the following description provides preliminary data on the immunologic relationship between ehv - 1 and ehv - 4 which led to the realization that a vaccine based on ehv - 4 would be most useful and that a combined vaccine based on ehv - 1 and ehv - 4 would more effectively control diseases caused by both viruses than the presently used monovalant vaccines based on ehv - 1 alone . equine fetal kidney ( efk ) cells were used at 5th passage . virus strains 438 / 77 and 405 / 76 were used as representatives of ehv - 1 and ehv - 4 , respectively . their origin , initial passage history , and typing have been described at pages 488 to 492 of the australian veterinary journal vol . 55 1979 and at pages 249 - 259 of archives of virology vol . 77 1983 . monolayers of efk cells in 1 , 200 cm 2 roller bottles were inoculated at a multiplicity of infection of 1 with either ehv - 1 or ehv - 4 . serum - free maintenance medium was added after adsorption . when cytopathic changes in the monolayers were complete , the roller bottles were frozen and thawed three times , the contents were clarified by low - speed centrifugation , and the supernatants were harvested and stored at - 70 ° c . titers of these virus stocks were measured by plaque assay . the titer of the ehv - 1 stock was 5 × 10 6 plaque - forming units ( pfu )/ ml , and that of the ehv - 4 stock was 7 . 5 × 10 6 pfu / ml , although titers & gt ; 10 7 . 5 are now being evaluated . virus stocks were inactivated by treatment with 0 . 2 %- propiolactone ( bpl ) for 27 hours at 4 ° c . and then for three hours at 37 ° c . to hydrolyze residual bpl . complete inactivation of infectivity was verified by three passages ( approximately seven days / passage ) of 0 . 4 ml of inoculum in efk cells . inactivated virus stocks were concentrated five times by ultrafiltration through a membrane system at 4 ° c . and 400 kpa . concentration was verified by protein estimation and particle counts . roller bottles of noninfected efk cell monolayers were similarly processed to produce control cell lysate antigen ( cl ag ). each antigen was dispensed in 2 - ml aliquotes and stored at - 70 ° c . three colostrum - deprived spf foals were used . foals were removed immediately after induced parturition or supervised natural birth and then transferred to an isolation unit where they were reared under spf conditions . freedom from infection with ehv - 1 and ehv - 4 was established before the foals were vaccinated by the absence of clinical signs of respiratory tract disease and the lack of detectable ( titer & lt ; 8 ) virus - neutralizing antibody ( vn ab ) against ehv - 1 and ehv - 4 . during the vaccination period , foal 2 remained free of clinical respiratory tract disease and detectable vn ab to ehv - 1 and ehv - 4 . foals 1 , 2 and 3 were 5 , 4 and 10 weeks old , respectively , on the day of the first vaccination dose was given ( day 0 ). the foals were vaccinated and challenge exposed according to the schedule in table 1 . vaccinations given on days 0 and 14 comprised im injections of 2 ml of antigen gently mixed with 0 . 5 ml of steril alhydrogel ( approx . 17 mg of aluminum hydroxide / ml ). before challenge exposure with ehv - 1 , foals 1 and 2 were separated from foal 3 by transfer to another isolation unit . intranasal ( in ) challenge exposures were conducted by swabbing the nasal and nasapharyngeal mucosa with virus diluted in 5 ml of maintenance medium ( table 1 ). rectal temperatures and presence or absence of signs of respiratory tract disease were monitored twice daily . blood samples for total and differential wbc counts and for lymphocyte blastogenesis ( lb ) assays and nasal swabs and blood samples for virologic examination were obtained twice a week , except that these collections were done three times in the first week after virus challenge exposures . serum samples for vn ab assay were obtained twice a week . total and differential wbc counts were measured by standard procedures . nasal swabs were transported ( 4 ° c .) in 5 ml of maintenance medium and filtered through a 450 nm ( apd ) membrane filter before inoculation onto efk cells . some swabs were frozen at - 70 ° c . before filtration and inoculation at a later date . the wbc from heparinized blood samples ( 10 iu of heparin / ml of blood were examined for infective virus . several processing techniques were attempted , including ( i ) inoculation of fresh or freeze / thaw - disrupted buffy coat cells onto efk cells , ( ii ) inoculation of fresh or freeze / thaw - disrupted mononuclear cells ( separated from blood by ficoll - diatrizoate density gradient centrifugation ) onto efk cells , and ( iii ) inoculation of mitogen stimulated mononuclear cells onto efk cells . all nasal swabs and wbc samples were passaged three times on efk cells with seven days / passage to detect viral cytopathic effects . positive samples were typed by plaque morphology analysis and restriction endonuclease fingerprinting of virus dna . for lb assays , separated triple - washed mononuclear cells were suspended in rpm1 1640 medium supplemented with 2 nm l - glutamine , 3 nm hepes buffer , penicillin ( 40 u / ml ), streptomycin ( 40 g / ml ), amphotericin b ( 1 g / ml ), and spf foal serum . two batches of spf foal serum were used : serum a for mononuclear cells from foals 1 and 2 and serum b for mononuclear cells from foal 3 . the medium was supplemented with either 15 % serum a or 10 % serum b : concentrations which elicited optimum lb responses during preliminary tests . mononuclear cells were suspended in supplemented rpm1 1640 medium at 2 concentrations : 1 × 10 6 cells / ml and 5 × 10 5 cells / ml . viability was assessed by trypan blue exclusion , and purity was assessed by examination of stained smears . six wells of a round - bottomed tissue culture multiwell plate , each containing 0 . 025 ml of 1 of the following were prepared : rpm1 1640 medium : 0 . 31 g phytohemagglutinin ; 1 : 2 dilutions of ehv - 1 antigen ( ehv - 1 ag ), ehv - 4 ag and cl ag ; 1 : 5 dilutions of ehv - 1 ag , ehv - 4 ag and cl ag ; 1 : 10 dilutions of ehv - 1 ag , ehv - 4 ag and cl ag ; and 1 : 20 dilutions of ehv - 1 ag , ehv - 4 ag and cl ag . mononuclear cells ( 2 × 10 5 and 1 × 10 5 ) in 0 . 2 ml of medium were added to triplicate wells of each ag or mitogen . preliminary results elicited optimum lb response with 2 × 10 5 mononuclear cells / well plus 1 : 5 dilution of each ag , and the range of variables ( in the preceding paragraph ) was chosen to allow for changes in the immunologic status of the foals , with respect to ehv - 1 and ehv - 4 , during the vaccinations and challenge exposures . mitogen cultures were incubated for three days and ag cultures , for four days , at 37 ° c . in a humidified 5 % co 2 atmosphere . all cultures were pulsed with 1 ci [ 3 h ] thymidine , during the last 18 to 20 hours of the incubation . at the end of incubation , cultures were harvested with a semiautomated cell harvester onto glass fiber filter paper , transferred to plastic scintillation vials , and processed for scintillation counting by addition of 0 . 5 ml of tissue solubilizer , 0 . 02 ml of glacial acetic acid , and 2 . 5 ml of scintillation fluid . scintillations were counted for one minute in a liquid scintillation counter and expressed for each well as counts per minute ( cpm ) minus background cpm . the cpm of wells incubated with rpmi 1640 medium ( nonstimulated wells ) and mitogen ( stimulated wells ) were used to verify the presence of functional mononuclear cells . stimulation indices ( si ) were calculated for each dilution of ag . e . g ., si for 1 : 2 dilution of ehv - 1 ag = mean cpm 1 : 2 dilution of ehv - 1 ag stimulated wells / mean cpm 1 : 2 dilution cl ag - stimulated wells . the highest of the 8 si ( 4 ag dilutions and 2 cell concentrations ) was taken as the peak response for a particular day . serum samples were heat inactivated ( 56 ° c . for 30 minutes ) and tested for vn ab to ehv - 1 and ehv - 4 by a microprocedure . rabbits were inoculated im with 1 ml of stock virus ( emulsified in 1 ml of freund &# 39 ; s complete adjuvant ) at 8 , 10 , and 12 weeks of age , and then iv with 1 ml of stock virus at 14 , 16 and 17 weeks of age . blood was collected 10 days after the last injection ; serum was prepared , heat inactivated , and tested for vn ab to both ehv - 1 and ehv - 4 . clinical signs of respiratory tract disease were not observed in any of the foals after vaccination or challenge exposure . the temperature chart for foal 3 ( fig1 ) is typical of the responses of the foals . rectal temperatures did not exceed 40 ° c . and fluctuations associated with vaccination or challenge exposures were small ( approximately 0 . 5 °). the temperature or 39 . 9 ° c . on day 29 in foal 3 was associated with the development of a small facial swelling of undetermined origin . total and differential counts for foal 3 are shown in fig2 . total wbc numbers varied directly with variations in neutrophil numbers . lymphocyte counts increased after each vaccination and after the first challenge exposure . later variations in lymphocyte counts could not be accounted for . similar changes were observed in foals 1 and 2 , although in foal 1 , a sharp decrease in lymphocyte count ( 2 . 7 × 10 9 cells / l to 1 . 85 × 10 9 cells / l ) occurred two days after the first challenge exposure , before a later increase in lymphocyte count at day seven . although virus was not isolated from the wbc preparation of any foal , virus was isolated from a single nasal swab - from foal 2 on day 83 ( two days after challenge with ehv - 4 ). the virus was typed as ehv - 4 . mononuclear cell suspensions had a median purity of 96 % mononuclear cells ( n = 110 ), with the main contaminating cells being neutrophils and basophils . average yield of mononuclear cells from blood samples was 50 %. viability was always & gt ; 95 % at the start of incubation . repeatability ( triplicate wells ) was satisfactory with se approximately 14 % of the mean . peak responses of all foals to ehv - 1 ag and ehv - 4 ag are shown in fig3 . foal 1 responded in vitro after vaccination with peak si that initially ( days 14 and 21 ) were specific for the vaccination type ( ehv - 1 ) by a factor of 2 or more , but all later peak si were of similar magnitude to both ehv - 1 ag and ehv - 4 ag , regardless of the type of the virus used in the challenge inoculum and the record of earlier exposure . foal 2 did not respond in vitro to either ehv - 1 ag or ehv - 4 ag after vaccination with control cl ag . the peak si after challenge exposure tended to favour the type of the challenge virus , but the differences were smaller than those in foal 1 . the response of foal 3 was similar to that of foal 1 , i . e . an early response at day 7 was specific for the vaccination type ( ehv - 4 ) followed by peak si of similar magnitude to ehv - 1 ag and ehv - 4 ag during the remainder of the vaccination and challenge exposure periods . the vn ab titers against ehv - 1 and ehv - 4 for all foal sera are shown in fig4 . foal 1 produced low concentrations of ehv - 1 vn ab after 2 vaccinations with ehv - 1 ag and these concentrations were increased by 2 ehv - 1 challenge exposures to a titer of 90 at day 73 . this response to ehv - 1 vaccination and challenge exposure was highly specific with only traces ( titers ≦ 9 ) of ehv - 4 vn ab present up to day 81 . subsequent challenge exposures with ehv - 4 on days 81 and 107 increased the titers of both ehv - 4 vn ab and ehv - 1 vn ab . foal 2 remained free of detectable vn ab ( titers & lt ; 8 ) after two vaccinations with control cl ag and one challenge exposure with ehv - 1 . a second ehv - 1 challenge exposure on day 60 elicited a small ehv - 1 vn ab response ( to a titer of 22 on day 73 ). challenge exposure with ehv - 4 on day 81 increased the ehv - 1 vn ab to high titers (& lt ; 256 ) and elicited detectable ehv - 4 vn ab titers after day 91 . the ehv - 4 vn ab titers increased after the second ehv - 4 challenge exposure on day 107 , whereas ehv - 1 vn ab levels were unaffected . foal 3 produced both ehv - 1 vn ab and ehv - 4 vn ab after ehv - 4 ag vaccination , with higher titers of ehv - 1 vn ab . challenge exposure with ehv - 4 increased the levels of both viral ab to comparable titers of ˜ 38 . subsequent challenge exposures with ehv - 1 had little effect on ehv - 4 vn ab titers and produced only slight increases in ehv - 1 vn ab titers . the vn ab titers of immunized rabbits against both ehv - 1 and ehv - 4 are shown in table 2 . sera from ehv - 1 immunized rabbits were highly specific for ehv - 1 and showed little cross - reactivity for ehv - 4 . sera from ehv - 4 immunized rabbits neutralized both ehv - 1 and ehv - 4 to abut the same titer . clinical signs of respiratory tract disease were not observed in any of the three foals . reduced clinical disease was reported for spf gnotobiotic foals inoculated intranasally with ehv - 1 or ehv - 4 and may be due to an atypical microbial flora in the respiratory tract of these foals . virus isolation from wbc samples after challenge exposures was unsuccessful , even though several processing techniques were attempted . the ehv - 4 was recovered from a nasal swab from foal 2 on day 83 ( or 2 days after intranasal ehv - 4 challenge exposure ) and may represent active infection of a nonimmune foal , but this was not confirmed . in vitro lb responses to ehv - 1 ag and ehv - 4 ag were used to determine the specificity of a cellular immune response of horses to ehv - 1 and ehv - 4 . type - specific responses , even though significantly cross - reactive , have been demonstrated for herpes simplex cirus type 1 ( hsv - 1 ) and type 2 ( hsv - 2 ), using this approach . except for the early responses of foals 1 and 3 when peak si after vaccination were specific for the immunizing type of virus by a factor of & gt ; 2 , the peak si to ehv - 1 ag and ehv - 4 ag were of similar magnitude , regardless of the type of the virus used and regardless of the record of earlier exposure of the foals . for primary hsv - 1 infections of persons , a similar pattern with an early type - specific lb response followed by a response cross - reactive to hsv - 2 has been described , but generally , the type specificities of the lb responses to hsv - 1 and hsv - 2 are consistently manifested by at least a 2 - fold difference in si . such a clear difference was not demonstrated for ehv - 1 and ehv - 4 . specificity of the cellular immune response of the horse to ehv - 1 and ehv - 4 may be further characterized by using other assays of cellular immunity . foals and rabbits exposed to ehv - 1 produced a specific ehv - 1 vn ab response , with little production of ehv - 4 vn ab ; animals exposed to ehv - 4 produced a cross - reactive response with equivalent titers of ehv - 1 vn ab and ehv - 4 vn ab . this relationship ( tables 2 and 3 ), using hyperimmune sera from rabbits and foals is similar to other studies , using hyperimmune rabbit antisera and gnotobiotic foal antisera to ehv - 1 and ehv - 4 . fig4 illustrates the maintenance of this relationship during the development of vn ab in spf foals after sequential exposure to ehv - 1 and ehv - 4 or vice versa . it seems that ehv - 1 possesses type - specific antigenic determinant ( s ), presumably in the envelope glycoproteins , that are important for the generation of vn ab to this virus , whereas antigenic determinants common to both ehv - 1 and ehv - 4 are the most significant determinants recognized during the vn ab response to ehv - 4 . further studies with monoclonal antibodies to ehv - 1 and ehv - 4 and preparations of individual glycoproteins from each type may define this relationship more closely . whether ehv - 1 and ehv - 4 are cross protective in horses remains unclear . the small number of spf foals used in the present preliminary study , together with the lack of clinical disease and virus isolations after challenge exposure , even from the control foal , prevented making full assessment of cross protection . the 3 spf foals were , nevertheless , invaluable for determining the specificity of certain immune responses to vaccination and challenge exposure with ehv - 1 and ehv - 4 . cross - reactive lb responses were demonstrated in all foals after exposure to either ehv - 1 or ehv - 4 and are noteworthy in comparison to the type - specific responses for hsv - 1 and hsv - 2 . however , the relative importance of this cellular immune response in vivo and its correlation with resistance to infection are not established . in contrast , the role of antibody in defense against herpesvirus infection both alone and in combination with complement and k cells is recognized , indicating that a highly specific antibody response to ehv - 1 , as demonstrated in the present experiments , may not cross protect against infection with ehv - 4 . the specificity of the antibody response raises questions about the use of vaccines based on ehv - 1 alone to control both ehv - 1 and ehv - 4 infections . although it is possible that repeated vaccinal doses of ehv - 1 may elicit some ehv - 4 vn ab , it would seem more appropriate to use a vaccine derived from ehv - 4 to achieve this . this preliminary study indicates that a monovalent ehv - 4 vaccine would elicit antibody and cellular immune responses to both ehv - 1 and ehv - 4 . however , it seems more likely that the most appropriate vaccine for the control of abortion and rhinopneumonitis should be vivalent , incorporating both ehv - 1 and ehv - 4 .
a vaccine for equine herpesvirus which includes the equine rhinopneumonitis virus type 4 in an inactivated or modified live virus form . also disclosed is a combined vaccine based on the equine abortion virus ) and equine rhinopneumonitis virus ) and equine rhinopneumonitis virus ) in an inactivated or modified live virus form . the viruses are present in a suitable adjuvant with a suitable preservative .