Some factors determining differences in the antigenicity of vaccinia virus strains*

Bull. Org. mond. Sante 1972, 46, 159-163 Bull. Wid Hith Org. Some factors determining differences in the antigenicity of vaccinia virus strains* S. S. MARENNIKOVA,1 K. L. tIMIgKJAN,2 L. S. 9ENKMAN,3 & G. R. MACEVIC 2 The causes determining differences in the antigenicity of different strains of vaccinia virus were investigated in rabbits. A relationship between the antigenicity of a strain and its capacity to multiply in immunized animals was established. Inactivated preparations of the viruses under investigation were capable of stimulating the production of virus neutralizing antibody in vaccinated rabbits. The virus showed the greatest accumulation and the longest persistence in animals vaccinated with the white clone Tashkent strain; similar results were obtained with the B-51 and Lister Institute strains. Strain EM-63 showed the smallest capacity to multiply. Previous studies in the Smallpox Prophylaxis Laboratory (Unanov et al., 1965; Taspulatov & Marennikova, 1968) and elsewhere (Noorda, 1964; Jaroslavskaja, 1968) have revealed differences in the antigenicity of various strains of vaccinia virus and have shown that such differences depend largely on the virulence of the vaccines. However, we know of no specific investigations of the causes underlying these differences. The aim of the present study was to investigate the possible factors that determine differences in the antigenicity of four vaccinia virus strains. MATERIALS AND METHODS Virus strains The source of the study material was dry smallpox vaccines prepared from the following strains: EM-63, lot 41; Byelorussian Epidemiology and Microbiology Research Institute strain B-51, lot 499; the Lister In- stitute, England, lot 4079; white clone Tashkent, lot 716. All these vaccines had been purified by treatment with a fluorinated hydrocarbon (Freon). The Lister Institute strain was adapted to calf skin (L-IVP) in the Institute for Research on Virus Prepa- rations, Moscow. * From the Smallpox Prophylaxis Laboratory, Institute for Research on Virus Preparations, Moscow, USSR (WHO International Reference Centre for Smallpox). 1 Chief, Smallpox Prophylaxis Laboratory. 2Junior Scientific Officer, Smallpox Prophylaxis Labora- tory. ' Aspirantura Student, Smallpox Prophylaxis Laboratory. Storage of virus suspensions The smallpox vaccines were stored at a temperature of - 40°C until used. Their potencies were tested by repeated titrations on the chorioallantoic membrane of 12-day-old chick embryos by the method of pock counting. Immunization of animals and collection of materials for the tests White-skinned chinchilla rabbits were used in the experiments and were immunized in one of two ways: by scarification of an area of skin 2.5 by 2.5 cm above the right scapula or by intradermal inoculation. The immunizing dose employed was 4-8 x 106 pock-forming units (PFU) contained in 0.1 ml of vaccine suspension. Four groups of rabbits were vaccinated by the scarification method, a dif- ferent vaccine strain being used for each group. Two rabbits from each group were killed 6 and 24 hours, and 2, 3, 4, 5, 6, 7, and 10 days after vaccina- tion, and their regional lymph nodes were collected. At similar intervals, beginning 2 days after vaccina- tion, scrapings were taken of lesions on the scarified area of skin. Four other groups of rabbits were vac- cinated intradermally with the different vaccines; two rabbits from each group were killed 4 hours, and 2, 3, and 4 days after vaccination and the areas of infiltrated skin were excised. From each specimen, a 10% (by weight) homogenate was prepared in medium 199 and the amount of virus was determined by titration on 12-day-old chick embryo chorio- allantoic membranes. 2789 -159- S. S. MARENNKOVA AND OTHERS In order to investigate the antigenicity of the vac- cine strains, groups of 3-8 rabbits were vaccinated by scarification using 0.1-ml doses of live vaccine con- taining 106PFU; other groups of rabbits were inoculated intravenously with 2-ml doses of inacti- vated vaccine. Vaccine suspensions containing 2.0-7.6 x 108PFU were inactivated by y-irradiation from a 60Co source in doses of 1.5 mrad. The com- pleteness of inactivation was tested by blind passage on chick embryo chorioallantoic membranes. The animals were bled 3-4 weeks after vaccination and their sera were tested by haemagglutination- inhibition using 4 units of haemagglutinin, and by virus neutralizing tests, with 50% pock reduction on the chorioallantoic membrane of 12-day-old chick embryos. RESULTS The results of determinations of the intensity of antibody production in rabbits after immunization by scarification are given in Table 1. It can be seen that there was variation in the antigenicity of the Table 1. Antigenicity of the four vaccine strains in rabbits vaccinated by scarification Geometric mean titre Immunizing 4 weeks after immunization Strain dose(PFU) Antihaem- Virus neutra- agglutinins lizing antibody EM-63 8.0 x 1 06 1:8 1:158 B-51 4.0 x 106 1: 23 1 :525 Lister Institute 7.6 x 106 1: 52 1: 832 White clone Tashkent 6.0 x 106 1: 52 1: 891 vaccine strains under study. The most marked anti- body responses were achieved after immunization with the white clone Tashkent and the Lister In- stitute strains. In the next series of experiments, the capacity of the strains to multiply in rabbits after vaccination by scarification or intradermal inoculation was tested, approximately equal doses of virus being used. Results from one series of experiments are shown in Fig. 1 and Table 2. It may be seen from these data that after vaccina- tion by both intradermal inoculation and scarifica- tion, certain differences can be detected in the capacities of the strains to multiply under these con- ditions. Thus, after application of the vaccine to scarified skin (Fig. 1), the highest virus titres were obtained in skin scrapings from the rabbits inocu- lated with the white clone Tashkent and the Lister Institute strains. The titre of the EM-63 strain in scrapings taken at the peak of reaction was lower, and declined much more quickly when the reaction subsided, than that of the other viruses. The B-51 strain was intermediate in its capacity to multiply. After intradermal inoculation (Table 2), the capacity of the strains to multiply was about the same as that following scarification. Once again, the maximum titre of strain EM-63 and its decline occurred earlier than in the other strains. A further series of experiments was carried out to determine the capacity of the various strains to invade and accumulate in the regional lymph nodes after vaccination by scarification. Fig. 2 shows that the most marked invasive capacity was found in the B-51 and the white clone Tashkent strains, which were detected in the lymph nodes 1 or more days before the two other strains. The curves for virus content of strain B-51 and white clone Tashkent in the regional lymph nodes were closely similar. Table 2. Accumulation of virus in the skin of rabbits inoculated intradermally with four vaccine strains 1Inoculation Amount of virus homogenates of infiltrated skin Strain dose (PFU/ (PFU/0.1 ml) after: 0.1 ml) | 4 hours 48 hours 72 hours 96 hours EM-63 2.7 x 106 < 10 5.4 x 105 1.4 x 105 3.5 x 103 B-51 2.5 x 106 < 10 3.2 x 105 1.9 x 106 1.2 x 10O Lister Institute 3.6 x 106 < 10 1.9 x 106 3.3 x 106 3.0 x 10I White clone Tashkent 3.7 x 106 < 10 1.6 x 106 1.4 x 107 2.6 x 106 160 ANTIGENICITY OF VACCINIA VIRUS STRAINS =1 -._ U- 0- 0) 00 0 -J 4 5 Time after vaccination (days) 161 10 Wtio 10774 Fig. 1. Accumulation of virus in the skin of rabbits vaccinated by scarification and intradermal inoculation. 1- U- a- a) 00 Time after vaccination (hours, days) WHO 10773 Fig. 2. Multiplication of virus in the regional lymph nodes of rabbits vaccinated by scarification. S. S. MARENNIKOVA AND OTHERS The white clone Tashkent strain, which had the greatest capacity to multiply, gave the maximum titre and persisted in the regional lymph nodes for the longest period (up to 7 days). The lowest capacity to multiply was found in the EM-63 strain, which could be found in regional lymph nodes for only 2 days (3-4 days after inoculation) and in con- siderably lower titres than the other strains. The Lister Institute strain began to accumulate in the regional lymph nodes at the same time as the EM-63 strain but persisted there for longer periods and in higher titres. In order to exclude the effect of additional anti- genic stimulation and other factors associated with the active multiplication of virus in immunized animals as possible causes of variations in the antigenicity of the various strains, further experi- ments were carried out with virus preparations inactivated by y-irradiation. The results of these experiments (Table 3) indicate that the antigenicity of three inactivated strains-EM-63, Lister Institute, and B-51-did not differ greatly, and the observed differences in the mean virus neutralizing antibody titre are not statistically significant (P > 0.1). Table 3. Antigenicity of four inactivated vaccine preparations Immunizing No. of Titre of virusdose ails neutralizingStrain (PFU/2 ml in theis antibodyzbefore inac- inrteuativation) group 3 weeks 4 weeks EM-63 7.0 x 108 7 1 :200 1 :400 B-51 2.0 x 1 08 3 1: 160 1: 250 Lister Institute 6.6 x 103 8 1 :160 1: 250 White clone Tashkent 7.6 x 1 08 7 1: 630 1: 800 At the same time, it should be noted that the titres of virus neutralizing antibody developing in response to vaccination with an inactivated prepara- tion of white clone Tashkent strain were higher than those obtained with the other three strains. These differences are statistically significant (P <0.01). DISCUSSION The present studies demonstrated a close associa- tion between the antigenicity of the various strains of vaccinia virus and their capacity to multiply in vac- cinated rabbits. The higher the rate of virus multi- plication in the skin and regional lymph nodes, the greater the active antibody response. These results confirm the observations made by Morozov & Konstantinova (1961) and by, Solov'ev and Mast- jukova (1956) that the antigenic and immunogenic properties of smallpox vaccines are determined not only by the number of infectious virus particles but also by their virulence. Investigation of the antibody response to vaccina- tion with inactivated preparations showed that the white clone Tashkent strain produced significantly higher levels of virus neutralizing antibody than the three other strains. This suggests that the intensity of antigenic stimulation may depend not only upon the capacity of the virus to multiply in the host but also on certain other properties. It is evident, how- ever, that the importance of these other factors is not very great, since inactivated preparations of the EM-63, B-51, and Lister Institute strains showed no significant differences in the levels of virus neutraliz- ing antibody produced, whereas live cultures of these three strains differed markedly from one another in their antigenicity. CONCLUSIONS Experiments in rabbits revealed differences in the antigenicity of four vaccinia virus strains-namely, EM-63, B-51, Lister Institute, and white clone Tashkent. The strains studied varied in their capacities to accumulate in the skin after vaccination by scarifica- tion of the skin or intradermal inoculation. The rate at which the four strains penetrated and multiplied in regional lymph nodes also varied. A direct correlation was established between the intensity of the antigenic response and the rate of multiplication of the different strains. Inactivated preparations of all four strains were capable of inducing the production of virus neutraliz- ing antibody in rabbits. 162 ANTIGENICITY OF VACCINIA VIRUS STRAINS 163 RESUME' QUELQUES FACTEURS RESPONSABLES DES DIFFERENCES D'ANTIGtNICITE ENTRE SOUCHES DE VIRUS VACCINAL Quatre lots differents de vaccin antivariolique ont servi a etudier les facteurs determinant les diff6rences d'anti- genicite entre diverses souches de virus vaccinal. Chaque vaccin a ete administre 'a des lapins soit par scarification, soit par voie intradermique. La r6ponse immunitaire (production d'anticorps inhibant l'hemag- glutination ou neutralisants) a varie suivant les vaccins inocules, les vaccins de Tachkent et du Lister Institute temoignant du pouvoir antigenique le plus eleve. On a aussi recherch6 dans quelle mesure les virus se multi- pliaient chez l'animal d'experience en prelevant par grattage ou par excision des fragments de tissus aux endroits de scarification ou d'injection. Dans les produits de grattage, les titres maximaux de virus ont ete deceles apres introduction par scarification des vaccins de Tachkent et du Lister Institute. Des r6sultats analogues ont 6td obtenus apres injection des preparations par voie intradermique. Enfin, on a recherch6 l'aptitude des differentes souches vaccinales a envahir les ganglions lymphatiques regionaux et a s'y multiplier. Le virus B-51 et le virus de Tachkent y ont ete deceles un jour ou plus avant les autres souches. La presente etude montre qu'il existe un lien tres etroit entre 1'antigenicit6 des souches de virus vaccinal et leur aptitude a se multiplier chez I'animal vaccin6. Cependant, d'autres facteurs interviennent, les divers vaccins utilises au cours de cette dtude ayant conserv6 leur pouvoir de susciter la production d'anticorps apres avoir ete inac- tiv6s par irradiation gamma. REFERENCES Cimiskjan, K. L. et al. (1970) Vop. Virus (in press) Jaroslavskaja, N. V. (1968) Collected papers of the TarasevicY State Control Institute, Moscow, Medgiz, p. 129 Morozov, M. A. & Konstantinova, V. I. (1961) i. Mikro- biol. (Mosk.), 4, 79 Noordaa, J. van (1964) Primary vaccination ofadults with an attenuated vaccinia virus, The Hague, van Stockum Solov'ev, V. D. & Mastjukova, Ju. N. (1956) Vop. virus, 1, 23 Taspulatov, G. M. & Marennikova, S. S. (1968) Materials of the All-Union Conference on Problems of Safety of Virus Vaccines and Vaccination Prophylaxis of Influenza, Smallpox, and Measles, and the Symposium on Genetics and Biochemistry of Human Viruses, Moscow, Medgiz, p. 32 Unanov, S. S. et al. (1965) Materials of the Tbilisi Scien- tific/Practical Conference on the Problems of Smallpox Prophylaxis and Vaccination, Moscow, Medgiz, p. 7