of dequte potency of ech of the component strins.

4 VOL. 23, 1972 TABLE 3. COMPONENTS OF INFLUENZA VIRUS VACCINE 949 Reltive composition of inctivted influenz virus vccines Immunodiffusiond Antigenic Hemgglutinin composition Actul composition Vccines CCA - AO Al A2 B Totl contentc No Al 2 tests A2 B Totl Experimentlf A B C D E F 1, ,200 1, G 0 1, ,000 1, H Ih ,000 1,000 1, J Commercil (Lot no.) ,400 1, , , i ,400 1, , DBS reference 67CPj Ck Key to bbrevitions: AO = AO/PR/8/34; Al = Al/AA/1/57; A2 = A2/Aichi/2/68; B = B/Mss/3/66; CCA = chicken cell gglutintion; DBS = Division of Biologics Stndrds. & CCA units per milliliter s determined from the CCA content of concentrtes from which the vccines were prepred. ctotl CCA units per milliliter by ctul determintion performed on finl vccines; expressed s the men corrected vlue of five determintions employing 71C s the reference. dimmunodiffusion employing immune chicken ser; vlues expressed s the men of the reciprocl of the highest ntigen dilution giving precipitin rection. There were no rections with norml chicken serum. e Antigen composition bsed on the immunodiffusion vlues relted to the 71C reference (see text). ' Vccine C prepred by mixing equl volumes of A nd B; vccine D by mixing equl volumes of A nd C; nd vccine E by mixing equl volumes of B nd C. Contins A2/Jpn/170/62 ntigen. h Contins B/Mrylnd/1/59 ntigen. I A2 component ws the A2/Aichi/2/68/X-31 strin. } NIH 67 CP (civilin polyvlent) reference vccine; contins 100 CCA units AO/PR/8/34, 100 Al/AA/1/57; 100 A2/Jpn/170/62, 100 A2/Tiwn/1/64, nd 200 B/Mss/3/66 per ml. k NIH 71C (civilin) reference vccine; contins 400 CCA units of A2/Aichi/2/68 nd 300 B/Mss/3/66 per ml. In our opinion, ppliction of the ID test s described in this report would serve s n interim mesure until improved tests re developed. Certinly, the use of stndrdized monospecific ntiser, pretretment of vccines, reproducibility, more precise expression of quntittion nd other test prmeters require dditionl investigtion. ACKNOWLEDGMENTS We wish to thnk Albert V. Hennessy for his helpful criticisms nd suggestions in the preprtion of this mnuscript. LITERATURE CITED 1. Berd, C. W Avin influenz ntibody detection by immunodiffusion. Avin Dis. 14: Berd, C. W Demonstrtion of type-specific influenz ntibody in mmmlin nd vin ser by immunodiffusion. Bull. W. H : Hn, L., nd L. Hoyle The disintegrtion of the internl ribonucleoprotein of influenz virus A with the production of serologiclly distinct components. Act Virol. 10: Miller, G. L., nd W. M. Stnley Quntittive spects of the red blood cell gglutintion test for influenz virus. J. Exp. Med. 79: O'Brien, T. C., S. Rstogi, nd N. M. Turso Sttisticl evlution of diluents nd utomtic di-

5 950 MYERS AND TAURASO luting nd pipetting mchines in influenz serology. Appl. Microbiol. 21: Smdieh, B., nd R. A. Bnkowski Identifiction of influenz A viruses by immunodiffusion test. Amer. J. Vet. Res. 32: Schild, G. C., nd H. G. Pereir Chrcteriztion of the ribonucleoprotein nd neurminidse of influenz A viruses by immunodiffusion. J. Gen. Virol. 4: Styk, B., nd L. Hn Immunodiffusion studies on the rection of influenz virus with specific ntibody nd non-specific serum bet-inhibitor. Act Virol. 10: Styk, B., nd L. Hn Antibody response of APPL. MICROBIOL. guine pigs nd white mice to nucleoprotein component of influenz virus s studied by gel double diffusion. Act Virol. 12: Styk, B., L. Hn, nd M. Sedilekov Antibody response to nturl influenz A2 infection of mn s studied by gel double diffusion. Act Virol. 12: Turso, N. M., T. C. O'Brien, nd E. B. Seligmnn, Jr Problems of influenz vccine stndrdiztion. Bull. W. H : Turso, N. M., F. A. Pedreir, S. L. Spector, nd G. M. Bernier Effect of vrious methods of removing nonspecific inhibitors to virus hemgglutintion upon serum proteins nd immunoglobulins. Arch. Gesmte Virusforsch. 34: