Zbl. Vet. Med. B, 22, 162- 168 (1975) @ 1975 Verlag Paul Parey, Berlin und Hamburg ISSN 0044-4294/ASTM-Coden:
ZVRBAZ
From the Instituto de Investigaciones Veterinarias, Maracay, Venezuela
Evaluation in Donkeys of an Inactivated Venezuelan Equine Encephalitis Vaccine
D A R ~PARRA O VILLASMIL, JULIETA
DE SIGER,MARIOPBREZ BARRIENTOS, MANFRED MUSSGAY" and RONALDB. MACKENZIE
W i t h I tables (Received for publication August 26, 1974)
Introduction In 1938, KUBESand RIOS (1939) isolated the virus of Venezuelan equine encephalitis (VEE) for the first time. During the same year they developed and began production of a formalin inactivated VEE vaccine prepared from virus grown in embryonated chicken eggs; it substituted for an inactivated vaccine against the Eastern and Western types of equine encephalitis which is reported as having not been effective against encephalitis which was prevalent in Venezuela at that time (VILLEGAS, 1971). Recent outbreaks of VEE in North, Central and South America (LORD, 1973) have resulted in the extensive use of a modified live virus (MLV) vaccine. While application of the MLV vaccine has produced favorable results (EDDYet al., 1972), some workers feel that the use of an inactivated VEE vaccine might be preferable under certain circumstances (BRYANS et al., 1971). With this in mind, an effort has been made to produce an inactivated vaccine of good immunogenic quality. Its method of preparation has been described previously (MUSSGAY et al.? 1972); the purpose of this publication is to report the immunogenic effect which it produced in a group of donkeys. Material and Methods Donkeys: the donkeys used for this vaccine evaluation were procured in the Venezuelan State of Bolivar, near the town of Manteco; they ranged in age from two to four years. Each was tested for the presence of neutralizing (N) antibody to VEE as well as for hemagglutination inhibition (HI) antibody to Venezuelan, Eastern (EEE) and Western (WEE) equine encephalitis viruses. ~
:$
Federal Research Institute for Animal Virus Diseases, Tiibingen, FRG.
Evaluation in Donkeys of an Inactivated Vaccine
163
Vaccine: the vaccine tested was produced from the “Guajira Strain” of VEE virus (MUSSGAYet al., 1972) in baby hamster kidney and it was inactivated with formalin, and Tween-80 Tri(n-buty1)phosphate (TNBP), and saponin was added. Virus: the virus used for post-vaccination challenge was strain P 676, which had been isolated in Venezuela by scientists from the Instituto Venezolano de Investigaciones Cientificas (IVIC). It has been reported by YOUNG and JOHNSON as belonging to subtype I, variant C of the VEE virus complex (YOUNGand JOHNSON, 1968). The strain was isolated from a pool of Aedes triannulatus mosquitos near Sotillo, Miranda (66’ 3’ W X 10’ 28’ N) during August, 1963. It had been passaged once in Vero cells and three times in suckling mouse brain. While the strain is frequently referred to as P 676 in published literature, an earlier passage level of the same strain has also been referred to as CBSI-9 (ZARATE and LOUISA,1972). O n a t least one occasion it has been erroneously reported as having originated from equine material (WALTON,1972). Vaccination and Challenge: of the 11 experimental donkeys, six received two ml of vaccine and one (donkey No. 13) received one ml all by the subcutaneous route. All vaccinated animals were observed for signs of any adverse reaction; twice daily rectal temperatures were taken of all of them during the entire course of the experiment. Fourteen days after vaccination, immediately after having been bled for blood and antibody studies, all 1 1 donkeys received subcutaneously, 50,000 suckling mouse intraperitoneal LD,, units (SMIPLD 50) of challenge virus. They were observed for a total of 10 days following challenge; viremia determinations were done daily. Neutralization Tests: sera were inactivated at 56 ‘C for 30 minutes, then mixed with 195 50 per cent lethal doses of challenge virus and incubated at 37 ‘C for one hour. After incubation each serum-virus mixture was inoculated into groups of six adult mice via the intraperitoneal route, each mouse receiving 0.025 ml. All sera, taken 0, 14 and 22 days post-vaccination were tested in a single test. Hemagglutination Inhibition Test: the technique used was a microand CASALS (1958). Antigen was technique modification of that of CLARKE prepared from suckling mouse brain treated by sucrose-acetone. Sera were acetone extracted. Eight units of hemagglutinin were used. Table 1 Immune response of vaccinated donkeys expressed in terms of mouse survival
::. ’:+
No. days after vaccination Reported as, No. of mice surviving/No. of mice inoculated
VILLASMIL, DE SIGER, BARRIENTOS, MUSSGAY and MACKENZIE
164
Table 2 Hemagglutination-inhibition antibodies in donkey serums after vaccination Serum taken on day’
Donkey No.
14
21
: 10 c 1 : 10 ( 1 : 10 e l : 10
c 1 : 10
: 10 1 : 320 cl : 10
c1:lO 1 : 320
0 ( 1 :
Cl
11
10 - = l : 10 e l : 10 c 1 : 10
12 13
c 1 : 10 c 1 : 10
(1
14
c 1 : 10
1
2 5
~~~~
c 1 : 10 c l : 10 c l : 10
cl : 10
’> No. days post-vaccination
Table 3 Temperatures of vaccinated and non-vaccinated donkeys, pre- and post-challenge Donkey
TemDerature
No.
Pre- challenge
*
-
Post challenqe**
Vaccinated
1 2 5 11 12 13 14 Non - vaccinated
15 17 18 20
38.1 38.1 38.1
38.6 38.1 38.1
38.1 7
39.0 1
}
37’8 38.3 38.2 J
38.1
R
}
39’7 39.0 39.2 J
39.2 =
R
’>Mean daily temperatures during 12 days, pre-challenge *’) Mean daily temperatures during 7 days, post-challenge X Overall mean temperatures of the group
Table 4 Post-challenge symptoms among non-vaccinated animals Symptoms
Donkey
No.
Conjunctivitis
17 , 18 , 20
Anorexia
15 , 17 , 18 , 20 15 , 17 , 18 , 20 17 , 20 15 , 17 , 18 , 20 17 . 20 17
Stupor Abdominal distention Tremors and muscular incoordination Diarrhea Prolapse of penis
Evaluation in Donkeys of an Inactivated Vaccine
165
Results Development of Specific Antibodies: there was no evidence of virus neutralizing activity among the sera taken from the seven donkeys immediately before vaccination; as may be seen in Table 1, only one of a total of 42 mice which were inoculated with seven serum-virus mixtures, survived the 195 LD,, units of virus which each received. However, sera taken two or more weeks after vaccination gave evidence of protection of the mice, mean survivorship among them being 38 and 36 per cent for sera taken 14 and 21 days post-vaccination, respectively. Previous to vaccination, donkey No. 17 reacted in HI test, at a dilution of 1 :20, with EEE antigen. All others were negative for VEE, EEE and WEE at the same dilution. As is shown in Table 2, only one donkey, No. 13, developed H I antibody after vaccination. Clinical Findings: Table 3 shows mean rectal temperatures of donkeys before and after receiving the challenge dose of strain P 676 virus. As may be seen, there was no febrile post-challenge response among vaccinated animals, whereas those which had not been vaccinated all developed fever. Neither did the vaccinated animals show signs of clinical illness, while those not vaccinated manifested the clinical findings noted in Table 4. One donkey died on the fourth post-challenge day with signs and symptoms of encephalitis. Viremia: as may be seen in Table 5, while all nonvaccinated donkeys were viremic after challenge, attempts to demonstrate post-challenge viremia among vaccinated animals were negative during 10 days. Table 5 Viremia among non-vaccinated donkeys
+ Virernia demonstrated
.- Viremia not demonstrated
Discussion None of the 11 animals in the test demonstrated the presence of HI or N antibody for VEE at the time it entered the experiment. There had been no known epizootic VEE activity in the region from whence the donkeys came during the previous 6 years; serological and epidemiological evidence both suggest, but do not eliminate the possibility, that the experimental donkeys had not had a previous natural infection with a virus of the VEE complex. It is not known whether other Group A arboviruses have been active during recent years in the region where the donkeys were obtained. I t was not by design but by a miscalculation, that donkey No. 13 received only 1 ml of vaccine. It is interesting to note that that same donkey showed the best serological response both in HI and N antibodies. The possibility exists that this sharp serological response of donkey No. 13 might have been due to its having experienced a previous, naturally occurring Group A arbovirus infection, thus showing an anamnestic response; and not related in any way to the quantity of antigen which received.
166
VILLASMIL, DE SIGER, BARRIENTOS, MUSSGAY and MACKENZIE
The administration of a challenge dose of VEE virus fourteen days after vaccination produced illness, fever and viremias among the four non-vaccinated controls and one of them died. The seven vaccinated animals remained well, and did not develop fever or viremia. These results suggest that VEE virus, inactivated by formalin and treated with Tween-80 and TNBP and saponin might be useful as a vaccine to protect equidae from YEE infection. Further experiments should be carried out in donkeys and horses which have assuredly not experienced previous Group A arbovirus infection during their lifetime in order to determine the degree and duration of protection. While this experiment suggests that an inactivated vaccine can be produced which retains considerable antigenicity, the problem of the existence of (and effective testing for) persistent, non-inactivated virus particles has not been dealt with in this experiment. However, since the virus was prepared in tissue culture media, it might well be readily susceptible to inactivating agents, and less likely to contain infective virus particles than a vaccine prepared in animal tissues. Furthermore, the use of two inactivation procedures - forand WEILAND, malin and TNBP - ensures safety of the vaccine (MUSSGAY 1973). It should be clear, then, that these results are very preliminary and that further, more extensive tests of antigenicity as well as safety in equidae are indicated.
Summary Seven donkeys were immunized with a Venezuelan equine encephalitis virus vaccine which had been prepared by inactivation of the virus with formalin and Tween-80-Tri(n-butyl)phosphateand addition of saponin. Fourteen days later they, and four non-vaccinated controls, each received 50,000 suckling mouse intraperitoneal LD50 units of challenge virus. None of the seven immunized donkeys demonstrated signs of clinical illness or viremia. Of the four non-vaccinated controls, all became ill and one died. These results suggest that more extensive experiments might well be carried out on this vaccine. Acknowledgements We are grateful to Dr. CARLOS QUIROZ for his helpful professional GRUBERand Mr. WILLIAMJ. L ~ P E for Z advice and to Miss ELVIRAPULGAR their fine technical assistance. Zusammenfassung Erprobung eines Impfstoffes auf der Basis inaktivierter ,,Venezuelan equine encephalitis"-Viren an Eseln Sieben Esel wurden mit einer ,,Venezuelan equine encephalitis"-VirusVakzine, die d u r h Inaktivierung des Virus mit Formalin und Tween-80-Tri(n-buty1)phosphat und mit einem Zusatz von Saponin hergestellt worden war, immunisiert. Vierzehn Tage nach der Immunisierung erhielten die Impflinge und vier nicht geimpfte Kontrolltiere jeweils 50 000 LD5,-Einheiten (die LD,, bezieht sich auf intraperitoneal belastete Babymause) des Belastungsvirus. Keiner der sieben immunisierten Esel zeigte Anzeichen einer klinischen Erkrankung oder eine Viramie. Von den vier ungeimpften Kontrolltieren erkrankten alle und eines starb. Diese Ergebnisse regen dazu an, umfassendere Untersuchungen mit dieser Vakzine durchzufuhren.
Evaluation in Donkeys of an Inactivated Vaccine
167
Rksumk Essai d’un vaccin sur l’iine avec le virus de l’enciphalite equine vknkzuilienne inactive Sept Snes ont ktk immunisks au moyen d’un vaccin du virus de l’enckphalite equine vknkzuklienne; le virus a ktk attknuk avec de la formaline, du Tween-80-tri(n-butyl)phosphate et fabriquk avec une addition de saponine. Les animaux vaccinks et quatre animaux de contrble ont r e p quatorze jours aprks l’immunisation 50 000 unitks LD50 du virus (la LD,, a ktk Ctablie par infection intrapkritonkale de souriceaux). Aucun des sept Snes immunisks n’a prksentk des signes cliniques ou une virkmie. Tous les animaux de contrble non vaccinks tombkrent malades et un mourut. Ces rksultats encouragent d’autres recherches plus ktendues avec ce vaccin. Resumen Ensayo en burros de una vacuna inactivada a base de virus de la uencefalitis equina venezolanan Se inmunizaron 7 burros con una virus-vacuna contra la c
ZVRBAZ
From the Instituto de Investigaciones Veterinarias, Maracay, Venezuela
Evaluation in Donkeys of an Inactivated Venezuelan Equine Encephalitis Vaccine
D A R ~PARRA O VILLASMIL, JULIETA
DE SIGER,MARIOPBREZ BARRIENTOS, MANFRED MUSSGAY" and RONALDB. MACKENZIE
W i t h I tables (Received for publication August 26, 1974)
Introduction In 1938, KUBESand RIOS (1939) isolated the virus of Venezuelan equine encephalitis (VEE) for the first time. During the same year they developed and began production of a formalin inactivated VEE vaccine prepared from virus grown in embryonated chicken eggs; it substituted for an inactivated vaccine against the Eastern and Western types of equine encephalitis which is reported as having not been effective against encephalitis which was prevalent in Venezuela at that time (VILLEGAS, 1971). Recent outbreaks of VEE in North, Central and South America (LORD, 1973) have resulted in the extensive use of a modified live virus (MLV) vaccine. While application of the MLV vaccine has produced favorable results (EDDYet al., 1972), some workers feel that the use of an inactivated VEE vaccine might be preferable under certain circumstances (BRYANS et al., 1971). With this in mind, an effort has been made to produce an inactivated vaccine of good immunogenic quality. Its method of preparation has been described previously (MUSSGAY et al.? 1972); the purpose of this publication is to report the immunogenic effect which it produced in a group of donkeys. Material and Methods Donkeys: the donkeys used for this vaccine evaluation were procured in the Venezuelan State of Bolivar, near the town of Manteco; they ranged in age from two to four years. Each was tested for the presence of neutralizing (N) antibody to VEE as well as for hemagglutination inhibition (HI) antibody to Venezuelan, Eastern (EEE) and Western (WEE) equine encephalitis viruses. ~
:$
Federal Research Institute for Animal Virus Diseases, Tiibingen, FRG.
Evaluation in Donkeys of an Inactivated Vaccine
163
Vaccine: the vaccine tested was produced from the “Guajira Strain” of VEE virus (MUSSGAYet al., 1972) in baby hamster kidney and it was inactivated with formalin, and Tween-80 Tri(n-buty1)phosphate (TNBP), and saponin was added. Virus: the virus used for post-vaccination challenge was strain P 676, which had been isolated in Venezuela by scientists from the Instituto Venezolano de Investigaciones Cientificas (IVIC). It has been reported by YOUNG and JOHNSON as belonging to subtype I, variant C of the VEE virus complex (YOUNGand JOHNSON, 1968). The strain was isolated from a pool of Aedes triannulatus mosquitos near Sotillo, Miranda (66’ 3’ W X 10’ 28’ N) during August, 1963. It had been passaged once in Vero cells and three times in suckling mouse brain. While the strain is frequently referred to as P 676 in published literature, an earlier passage level of the same strain has also been referred to as CBSI-9 (ZARATE and LOUISA,1972). O n a t least one occasion it has been erroneously reported as having originated from equine material (WALTON,1972). Vaccination and Challenge: of the 11 experimental donkeys, six received two ml of vaccine and one (donkey No. 13) received one ml all by the subcutaneous route. All vaccinated animals were observed for signs of any adverse reaction; twice daily rectal temperatures were taken of all of them during the entire course of the experiment. Fourteen days after vaccination, immediately after having been bled for blood and antibody studies, all 1 1 donkeys received subcutaneously, 50,000 suckling mouse intraperitoneal LD,, units (SMIPLD 50) of challenge virus. They were observed for a total of 10 days following challenge; viremia determinations were done daily. Neutralization Tests: sera were inactivated at 56 ‘C for 30 minutes, then mixed with 195 50 per cent lethal doses of challenge virus and incubated at 37 ‘C for one hour. After incubation each serum-virus mixture was inoculated into groups of six adult mice via the intraperitoneal route, each mouse receiving 0.025 ml. All sera, taken 0, 14 and 22 days post-vaccination were tested in a single test. Hemagglutination Inhibition Test: the technique used was a microand CASALS (1958). Antigen was technique modification of that of CLARKE prepared from suckling mouse brain treated by sucrose-acetone. Sera were acetone extracted. Eight units of hemagglutinin were used. Table 1 Immune response of vaccinated donkeys expressed in terms of mouse survival
::. ’:+
No. days after vaccination Reported as, No. of mice surviving/No. of mice inoculated
VILLASMIL, DE SIGER, BARRIENTOS, MUSSGAY and MACKENZIE
164
Table 2 Hemagglutination-inhibition antibodies in donkey serums after vaccination Serum taken on day’
Donkey No.
14
21
: 10 c 1 : 10 ( 1 : 10 e l : 10
c 1 : 10
: 10 1 : 320 cl : 10
c1:lO 1 : 320
0 ( 1 :
Cl
11
10 - = l : 10 e l : 10 c 1 : 10
12 13
c 1 : 10 c 1 : 10
(1
14
c 1 : 10
1
2 5
~~~~
c 1 : 10 c l : 10 c l : 10
cl : 10
’> No. days post-vaccination
Table 3 Temperatures of vaccinated and non-vaccinated donkeys, pre- and post-challenge Donkey
TemDerature
No.
Pre- challenge
*
-
Post challenqe**
Vaccinated
1 2 5 11 12 13 14 Non - vaccinated
15 17 18 20
38.1 38.1 38.1
38.6 38.1 38.1
38.1 7
39.0 1
}
37’8 38.3 38.2 J
38.1
R
}
39’7 39.0 39.2 J
39.2 =
R
’>Mean daily temperatures during 12 days, pre-challenge *’) Mean daily temperatures during 7 days, post-challenge X Overall mean temperatures of the group
Table 4 Post-challenge symptoms among non-vaccinated animals Symptoms
Donkey
No.
Conjunctivitis
17 , 18 , 20
Anorexia
15 , 17 , 18 , 20 15 , 17 , 18 , 20 17 , 20 15 , 17 , 18 , 20 17 . 20 17
Stupor Abdominal distention Tremors and muscular incoordination Diarrhea Prolapse of penis
Evaluation in Donkeys of an Inactivated Vaccine
165
Results Development of Specific Antibodies: there was no evidence of virus neutralizing activity among the sera taken from the seven donkeys immediately before vaccination; as may be seen in Table 1, only one of a total of 42 mice which were inoculated with seven serum-virus mixtures, survived the 195 LD,, units of virus which each received. However, sera taken two or more weeks after vaccination gave evidence of protection of the mice, mean survivorship among them being 38 and 36 per cent for sera taken 14 and 21 days post-vaccination, respectively. Previous to vaccination, donkey No. 17 reacted in HI test, at a dilution of 1 :20, with EEE antigen. All others were negative for VEE, EEE and WEE at the same dilution. As is shown in Table 2, only one donkey, No. 13, developed H I antibody after vaccination. Clinical Findings: Table 3 shows mean rectal temperatures of donkeys before and after receiving the challenge dose of strain P 676 virus. As may be seen, there was no febrile post-challenge response among vaccinated animals, whereas those which had not been vaccinated all developed fever. Neither did the vaccinated animals show signs of clinical illness, while those not vaccinated manifested the clinical findings noted in Table 4. One donkey died on the fourth post-challenge day with signs and symptoms of encephalitis. Viremia: as may be seen in Table 5, while all nonvaccinated donkeys were viremic after challenge, attempts to demonstrate post-challenge viremia among vaccinated animals were negative during 10 days. Table 5 Viremia among non-vaccinated donkeys
+ Virernia demonstrated
.- Viremia not demonstrated
Discussion None of the 11 animals in the test demonstrated the presence of HI or N antibody for VEE at the time it entered the experiment. There had been no known epizootic VEE activity in the region from whence the donkeys came during the previous 6 years; serological and epidemiological evidence both suggest, but do not eliminate the possibility, that the experimental donkeys had not had a previous natural infection with a virus of the VEE complex. It is not known whether other Group A arboviruses have been active during recent years in the region where the donkeys were obtained. I t was not by design but by a miscalculation, that donkey No. 13 received only 1 ml of vaccine. It is interesting to note that that same donkey showed the best serological response both in HI and N antibodies. The possibility exists that this sharp serological response of donkey No. 13 might have been due to its having experienced a previous, naturally occurring Group A arbovirus infection, thus showing an anamnestic response; and not related in any way to the quantity of antigen which received.
166
VILLASMIL, DE SIGER, BARRIENTOS, MUSSGAY and MACKENZIE
The administration of a challenge dose of VEE virus fourteen days after vaccination produced illness, fever and viremias among the four non-vaccinated controls and one of them died. The seven vaccinated animals remained well, and did not develop fever or viremia. These results suggest that VEE virus, inactivated by formalin and treated with Tween-80 and TNBP and saponin might be useful as a vaccine to protect equidae from YEE infection. Further experiments should be carried out in donkeys and horses which have assuredly not experienced previous Group A arbovirus infection during their lifetime in order to determine the degree and duration of protection. While this experiment suggests that an inactivated vaccine can be produced which retains considerable antigenicity, the problem of the existence of (and effective testing for) persistent, non-inactivated virus particles has not been dealt with in this experiment. However, since the virus was prepared in tissue culture media, it might well be readily susceptible to inactivating agents, and less likely to contain infective virus particles than a vaccine prepared in animal tissues. Furthermore, the use of two inactivation procedures - forand WEILAND, malin and TNBP - ensures safety of the vaccine (MUSSGAY 1973). It should be clear, then, that these results are very preliminary and that further, more extensive tests of antigenicity as well as safety in equidae are indicated.
Summary Seven donkeys were immunized with a Venezuelan equine encephalitis virus vaccine which had been prepared by inactivation of the virus with formalin and Tween-80-Tri(n-butyl)phosphateand addition of saponin. Fourteen days later they, and four non-vaccinated controls, each received 50,000 suckling mouse intraperitoneal LD50 units of challenge virus. None of the seven immunized donkeys demonstrated signs of clinical illness or viremia. Of the four non-vaccinated controls, all became ill and one died. These results suggest that more extensive experiments might well be carried out on this vaccine. Acknowledgements We are grateful to Dr. CARLOS QUIROZ for his helpful professional GRUBERand Mr. WILLIAMJ. L ~ P E for Z advice and to Miss ELVIRAPULGAR their fine technical assistance. Zusammenfassung Erprobung eines Impfstoffes auf der Basis inaktivierter ,,Venezuelan equine encephalitis"-Viren an Eseln Sieben Esel wurden mit einer ,,Venezuelan equine encephalitis"-VirusVakzine, die d u r h Inaktivierung des Virus mit Formalin und Tween-80-Tri(n-buty1)phosphat und mit einem Zusatz von Saponin hergestellt worden war, immunisiert. Vierzehn Tage nach der Immunisierung erhielten die Impflinge und vier nicht geimpfte Kontrolltiere jeweils 50 000 LD5,-Einheiten (die LD,, bezieht sich auf intraperitoneal belastete Babymause) des Belastungsvirus. Keiner der sieben immunisierten Esel zeigte Anzeichen einer klinischen Erkrankung oder eine Viramie. Von den vier ungeimpften Kontrolltieren erkrankten alle und eines starb. Diese Ergebnisse regen dazu an, umfassendere Untersuchungen mit dieser Vakzine durchzufuhren.
Evaluation in Donkeys of an Inactivated Vaccine
167
Rksumk Essai d’un vaccin sur l’iine avec le virus de l’enciphalite equine vknkzuilienne inactive Sept Snes ont ktk immunisks au moyen d’un vaccin du virus de l’enckphalite equine vknkzuklienne; le virus a ktk attknuk avec de la formaline, du Tween-80-tri(n-butyl)phosphate et fabriquk avec une addition de saponine. Les animaux vaccinks et quatre animaux de contrble ont r e p quatorze jours aprks l’immunisation 50 000 unitks LD50 du virus (la LD,, a ktk Ctablie par infection intrapkritonkale de souriceaux). Aucun des sept Snes immunisks n’a prksentk des signes cliniques ou une virkmie. Tous les animaux de contrble non vaccinks tombkrent malades et un mourut. Ces rksultats encouragent d’autres recherches plus ktendues avec ce vaccin. Resumen Ensayo en burros de una vacuna inactivada a base de virus de la uencefalitis equina venezolanan Se inmunizaron 7 burros con una virus-vacuna contra la c
