ATTENUATED REVERTANT SEROTYPE 1 MAREK'S DISEASE VACCINE

Abstract: A Marek's disease vaccine comprising an attenuated revertant virus derived as a clone of Md11/75/R2 or an antigenic component of the virus is characterized by a markedly reduced pathogenicity compared to the parent virus, without a comparable loss in immunogenicity. Attenuated revertants of the invention are exemplified by Md11/75/R2/23 and Md11/75/R2/29. These viruses or immunogenic components thereof can be formulated into monovalent and polyvalent vaccines.

Description: Attenuated evertant Seirotype 1 Marek's Disease Vaccine Εackar-mΔ of the mventiαn Field of -flt T^ *nion Marek's i-.mfie (MD), a highly prevalent and itαportant ly_Tτhcpr * ol__fe_ative disease of chickens , is ccπtrolled in ccππercial chickens by live virus vaccines consisting of attenuated or naturall aviruleπt MD-related hexpesvixuses. Although vaccinatictri μruj dius have been cexisidered highly effective overall, the poultry industry centimes to experience losses due to MD. Given the tendency of MD virus to bernnp sere virulent with time coupled with the econαnic pressures _xτ___πxr_ing the poultry jjndustry, there is still a LIUHJ incentive to develop even more efficacious products that will protec better in the face of early challenge with very virulent field strains. This j veπtion relates to a novel vaccine against MD which does in fact provide ssperiσr protection over the existing oconercial vaccines. ____g?rigtt9B of ttw Prior art _3_tere are three distinct serotypes of MD virus found in chickens: (1) sen-type 1, the onoogenic form responsible far the disease, imlnrling high- and lσw-virulencse MD virus and their attenuated variants; (2) serotype 2, a ncnαnoogenic MD virus; and (3) serotype 3, ____-p_βvirus of turkeys (HVT). The prututype MD v_ * ccine consists of the serotype 3 virus originally isolated from turkeys as reported in Witter et al. I [Am. J. Vβe. Res. 31: 525-538 (1970)] and Qkazaki et al., U.S. Patent No. 3,642,574. Its lack of onccgenicity, εβlf-liπ ting infection, good replication in vivo and in vitro , av2_i_L__bility as cell-free and cell-associated preparations, and high protective efficacy have established HVT as a standard for MD vaccines th__oug__out the world. A (_-.iiii-.H_ly used strain of HVT is P026. Vaccines produced from the naturally avirulent SB-1 strain [Scha et al., J. Nad . Cancer Insc. 60: 1075-1082 (1978) and U.S. Patent No. 4,160,024], an isolate of a serotype 2 MD virus, have bee licensed in the United States since 1984. The SB-1 strain is poorly protective against the highly virulent M__5 str__in. It is .usually used in ccnbination with HVT as a bivalent -vaccine since the two viruses together pr ___ce greater protection than does either one alone [Schat et al., Avian Pachol . U: 593-606 (1982); Witter, Avian p&chol . 11: 49-62 (1982), herein inccxporated by reference]. This p___n___enon has been ermpf- "protective synergisn. n The SB-1 + HVT bivalent vaccine represents about 18% of the United States sarket far MD vaccines at present and is considered to be the most efficacious of the -various MD products available. However, sporadic losses occur despite its use. __nσther MD vaccine prc»:-uc_d from strain CVT988 done C (CVI988/C) has recently been licensed for rrπwy ri.. use in the United States. This vaccine is a mildly virulent serotype 1 MD virus attenuated by serial rjnfvstrj in tifwne culture and has been rgxn ed by deBner et al. [Avian Dis. 30: 276-283 (1986)]. An espe_rin__nt__L vaccine derived frαa Mail, a very -virulent serotype 1 MD field solate, was reported by Witter, supra. Mill was attenuated by 75 serial passages in cell c_ t_tre, and the resultant vaccine designated M_U__/75C. This vaccine has been shown to provide good protection against challenge with M__5 and most other highly virulent MD viruses tested; but it was less efficacious against challenge with the JIV102W strain, a prototype MD virus effectively protected against fay HVT and SB-1 vaccines. Furthermore, its efficacy was consistently lower in chicks with HVT antibody. In U.S. Patent No. 4,895,717, Witter discloses a revertaπt derivative of M____1/75C which has been referred to as M__ L/75CR2. M-_1_L 75Q/R2 was shewn to be superior to several other monσvalent vaccines and was the equal of a bivalent (HVT + SB-1) vaccine [Witter, Avian Dis. 31: 752-765 (1987)]. However, the inherent pathogenicity of serotype 1 viruses and the potential of attenuated strains to revert to greater pathogenicity (Witter et ad., Avian Pat±ol . 13: 75-92 (1984)] are factors to be considered in the licensing of such products. Thus, although HVT, SB-1, CVI988/C, M____L/75C, and M__11/75C/R2 ar all effective against certain MD -viruses, none of these vaccines protect optimally against all MD challenge viruses in all chickens. In an effort to avert any large-scale outbreaks of MD in the future, the search for improved vaccines has continued. An ideal serotype 1 vaccine probably should be derived from a very virulent MDV pathotype to optimize honology with the most pathogenic field strains likely to be encountered. It should be attenuated to the point of safety when applied to < πnp-τ:ia1 chicikens and either should be incapable of reversion or should not spread horizontally. It -shci-ld replicate readily in vitro and in vivo. Finally, it sshould induce a high level of protection against early challenge with very virulent field srtrains in the presence of maternal azitibodies against any of the viral serotypes. ff ._mrT.TT f * n JW"**"* I have now discovered two novel MD vaccines derived as clones of }_11/75C KZ. The clones are designated Mm/75/R2/23 and M__U 75 B2/29 and were selected from a pool of about 40 M_ 11/75C/R2 clones evaluated. The subject clines are uniquely characterized by a markedly reduced pathogenicity compared to the parent virus, without a comparable loss in immunogenicity. In experiments with a highly susceptible line of chickens, both clones failed to induce gross lesions of MD. Moreover, both clones induced immunity against challenge with very virulent strains of MDV that were nearly comparable to that of the parent virus. The observed level of immunity was not significantly different from the immunity provided by the bivalent (HVT + SB-1) vaccine. In accordance with this discovery, it is an object of the invention to provide a novel, highly protective, serotype 1 vaccine against MD in chickens. It is also an object of the invention to provide both onovalent and polyvalent vaccines against MD which are more efficacious than those presently in commercial use. it is another object of the invention to improve the viability and productivity of chickens, particularly broilers and layers, and to reduce economic losses in the poultry industry caused by MD. Another object of the invention is to provide an MD vaccine which is characterized by a rapid rate of replication, and thereby enhanced protective efficacy and in vitro production efficiency. Other objects and advantages of this invention will become readily apparent from the ensuing description. Deposit of Biological Material Attenuated revertant serotype 1 Marek's disease virus clones Mdll/75C/R2/23 and Mdll/75C/R2/29 have been deposited under the provisions of the Budapest Treaty in the American Type Culture Collection in Rockville, MD on May 22, 1991, and have been assigned Accession Nos. ATCC VR2328 and ATCC VR2327, respectively. Detailed DescE-Jt,*"-"- * <->f » * Invention "Vaccine" is defined herein in its broad sense to refer to any type of biological agent in an adminJLstratable form capable of stimulating an irrπαne response in an animal inoculated with the vaccine. For purposes of this invention, the vaccine may ■ ■ «-.n*rig either the virus itself or an iππunogenic (antigenic) <-nnxH__aιL of the virus. The vaccine may also be produced frαn a vector having inserted therein a gene which encodes an isnυnogenic cαrpcneπt of t virus. The term "revertant" is intended to refer to a subculture of an attenuated virus, the subculture being characterized by increased virulence and increased in vivo replication as ccαpared to the attenuated form. The term is used herein generically to enoαtπpass both true revertants and apparent revertants, the latter being derived from a population of viral particles rather than frαn a cloned virus. As previously mentioned, the M__L275QR2/23 and M___V75CP2/29 clones are derived from the M311/75ςP2 (R2) revertant virus of Witter (U.S. Patent No. 4,895,717), supra. They were obtained by serially passing R2 in chicken embryo fibroblasts (CEF) , and at passage 100 isolating cell-free virus. As with the R2, there appear to be a positive correlation between increase in replicative ability in vivo and increase in protectivity against challenge viruses. Thus, replicative rate may be used as an indicator of protectivity against challenge viruses. As derivatives of the very virulent Mdll strain, both R2 clones should have good honology with other -viruses of this pathotype. The are attenuated to the point where, except on rare occasion, no gross or histαlogical lesions are induced even in genetically susceptible, maternal antibody negative chickens. Both reverted to greater virulence upon backpassage in chickens, but the danger associated with such reversion is __inimized by the failure of either virus to to unj ioculated chicCkens. The concern for pa * t±__*genicity of the clones is especially minimized far chickens protected either by xaaternal aπtihπiies or by genetic resistance. The viruses replicate well in vitro and induce readily detectable viremias in vivo . Both induce levels of protection against early challenge with very virulent MD viruses that exceed that of HVT alone and do not differ significantly from those of HVT + SB-1 bivalent αr R2 vaccines. A cell-associated vaccine can be prepared directly frαn in vitro culture cloning medium. To prepare cell-free virus inocula, cells frαn infected host tissue or cell culture are sonicated αr otherwise disrupted. The cellular debris is removed by ce_τtri____gation and the atrifugate .recovered as the inoculum. For a cell-free inoculum, the virus can be isolated as previously described. It is also an enbodimeπt of the invention to prepare vaccines from the killed virus αr frαn __n___n_genic ccqponeπts separated frαn the virus. Far example, a subunit vaccine can be prepared by sep__rating frαn the killed virus one αr more puri ied viral proteins identified as having i_ιτιmogenic properties. It is esvisioned within the aπbit of the invention that the gene or genes encoding the jjαπunogenic csxpcnent αr cαπponeπts respcnsible far the protective ability of the cloned -viruses could be inserted into a suitable vector system by xreccnbinant t____hnigues as known in the art. The __βthcdology involving _r__x__binant DNA t_e__hnigues has now becαne routine in science and has been successfully _k___c__strated in analogous applications [E. Paoletti et al., Proc. Nacl. Acad. Sci. U.S.A. 81: 193-197 (1984)]. Specifically, the process would first involve the identification of proteins αr other cc_πponents of the clone that are critical to the induction of protective iπmmity. Next, specific ∑regiαns of the viral genαne (genes) along with an endogenous pzrαnoters would be identified and c_____acterized through mapping with restriction endαnucleases and det-er ination of the • πuclβσtide sequences. The identified gene αr genes would then be spliced into expression vectors such as -V.fτteria1 pl____nids (to prndι,iCT- a killed protein product) or live viruses such as avian herpesvi uses αr avian pcxviruses (to piTrlir-p a live rexxEαbύiant DMA vaccine virus) . Other types of expression vectors could also be used. Once properly uuns ucted with the necessary p iπuler sequences, the expression vector will pn»live the product of the inserted gene; namely, the critical ύnυnizing protein αr proteins o the clone. If produced by a vector grown in vitro, the __a___r_i__ing protein will be obtained frαn the culture medium, purified, and used with appropriate adjuvants and diluents as a killed -vaccine far the i____ur____atian of chickens. Other vectors, chosen for their natural i___fectivity far ctiickens, will be i__oa_late directly into ___i___ens as a reccαbinut live virus vaccine. The vaccine will then pro___oβ the __n__ιnizing protein in vivo, thus causing protection drectly and without the need far additional ____xa_lations. Thus, it is apparent frαn the above discussion that viral agents contemplated within the scope of the " invention ct-iginating frαn the R2/23 and R2/29 clones include both ceil-free and cell-associated virus and also live and killed virus. Also cxxtteπplated as the vira agent are .antigens which are effective to elicit an ___n_tne response in chidoans to Marek's difMviise virus, whether those antigens have been derived directly frαn the R2/23 αr R2/29 clones, αr expressed b a recaΛ.iveaπt virus as described, supra. The viral agent is prepared for adm__-____t_ " aticn by fdπaαlatiαn in an effective i_π___ι____at_ic_n dosage with a p_______aceutically acceptable carrier αr diluent, such as physiological saline αr tiissne c_dture medium. The expression "effective iπmuni aticm dosage" is defined as being that amount which will induce _______nity in a chicken arp.inst challenge by a -virulent strain of MD. _____unity is considered as having been ___duced in a population of chickens when the level of pzn-itecticn far the population is signi_ficantly higher than that of an uπvaccinated control group. One measure of the level of protecticn is the protective index (PI), which is πalmlated as the MD in unvacsinated, MD virus challenged controls minus the MD in -vaccinated, MD virus challenged groups, and the difference divi ed by the percent MD in _ατvaccinated, MD virus challenged controls, with the result multiplied by 100. Typically, the vaccine will contain at least about 1500 PFU (plaque-forming units) of the virus, and preferably between 2000 and 5000 PHJ. The vaccine can be effectively administered aπytirne. after the chicken attains iiiimi__xιπpetence, which is at about the 18th day of incubation (3 days prehatch); but it is normally ________ _stered by jncculation within 24-48 hrs after hzEtching. Appropriate adjuvants as known in the art may also be included in the vaccine formulation. In many cases, the vaccinal efficacy can be enhanced by cs___bi____ng the R2/23 αr R2/29 with other viral agents into bivalent αr polyvalent vaccines. The following examples are intended only to further __ll___rtrate the invention and are not intended to limit the scope of the invention which is defined by the claims. Bnφl 1 Two clones useful as vaccines in aαrordance with this iπveπticn were pn:»Tnred frαnM_U_l/75ςR2 (designated hereafter as R2) by serially passing the R2 revertant in CEF. At passage 100, cell-free virus was iftolafed frαn sonicated, heavily-infected CEF culture and used to initiate 30 clones, each of which was grown into stocks far _rv_ uaticn at passage 101. These clones were designated R2/1-E2/30. 2 The 30 clones obtained in Exaπple 1 were split into groups and each evaluated in one of six "trials far inducement of v__r__nia, gross lesions and histological lesions in chickens. The chickens used fo this __p___imeπt were F. progeny (15x7) of Regional Poultry __-d_oratαry line 15I« males and line 7. females. The parent females were unvaccinated breeder hens that were free of antibodies to MD virus and HVT and the progeny chickens were cxxsidered negative far maternal antibodies (ab-) . The chickens wer inoculated int____bdcmin__lly with 20,000 EFU of virus at 1 day of age. After being held in isolators far 8 wks, they were killed and _____oined for gross lesions. Blood samples were obtained for me____ring viremia, and i,«w»- i- were examined far histological lesions. Controls for each trial included two lots inoculated with R2 (used at the 87th passage) , one lot inoculated with Md5, and one uninoculated lot. The Md5 virus stock was a suspension of infected tiss__a^culture cells cryopreserved with 10% d_____thy___ulfoκide at -196* C. The results of the evaluations are given in Table I below. Nine preparations induced detectable viremia in the absence of gross MD lesions, c____racteristics cxrβidered desirable for candidate -vaccine viruses. The absence of gross lesions was considered significant because the parent R2 vi_-__3-induced gross lesions in one αr more c__ickens in each of 12 replicate trials. ___a__pl« 3 denes R2/13, R2/22, R2/23, and R2/29 were selected for further evaluation as candidate vaccine viruses. Chickens (15x7 ab-) as daac ibod in Example 1 were inoculated _n__^___b_k_____nally with 20,000 PFU virus at 1 day of age. After being held in isolators for 2, 4, 8, αr 18 wks, the chi-doens were killed and eaeamined for viremia, gross lesions, and histological lesions as in Example 2. The reported viremia values are mean PFU counts of two white blood cell (WBC) pools (three birds each) , 10 β buffy coat cells/culture. For each treatment, body weights were determined as the means of all survivor birds at 17 wks, adjusted for sex. The results are reported in Table II. In contrast to the trial in Example 1, clones R2/13 and R2/22 produced gross and histological lesions at frequencies similar to that of the R2 parent virus. However, clone R2/23 induced no gross or microscopic lesions. Clone R2/29 also appeared relatively apathogenic although one chicken died at 17 wks, apparently with gross MD lesions. This chicken was not examined histologically and may have been misdiagnosed, considering the absence of gross or histological lesions in cagemates killed at 18 wks. Both R2/23 and R2/29 induced viremias, but at lower titers than R2 or the other clones. . Body weights of the groups did not differ from that of the uninoculated controls. Clones R2/23 and R2/29 were deposited under the Budapest Treaty, in the American Type Culture Collection in Rockville, MD, and assigned Accession Nos. ATCC VR2328 and ATCC VR2327 respectively. Data were analyzed by Bonferroni's modification of Student's t-test, in which the value of t for two groups was based on a variance computed from all groups, and the values of _. required for statistical significance were adjusted for the number of paired observations made. Example 4 A series of experiments were conducted to evaluate the ability of R2/23 and R2/29 to immunize chickens against challenge with virulent MD viruses. The chickens were 15x7 as used in Example 2 except they were obtained from breeder hens vaccinated with all three MD Serotypes, i.e., Mdll/75C (serotype 1), SB-1 (serotype 2), and FC126 (serotype 3) at a dose of 1000 PFU for each virus and were considered 10A positive for maternal antibndi.es (afcH-) . In the first experiment, each chicken was inoculated at 1 day of age with 2000 PFU of R2, R2/23, R2/29, PCL26/2, or FCL26/2 + SB-1 (2000 PFU of each) and placed in a "^-i-Pi . Hαrsf___l-__suer isolator. The PCL26/2 is a clon of P026. At 5 days post -vaccination, each chicken was _ha_ .enged with 500 PFU of M_5 virus. The birds were held far about 56 days post challenge, killed and necropsied, and the presence of gross lesions was recorded. Birds dying during the es eriment were examined far gross lesions. The birds considered to be at risk were those positive for MD lesions plus those survivors without lesions. The percent protection was calculated as the percent MD in unvaccinated, challenged controls minus the percent MD in vaccinated, challenged grope divided by the percent MD in -unvaccinated, ■challenged controls lαultiplied by 100. Three replicate treats were run, and the pooled results are reported in Table HI below. Both the R2/23 and R2/29 clones provided prσtecticn that was greater than that of HVT (FC126/2) alone, and was not _rtat____tic__Lly different from that of R2 or HVT + SB-1 vaccines. Eβople 5 A second challenge experiment was πrriiirtpri _a±_rt___ntially as described in __xa__ple 4, except serotype 1 challenge viruses RB1B, 295, and 287L were substituted far Md5. The results are reported in Table IV below. __B___ple 6 A -third, challenge experiment was conducted sajbstaπtially as described in ____ample 4, except protection was evaluated in various chicken strains. The strains used were 15x7 ___*• and two cσπnercial White Leghorn stocks, WL-A, and WL-B. Both WL-A and WL-B were frαn parents that received serotype 2 and 3 MD -vaccines and, consequently, - 11 - were considered positive for materncϋ. aπtihndies. The results are reported in Table V belcw. Esaxαple 7 Clcnes R2/23 and R2/29 were evaluated to determine if protection was diminished by additional passage of the virus stocks in cell culture. The procedure of Example 4 was repeated, except the clones were carried through to either passage 105 αr passage 115. The results are reported in Table VI below. Enoplβ 8 The R2 clones, plus lew passage Mdll as a positive control, were backpassaged in ab- chickens as described in Example 2 to monitor th ability of the viruses to revert to greater virulence. The inocula for passages 2, 3, and 4 were derived frαn an inoculated chicken in the previous passage. In addition, each group contained ccntact control chickens to mcnitαr the ability of the viruses to spread by natural route. Viremia was determined frαn buffy-coat cells frαn one 0.2-ml sample of blood pooled frαn two zrepresentative chickens. The blood was collected at 2 wks in each backpassage. Assays were conducted i t____plicate by standard procedures on chicken αr duck enbryo fibroblast cultures. Antihodies were detected in sera of test chickens by an indirect __ii__j__jfluo_."esceπoe test using antigen ccrisisting of cells infected with R2 virus. The results of b____passaging are shown in Table VH. ftri___ogenicity and -viremia titers of both R2 clones increased with backpassage; most of the increase uα-uiiMd between the first and second backpassages. Based an the frequency of gross MD lesions in ■chickens inoculated in _s____qpassages 2-4, the virulence of clone backpassaged R2/29 (16/36 positive) appeared greater than the virulence of backpassaged clone R2/23 (6/36 positive) (?<0.05) . ___pαrt__πtly, neither R2 clone was spread ___∑r__zcπtally as indicated b - 12 - the lack of gross lesions or antibodies through 8 wks in contact control c_ιick__ns at airy backpassage. Exaπple 9 A trial was canducted to test whether additional in vitro passages would change the avirulent nature of the R2 clones. Abt- ciiickens were inoculated and challenged as described in Example 4. No di__Xerences between passages 105 and 115 were observed for either -virus in gross or histologic lesions, body weights, or viremias. Both viruses were less pathogenic and induced lower viremia titers than the parent R2 virus. Curiously, body weights were higher in v__π_s-inoculated ciiickens than in ur ncculated controls. The results are reported in Table vm below. Exaxple 10 The growth rate of R2, R2/23, and FC126/2 in chicken embryo fibroblast cultures was conpared. Cultures were inoculated with about 100 FEU of each virus, and six replicate cultures per virus were assayed for cell-associated infectivity on days 1, 3, 5, 7, and 9. The results were expressed as a ratio of PFU to the input PFU, as detexxnined by titration of the original inoculum. On day 7, an additional 6 replicate cultures per virus were assayed for cell-free infectivity. Far cell-free virus assay, the designated plates were trypsinized and the cell harvest was resuspended in 1.0 ml of SPGA buffer, treated in two cycles (15 sec and 5 sec) with a scnifier (Heat Systems Ultrasonic, F_____ingdale, NY) at a power setting of 2 and a wattage setting of 40, and ceπt_rifuged at 6000 x g far 10 min. The sapernate was removed and assayed by inoculation of drained cultures with 0.1 ml. Medium was replenished after absorption for 30 min. The ccoparative rates of in vitro replication of R2/23, R2, and PC126/2 are given in Table IX. Titers (PEU/iπput PFU) of R2/23 were somewhat lower than that of R2 on days 1, 3, and 5 (P<0.05) , - 13 - but did not differ frαn R2 on days 7 and 9. Neither serotype 1 viru at any time replicated to as high a titer as did FCL26/2. However, it is likely that differences in PFU per input PFU may reflect differences in infection efficiency than in rate of replication. Cell-free virus recovery by scnication of infected cultures 7 days post inoculation was negligible far both serotype 1 viruses and was considerably less cαopared to that of F026/2 (?<0.05) . Bαnple U CEF cultures in 96-well plates were infected with selected MD -viruses of serotypes 1, 2, and 3 and fixed in cold ac_rtone-_ cx_hol when pla fies became apparent. The cultures were stained by indirect i_____ιr_ofl__3rescencε with serial dilutions of serotype specific xαencclcnal antibodies H19 and 2EN90 (serotype 1) , Y5 (serotype 2) , and L78 (serotype 3) . Titers were the highest ■dilution of antibody that produced a positive reaction en the infected cell antigen. Antigenic analysis by serotype-specific monoc onal antibodies as reported in Table X showed that R2/23, R2/29, and the parent R2 viru gave similar results; each reacted strongly with serotype l-specific antibodies H19 and 29190 but not with antibodies of other serotypes. In accord with other reports in the __it___ature, CVI988/Rispens and CVI988/C both lacked reactivity with H19 antibody but could be classified as serotype 1 srtrains by their reactivity with 2BN90. Cu.-iul arrtLbodies Y5 and L78 stained only serotype 2 and 3 antigens, respectively. It is understood that the foregoing detailed description is give merely by way of illustraticn and that modificatiαn and variations may be made therein without departing frαn the spirit and scope of the invention. - 14 - Table I Evaluation of R2 Derivative Clones in Ab- Chickens Gross Histo Vir_mj * fl lesions lesicns done Passage +/Tot Mean PFU +/-tot +/tot Randan denes R2/1 101 R2/2 101 R2/3 101 R2/4 101 R2/5 101 R2/6° 101 R2/7 101 R2/8 101 R2/9 101 R2/10 101 R2/11 101 R2/12 101 R2/13 ca 101 K2/14 101 R2/15 101 R2/16 101 R2/17 101 R2/18 101 B2/19 101 R2/20 101 52/21°. 101 " 82/22°°: 101 ΕQ/T- 01 101 R2/24 101 R2/25 101 R2/26 101 R2/27° 101 32/28°. 101 R2/29 aa 101 R2/30° 101 - 15 - Table I (Qantinued) Clone Passage Uhinoculated Controls a Chickens negative far maternal antibodies inoculated with 20,000 PfU of -virus at 1 day of age. Observations made 8 wks poet inoculation. A series of six trials was ccnώcted; ooπtrols for each trial inclτaded two lots inoculated with R2, one lot inoculated with M_5 and one uninoculated lot. b ND - not done. c Clones inducing viremia in absence of gross lesions. d Clones selected for further study. - 16 - Table II Pathogenicity of Four R2 Clcnes a with 20,000 PFU of each virus. Viremias are mean PFU counts of two WBC pools (three birds each) , 10« uffy coat cells/culture. Body weights are means of all survivor birds at 17 wks, adjusted fo sex. Means with dif ereπt letter designations differ by Bαnf ei-τoni t-test (P<0.05) . Bird died at 17 wks with enlarged vagus, sciatic, and brachial nerves. No histology was done. - 17 - Table III Ocπparative E icacy of Vaccines fran Attenuated R2 Clones 9 ∞ I Chickens positive for maternal antibodies were inoculated at 1 day of age with 2000 PFU of each virus, challenged with 500 PFU of Md5 virus at 5 days post vaccination, and evaluated at 8 wks post challenge. Statistics by Chi-Squaire (protection) , Bonferrcmi t-test (wei ts). Values with different letter designations differ (P<0.05). Table IV Protection by R2 Clones Against Different Challenge Viruses 3* % Pro- Vaccine Challenge +/Ttot % MD tection R2/23 RB1B R2/29 FCL26/2 PC126/2 + SB-1 Ncne R2/23 295 R2/29 PC126/2 PC126/2 + SB-1 Ncne R2/23 287L R2/29 F026/2 FC126/2 + SB-1 None R2/23 total R2/29 PC126/2 P026/2 + SB-1 Ncne Chickens positive for maternal antibodies were inoculated at 1 day of age with 2000 FEU of each virus, challenged with 500 FEU of the appropriate virus at 5 days post vaccination, and evaluated at 8 wks poet challenge. Statistics by Chi-Square. Values with different letter c_ιβignations differ (?<0.05) . - 19 - Table V Protection by R2 Clones Against MD in Different Chicken Strains a -___id____s positive for maternal antibodies were __noa_lated at 1 day of age with 2000 FEU of each virus, challenged with 500 FEU of Md5 virus 1 at 5 days post vaccination, and evaluated at 9 wks post challenge. Statistics by Chi-Square. Values with different letter designations differ (P<0.05). - 20 - Table VI Effect of Additional Cell Culture Passage on Protection 3 Chickens positive for maternal antibodies were inoculated at 1 day of age with 2000 PFU of each virus, challenged with 500 FEU of _£_5 virus at 5 days post vaccinaticn, and evaluated at 8 wks pest challenge. Data pooled from two replicate trials. Statistics by Chi-Sguare. Values with different letter d_____g___ticπs differ (F<0.05). - 21 - Table VII E)ackp__3sage of R2 Clones PRJ from 10 buffy coat cells from one sample of blood pooled from two representative chickens and assayed in triplicate. Blood collected at 2 wks (backpassage 1-4) . Antibody detected lay indirect __nιnuι_ofluor_s_u_k_noe on antigen consisting of cells infected with R2 virus. Sera collected at 8 wks. Number positive/number tested. Gross MD lesions at 8 wks. Number positive/number _____mined. Number dead in Table VIII Effect of Additional Tissue Culture Passage on Pathogenicity and Replication of R2 Clones 3 Gross Lesions Histo Lesions Body Wt. Viremia Virus Passage +/tαt % • /tot % 9 PFU to Chickens positive for maternal antibodies were inoculated at 1 day of age with 2000 PFU of each virus, challenged with 500 PFU of Md5 virus at 5 days post vaccination, and evaluated at 8 wks post challenge. Statistics by Bonferroni t-test. Values with different letter designations differ (P<0.05). Table IX Replication of R2 Clones values for same day followed fay different letter are different (P<0.05) by Bcnferroni t-test. Values are means of six replicates and are expressed as PFU per input PFU. Table X Antigenic Analysis of R2 denes by M_m_clcnal Antibodies Mcr__clc____L antibody Virus KL9 2EN90 Y5 L78 reacting with viral antigen in indirect i_n_uno-_luαrescence - 25 -

Claims: What is claimed: 1. A vaccine comprising an effective immunization dosage a viral agent selected from a clone of an attenuated revertant virus having the essential identifying c iracteristics of Mdll/75C/R2/23 or Mdll/75C/R2/29 - * d/or antigens of Mdll/75C/R2/23 or Mdll/75C/R2/29 <_ ective to elicit an immune response to a Marek's disease virus together with a pharmaceutically acceptable carrier or diluent therefor. 2. A vaccine according to Claim 1 characterised in that said viral agent is a live virus in a cell- associated preparation. 3. A vaccine according to Claim 1 characterised in that said viral agent is a live virus in a cell-free preparation. 4. A vaccine according to Claim 1 characterised in that said viral agent is a killed virus in a cell- associated preparation. 5. A vaccine according to Claim 1 characterised in that said viral agent is a killed virus in a cell-free preparation. 6. A vaccine according to Claim 5 characterised in that said viral agent is a subunit vaccine. 7. A vaccine according to Claim 1 characterised in that said viral agent is a product of an expression vector. 26 8. Use of the vaccine according to any one of the claims 1-7 for the protection of a chicken against Marek's diseases. 2 7

Inventors: WITTER RICHARD L, WITTER, RICHARD, L.
Applicants: US AGRICULTURE, THE UNITED STATES OF AMERICA AS REPRESENTED BY THE
Application year: 1992
Main classification: A61K39
IPC codes: A61K39, A61P31, C07K14