Comparison of the immune response of the rabbit to virulent and attenuated strains of type 1 poliovirus*

Bull. Org. mond. Sant } 1974, 50, 479-485Bull. Wid Hithi Org. Comparison of the immune response of the rabbit to virulent and attenuated strains of type 1 poliovirus* 0. G. GAUDIN,1 J. VIAC,2 D. THOUVENOT,3 & R. SOHIER 4 Rabbits were immunized with the attenuated LSc, 2alb or the virulent Mahoney strain of type 1 poliovirus, or with a partially purifiedpreparation of the latter. It wasfound that the animals responded in a heterogeneous manner to antigens with a homogeneous virion population, such as LSc,2a,b and, to a lesser extent, the partially purified Mahoney antigen. This heterogeneous immune response permits the preparation of specific antisera for intratypic serodifferentiation tests. However, the unpurified Mahoney strain, which has a heterogeneous virion population, induced a homogeneous immune response; all the antisera had a wide spectrum of antibodies and could not distinguish the wild from the attenuated strain. The differentiation capacity of the specific antisera was found to be linked to either the IgM or the IgG. The possible implications of these findings for human vaccination against poliomyelitis are discussed. The intratypic identification of poliovirus by means of immunologic markers (7, 8, 14) requires the use of strain-specific antisera that differentiate clearly between the " wild " and attenuated strains. It is well known (2, 13) that specific antisera to the attenuated LSc,2a,b strain of type 1 poliovirus are easily prepared, but -the preparation of specific antisera to the wild Mahoney strain usable for the McBride kinetic neutralization test is far more difficult. As a result, some authors have claimed (13) that the Mahoney and LSc,2a1b strains cannot be validly differentiated. We have shown previously (5) that the determina- tion of the Mahoney antigen character in a reverted attenuated vaccine strain may be extremely helpful for identifying strains of poliovirus isolated in cases of post-vaccinal paralysis following oral vaccine administration. In order to be able to perform antigenic analysis by kinetic studies, one must have specific anti-Mahoney antisera that do not cross- react with the LSc,2a1b strain. The present paper will * From the Virology Section, Public Health Laboratory, Lyons, France. I Formerly Chief, Virology Section. Now Professor of Microbiology, Department of Virology, Institut Pasteur d'Algerie, Algiers, Algeria. 'Assistant, Virology Section. Assistant, Department of Bacteriology, Virology, and Immunology, Faculty of Medicine, Lyons, France. ' Director, Virology Section, and Professor of Bacterio- logy, Virology, and Immunology. describe a technique for preparing such antisera and will discuss some of the factors that may influence the immune response to various type 1 poliovirus antigens in the rabbit. MATERIALS AND METHODS Tissue cultures Primary monkey kidney (MK) cells were used (Macaca irus). Viruses The Mahoney strain of type 1 poliovirus was maintained in the laboratory by 23-25 passages in MK tissue cultures. The attenuated LSc,2a,b strain was passaged twice under the same conditions from the SOM+ 1 virus. Chromatography A portion of the Mahoney strain was chromato- graphed on calcium phosphate as described by Gaudin et al. (3). The phosphate buffer used for elution (6) was a 4: 1 mixture of 0.5 M NaH2PO4 and 0.5 M KH2PO4; diluted, this buffer was also used for elution at concentrations of 0.01-0.5 M. The infectious titres of the fractions eluted were determined in plaque-forming units (PFU). The elution diagram was drawn as shown in Fig. 1. The " partially purified " Mahoney antigen used for immunization was a mixture of the three fractions eluted respectively at 0.03, 0.04, and 0.05 M. 3214 - 479 - 0. G. GAUDIN ET AL. Fig. 1. Elution pattern of Mahoney strain. Antigens After preliminary titration, the LSc,2a,b, unpuri- fied Mahoney, and partially purified Mahoney (see above) viruses were either diluted or concentrated by dialysis to yield antigens with a titre of 108 PFU/ml. Immunization Each antigen was used to immunize 6 adult rabbits each weighing 2-2.5 kg. The first step of the immunization procedure, carried out according to Siboni & von Magnus (10), consisted in injecting 7 ml of antigen subcutaneously, 1 ml intradermally at several different sites, and 2 ml intravenously on day 0. A booster dose (2 ml intravenously plus 1 ml subcutaneously) was given on day +35. The animals were bled on day 0 prior to immunization and again on days +3, +7, +12, +18, +39, and +45. Kinetic neutralization tests These tests were performed according to a modifi- cation of the McBride technique (7) described else- where (4). 8 U of complement was added to the virus-antiserum mixture after the determination of 1 U of complement by the standard complement fixation test. (The addition of this amount of complement has been shown (11) to be necessary for good reproducibility of the McBride test in heterolo- gous systems.) The virus dilution was-chosen so as to contain 10k-106 PFU/ml. Antisera were diluted so as to be able to neutralize 102 PFU of homologous virus in 10 min. The NK values were calculated with the formula of McBride (7). Classical neutralization tests Monolayers of primary MK cells in culture tubes were used. The maintenance medium was Parker No. 199. The virus-antiserum system was completely homologous. Virus was used at 100 TCID50. The neutralization process took place at room tempera- ture for 1 h. The test was read after 7 days of incubation at 37°C. Fractionation of antisera on sucrose gradients 27 LSc,2a1b antisera were separated on sucrose gradients of 12-33%. Sucrose was dissolved in phosphate buffer solution without Ca++ or Mg++. The volume of the gradient was 8 ml; the volume of diluted antiserum added was 0.3 ml. The tubes were spun (50 000 rev/min) on an MSE superspeed centrifuge, 10 x 10 rotor, for 17 h at 4°C (120 000g). The fractions were collected in volumes of 8 drops and tested for protein contents, IgM and IgG (by immunodiffusion), and neutralizing activity. RESULTS Potency of antisera Antiserum potency was evaluated by the classical neutralization test. Table 1 shows the titres of the individual antisera obtained at each bleeding, the mean titre for each bleeding, and the variance. Antibody titres were lower in antisera to unpurified Mahoney than in antisera to LSc,2a1b but the immune responses of the animals were more homo- geneous, i.e., the variance was smaller (a2 = 0.01-0.08). With the partially purified Mahoney virus, theboosterdosewasneeded to obtainahigherantibody response, which was more homogeneous (a2 = 0.01). When the LSc,2a1b strain was used, however, the booster dose was followed by a heterogeneous response (a2 = 0.23 and 0.21). Strain specificity (differentiation capacity) ofantisera We consider an antiserum to be specific if, when diluted to more than 1: 5, it neutralizes in 10 min 102 PFU of homologous virus and less than 101 PFU of heterologous virus. Under these conditions, a specific antiserum will yield an NK value of less than 50 against heterologous virus. On the basis of this criterion, the antisera obtained can be classified according to whether or not they are specific (Table 2). In conformity with previous reports, the preparation of strain-specific Mahoney antisera was found to be far more difficult than the 480 RABBIT RESPONSE TO POLIOVIRUS Table 1. Potency of antisera * Rabbit Antigen Bleeding 1 2 3 4 5 6 m a2 Mahoney 4 (day + 12) 2.50 2.05 2.80 2.80 2.50 2.80 2.58 0.09(unpurified) 5 (day + 18) 2.65 2.35 2.65 2.95 2.95 2.80 2.73 0.05 6 (day + 39) a 3.40 - 3.25 3.50 3.55 - 3.43 0.02 7 (day + 45) a 3.80 3.60 3.30 3.60 3.60 3.80 3.62 0.03 Mahoney 4 (day + 12) 1.45 1.75 2.35 2.35 2.05 2.20 2.03 0.13(partially purified) 5 (day + 18) 1.45 1.90 2.35 2.35 1.75 2.50 2.05 0.17 6 (day + 39) a 3.20 3.25 3.20 3.40 3.40 3.20 3.28 0.01 7 (day + 45) a 3.25 3.20 3.40 3.50 3.40 3.30 3.34 0.01 LSc,2a,b 4 (day + 12) 3.61 3.01 3.01 2.85 3.15 3.31 3.16 0.07 5 (day + 18) 3.15 3.31 3.15 2.85 3.31 - 3.15 0.04 6 (day + 39) a 4.51 4.51 4.35 3.75 3.45 - 4.11 0.24 7 (day + 45) a 4.51 4.51 4.35 4.51 3.45 3.75 4.18 0.21 Antibody titres are expressed as log. 10. They were determined by the classical neutralization test using homologous virus. a After booster dose (day + 35). preparation of specific antisera to LSc,2a1b, but it was nevertheless feasible. As would be expected, the fractions of the Mahoney viral population that elute at low molarity showed less cross-reactivity with LSc,2a1b than the total unpurified virus. Kinetics of the differentiating antibodies Fig. 2 illustrates the kinetics of the specificity (differentiation capacity) of the antisera obtained in the course of immunization. The figure underscores Table 2. Specificity (differentiation capacity) of antisera* No. of strain-specificAntigen used Total no. of (differentiating) for immunization antisera antisera a LSc, 2alb 36 11 Mahoney (unpurified) 36 0 Mahoney (partially purified) 23 3 * Determined by the kinetic neutralization test. a Defined as having an NK value of less than 50 against heterolo- gous virus (NK = 100 against homologous virus). the relative ease with which specific LSc,2a1b antisera can be obtained, as mentioned earlier. The immune response was far more heterogeneous with the attenuated strain. The NK values obtained for each of the three heterologous systems are shown in Table 3. On the basis of these data, an attempt was made to determine, for each group of antisera, the probability that the immune response had been influenced by two factors-time after immunization and individual variability. The results of an analysis of variance (9) of the NK values in terms of time and individual animal response are summarized in Table 4. It will be noted that the results for the antisera to partially purified Mahoney antigen are highly significant for both factors. Time after immunization thus appears to be a significant factor in the variance of the immune response to Mahoney antigens. In so far as the selected LSc,2a,b and the partially purified Mahoney antigens are concerned, however, the response is dependent on the individual animal's immunologic behaviour; in other words, the animal responds well or poorly from the outset. This supports the findings reported above under " Strain specificity of antisera ". 481 0. 0. GAUDIN ET AL. Fig. 2. Kinetics of specificity (differentiation capacity) in three types of antisera raised in rabbits. Antisera were considered to be specific if they had an NK of less than 50 against heterologous virus. Correlation ofspecificity (differentiation capacity) of antisera with immunoglobulin class In an attempt to determine the respective roles of the IgM and IgG components in antiserum specifi- city (differentiation capacity), the two classes of immunoglobulins were separated in 27 LSc,2a1b antisera. Fig. 3 shows the sucrose gradient curve and the fractions collected for the kinetic neutralization test. Table 5 permits a comparison of the NK values of total LSc,2a,b antisera with the NK of their IgG and IgM components. Wherever the IgG have an NK of under 50, the total antiserum is sufficiently specific and can be used as is. In some cases, however, where it is the IgM whose NK is less than 50, no dif- ferentiation capacity can be detected in the whole antiserum (as for rabbits 2 and 4). The reason is presumably that the specificity of the IgM is diluted by the nonspecificity of the IgG, which predominate. DISCUSSION As we have noted elsewhere (12), the immunologic identification of poliovirus strains isolated from vaccinated subjects is more easily performed when kinetic neutralization tests are done with anti- Mahoney antiserum as well as LSc,2a,b antiserum. It NK 100 _ 751 E I I i V 50 f| LSc,2a1b antisera 25 1i 0 NK 100 _ Liliti II T tMahoney antisera50 F 11} |I| (unpurified Mahoney) 25 - 0 _ NK 100 _ 75 -I 750 lITi [i Mahoney antisera50[itE 1 } (partially purified Mahoney) 25 0 days after immunization t 3 7 12 18 35 39 45 first t immunizatioh booster WWO 4OS26 482 483RABBIT RESPONSE TO POLIOVIRUS Table 3. NK values of antisera Bleeding a Rabbit 2 3 4 5 6 7 day + 3 day + 7 day + 12 day + 18 day + 39 day + 45 Immunized 1 67 b 58 b 48 43 33 44 with LSc,2aib 2 46 56 50 58b 81 75 3 58 b 58 b 73 84 72 76 4 62 b 61 b 64 b 71 b 69 66 5 52 47 40 28 25 24 6 70 89b 57 40 39 Immunized with 1 92 b 57 b 72 54 53 51 unpurified Mahoney 2 93 b 84 b 72b 58 61 70 3 93 b 65b 70 54 52 72 4 76b 66 b 70 50 75 68 5 69b 82 b 57 b 58 70 72 6 79b 66 b 65 57 67 64 Immunized with 1 82 52 53 partially purified Mahoney 2 59 59 65 57 3 69 65 81 62 4 74 53 66 59 5 53 38 52 57 6 80 53 41 32 N K against heterologous virus; N K = 100 against homologous virus. Each value shown is the average of three kinetic neutralization tests. a Bleeding 1 was done on day 0 before inoculation. b NK value from one test only. is thus important to be able to prepare Mahoney antisera that do not cross-react with the LSc,2a,b strain. While this is feasible, there are technical difficulties stemming from the heterogeneity of the virion population of the wild strain. From the pres- ent study it is evident that immune responses are more homogeneous, i.e., show greater variance, when the antigen used is a total heterogeneous virus. This has already been demonstrated in the chicken with a type 11 ECHOvirus (4). If, however, animals are Table 4. Significance of two factors in immune response, as assessed by analysis of variance of NK values of three groups of antisera Factor Significance of factor possibly A Antisera Antiseraanfluencing Antisera to unpurified to partiallyvariance to LSc,2aib Mahoney purified Mahoney Time after Not significant at 2.5 % Significant at 0.1 % prob- Significant at 0.1 % prob- immunization probability level ability level ability level Individual Significant at 1.0 % Not significant at 2.5 % Significant at 0.1 % prob- animal probability level probability level ability level 0. G. GAUDIN ET AL. Fig. 3. Optical density of the fractions of an LSc,2ai b antiserum obtained by ultracentrifugation on sucrose gradients. The IgM are in fractions 1-5, the IgG in fractions 6-10. immunized with only a fraction of the total virus, e.g., with partially purified Mahoney virus or the attenuated LSc,2a,b strain, some animals will pro- duce antibodies selectively against the virions in- jected. The attenuated LSc,2a1b strain elicits the most selective antibody response in this respect. The selectivity of the immune response depends on the individual animal and is not explained by the dif- ferent classes of immunoglobulins secreted during immunization. These findings in the rabbit might shed some light on the special immunogenicity of the two virus strains and might possibly be extrapolated to man. The question that arises concerns the antibody spectrum in vaccinated subjects. As shown by Bottiger (1) for type 3 poliovirus, the response of vaccinated subjects may vary depending on the nature of the vaccine administered. The antigenic structure of the LSc,2a1b strain seems to be simpler than that of the wild Mahoney strain because it has been selected and is homogeneous. If this is true, however, vaccinated subjects will respond in a heterogeneous manner, and some will develop only a narrow antibody spectrum. Thus, the partial reversion of the vaccine strain in the intestinal tract Table 5. Roles of IgM and IgG in the specificity (differentiation capacity) of LSc,2alb antisera' N K values a Rabbit Bleeding Total antiserum 1gM IgG 1 4 48 49 58 5 43 84 43 6 33 27 32 7 44 21 36 2 3 55 57 38 4 50 46 56 5 58 42 50 6 81 35 70 7 75 45 79 3 4 73 65 82 5 84 76 95 6 72 74 72 7 76 79 86 4 4 64 73 69 5 71 b 69 6 69 32 65 7 66 33 61 5 3 47 49 b 4 40 50 44 5 28 54 37 6 25 b 23 7 24 b 24 6 3 70 77 92 4 89 89 81 5 57 60 53 7 39 29 31 Determined by the kinetic neutralization test. a Specificity (differentiation capacity) is indicated by an NK value of less than 50. b Not determined. appears to be a favourable event. As partial rever- sion causes the vaccine to resemble more closely its parent strain, it would hence act to widen the spectrum of antibodies secreted in the course of immunization and would better enable the vac- cinated subject to counteract a wild poliovirus. O.D. 484 RABBIT RESPONSE TO POLIOVIRUS 485 REiSUME COMPARAISON DE LA REPONSE IMMUNITAIRE DU LAPIN A DES SOUCHES VIRULENTES OU ATTENUEES DE POLIOVIRUS DE TYPE 1 L'identification dite intratypique des virus poliomyeli- tiques par les methodes immunologiques exige qu'on dispose d'immunoserums permettant d'obtenir une diffe- renciation nette des souches dites (i sauvages * et des souches attenuees. D'apres les travaux publies, il est difficile d'obtenir des immunoserums anti-virus polio- myelitiques type 1 sauvages (Mahoney) utilisables pour la mise en ceuvre du test de McBride, alors que les serums anti-souches attenuees (LSc,2a,b) sont aisement obtenus. Des immunoserums ont ete prepares sur trois lots de lapins avec respectivement une suspension de virus LSc, de virus Mahoney et une suspension de virus Mahoney selectionnee par chromatographie. Les lapins immunises avec la souche LSc et la souche Mahoney selectionnee fournissent des immunoserums de qualite differente selon les animaux, mais dont certains (31 % pour la souche LSc et 13% pour la souche Mahoney selectionnee) permettent la mise en ceuvre d'un test de McBride satisfaisant. Par contre, les lapins immunises avec la suspension de virus Mahoney non purifiee ne fournissent aucun serum utili- sable, en raison du trop large spectre des anticorps elabores. L'activite anticorps permettant la differenciation des souches attenuees et sauvages dans le test de McBride est trouvee dans la classe des immunoglobulines M et G. Toutefois, si tous les serums dont l'activite anticorps permettant la differenciation se trouve dans la classe des IgG sont utilisables tels quels, il est possible, a partir de ceux qui n'ont pas cette activite differenciatrice asso- ciee aux IgG, d'isoler des IgM ayant cette activite. 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