DE2505066A1 - PARAINFLUENZA VIRUS VACZINE FOR THE CONTROL OF THE TRACHEOBRONCHITIS OF DOGS - Google Patents

Description

The invention relates to the production of a parainfluenza · live virus yakzine which confers immunity against sracheobronehitis ("stable cough ¹¹ ) of dogs.

Diseases of the respiratory organs occur in closed colonies of those united in kennels and similar institutions Dogs quite often. In such a colony of dogs, for example, respiratory diseases were for death of more than half the for 6 weeks in the Kennel-left dogs responsible. Although several "viruses considered pathogens were isolated

Dr Pi / M.

509832/0963

ORIGINAL INSPECTED

recently in various places of the V.St.A. a single SV ^ -like parainfluenza virus from diseased dogs. Table I gives information on the different types of dogs and the extent of the geographic spread of the virus mentioned. From Tables II and III it can be seen that SY _K -älmliehe canine parainfluenza isolates of various origins? future are identical in terms of antigenic character. Although other viruses, such as canine distemper, ICH and the related adenovirus, A / Toronto 26 and golGgcntlicli Gin Herpesvirus, can also cause symptoms of diseases of the respiratory organs, which are clinically difficult to differentiate from the clinical picture that can be traced back to the parainfluenza virus the facts suggest that tracheobronchitis is to a considerable extent due to the latter virus; this has been particularly true since the availability of the ICH and canine distemper vaccines.

Studies have shown that the aforementioned SVc-like parainfluenza virus can lead to an infection of almost 100 $> in dogs that do not possess antibodies, that the disease can be transmitted to other antibody-free dogs one after the other through contact after infection, and that recovered dogs, which Are challenged with the same virus 2 1/2 months later and are resistant to re-infection. These results were corroborated by field studies which showed that the SVc-like parainfluenza virus has a greater tendency to spread than other viruses causing similar respiratory symptoms, and that the natural onset of tracheobronchitis uniformly immunizes surviving animals against the similar parainfluenza infection. Tables IV and V list ¥ erte that by the inventor et al. on the occasion of the study of a spontaneous outbreak of a disease of the respiratory system of laboratory dogs were determined at the University of Pennsylvania. From these values it can be seen that the extent of

509832/0963

Parainfluenza infection is significantly higher than that of adenovirus infection associated with similar disease symptoms. Table TT illustrates the resilience of
dogs recovered from parainfluenza infection versus disease due to natural heating. Table YII illustrates the immunity developed in dogs following a natural or experimental infection, based on the fact that the dogs did not develop the Y virus when experimentally challenged ^{with a virulent strain;} "retire, still fall ill"

509832/0963

TABLE I

Literature on the isolation of an SVe-like parainfluenza virus from those with epizootic diseases of the respiratory organs Dogs

(epidemic)

location Dog type Washington, D.O. Laboratory dogs 5098 Lackland APB, Texas Guard dogs ro Pt. Benning, Ga. Military dogs O
CD New York and
New Jersey Lap dogs from the
Pet shop and dogs
from the kennel New Jersey domestic lap dogs Ithaca, N.T. Cornell Dogs
Dog farm Philadelphia, Pa. Dogs, from the animal
hospital Laboratory dogs Quakertown, Pa. Dogs from the kennel Dublin, Va. Dogs from the breeding

reference

Binn et al, Proc. Soc.Bxp.Biol. and Med., 12S (1967), HO

Crandell et al, Am. J. Vet. Res. 29 (1968), 2141

Binn et al, Am. J.Vet .Res. 29, (1968), 1809

Appel unl Percy / J. A.V.M.A. 156 (1969), 1778

Il

ti

Rosenberg et al, Am.J.Bpid. 94: 147 (1971)

Lief and Rosenberg (unpublished)

Ran (unpublished) kennel of the Plow Laboratory

TABLE II.E II

Results τοη reciprocal neutralization tests on some Canine parainfluenza isolates of various origins

liter with isolate

origin

Dog serum *

Waiter Reed G 95833 (Binn) ATCC VR Penn / 343 Penn / 406 Penn / 103 Penn / 268 Penn / 121 Binn's C95833

01 SÜerhospital Penn / 343 / Λ the üniv, of OO Pa, (1967) Qu £ ä3cer t own, Pa. Penn / 406 O ^ wj.nger
(May 1970) co Sabor the Penn / 103 UaiT.Ton Pa. (ATCC VE 666) (JiLLi 1970) Penn / 268 Penn / 121

16

64

16

16 SA-
SA 64 I. 2505066 128 *
128 128 VJl 16 * 64
128

TABLE III

Antigen analysis by Kf ^ Süa hybrid dogs (in Virginia and Pennsylvania) isolated viruses

Isolate Va / 7527 HI titer,
with Penn / 343 serum Va / 7526 32 Va / 7522 32. Penn / 343 32 Penn / 103, ATCC VR 666 32-64 64

509832/0963

! PABELIE IV

Parainfluenza and adenovirus infections on the occasion of epizootic disease "in laboratory dogs at the University of Pennsylvania ■

Dog virus isolate H-I disease mortality after no

PI adeno- PI Adeno- (a) 64/32 + 16/8. + virus virus 8/8 16/8 102 + ■ 0/64 (c) o / o J 16/128 ί 16/8 103 4- 0/64 0/0 - ± 16/0 64/8 OK4 0/0 0/0 + 64/64 + 64/16 115 4th 0/128 0/0 + 0/128 (d) + 64/32 119 4th 0/16 8/8 + 0 / ND + euthanized 8/128 ί " 121 + 0/256 16/8 + after 1 week 512/256 178 0/128 32/16 + 0 / ND only fever dies after
1 ΧιΤλλΤιο 3/28 20/28 4/28 216 0/0 16/8 + I nUvJUu
0/64 i (10 *) (71 *) (14 *) 224 0 / ND 0 / ND + dies after 16/16 + 2 weeks 16/16 died after 268 0/256 5 weeks 292 + 0/256 294 + + 0/128 396 0/0 405 + 0/64 411 0/0 412 0 / ND 413 + 0 / ND 423 + + 0/64 424 + 8/16 426 0/0 575 + 0/128 655 0/8 667 256/256 699 256/32 700 0/0 701 64/64 818 0/64 824 0/0 ge 15/28 4/28 15/28 velvet (54 #) (149 &) (54 *)

(a) Against the virus isolated from dog 294

(b) ± = UEI weak;

+ = stronger signs

(c) 0 = <1: 8

(d) only against ME

PI as parainfluenza

509832/0963

TABBIIE V

Occurrence of parainfluenza and adenovirus infections
on the occasion of an epizootic respiratory disease

o for laboratory dogs

virus before
handene
antibody number
the
dogs with
Increases ¹ (62 *)
0 with virus
isolations (62 *)
0 with increases.
or insulation sick dogs
with virus isolate
lations or
Antibodies
increases in total
velvet
ill (58 *)
0 οι
ο Para
influenza no
£ 1: 8 24
4th ι ₅ ω 54 *) 15th (54 *) 16 (67 *)
0 14/16 (87 *)
0 14th (50 *) 983 all 28 15 (p ¹ (22 *)
(10 *) 15th (33 *)
(2 *)
(14 *) 16 (57 *) 14/16 (87 *) 14th (33 *)
(14 *) 2/0963 Adeno
virus no
> 1: 8
all 9
19th
28 1
3 3
1
4th • 3 (33 *)
1 (2 *)
4 (14 *) .3 / 3 (100 *)
. 1/1 (100 *)
4/4 (100 *) 3
1
4th (750 not
identi
fied 28 2

(a) No convalescent serum available from a dog that shed viruses and died; in another dog there was an increase in antibodies, they were
however, no viruses isolated from the dog.

(b) No convalescent serum obtained from a dog which shed viruses and
perished

cn ι O

! TABLE YI

Results of natural irritation of dogs by the
epizootic disease have recovered in the laboratory

Yirus

previous
Suspension* Number of dogs Number dsr
irritation Isolations at • PI Α (3 * ?: η, 0νΛτΉ $ "RrTTTiaZftTcttTlg 18th
6 "
20th
4th 0
3 (50 *)
3
0 H- 8th
2 +
4 (1007 &)

xisaESAia? . ■ '. ■ 24 ■ 3 ** 10 **

Sees Siere with antibodies before the onset of epizootic disease in the laboratory

vo 1

- »The virus isolations were carried out on different dogs; it cn

. «, ... igg ^ g Soppe infections found. 'CD

■ '' ■. ■■ ■ ■. . ■ '' -, cn

¹ ■■ ■ '■. ■ ·. ■ ■ · ■, ■. , ■ ■. ■ ο

CD

IABBLLE VII

Resistance to experimental Penn / 343/67 canine parainfluenza virus irritation in dogs previously infected naturally or experimentally with the same virus

Goal story

Number of pre-existing dog serum antibodies

- Reaction to the irritation
Number of dogs:

virus from diseased with serum separating antibody

increases

natural action

experimental input

(G.M. = 1: 114)

(G.M. = 1:89)

5 **

aesative

21

13 *

21

Beepiratory symptoms confirmed by positive radiological results and / or histopathological reads. '

Me suvor existing. Tit he was low enough to face amplification reactions virus antigen introduced into the chamfer and throat even without multiplication

seigen.

The invention provides a SV, - similar dog-Parainflueiiza JLebendvirusimpfstoff to Terfügung that can be / stored in a freeze-dried form for months at 4 ⁰ C. It has been found that this vaccine is safe, immunogenic and suitable for the containment and control of tracheobronehitis in external conditions. Intramuscular tracking is preferred. There are no side effects with this mode of administration and the vaccinated animals rarely or never excrete Yiren. When the vaccine is administered naturally (in the nose and throat), the vaccinated animals excrete viruses for about 1 week, but just as little become ill, as do the animals that come into contact with them (Table VIII). The use of a series of blood kidney cell cultures, eg Madin Darby dog kidney cell cultures (MDCK cell cultures), ... for the original isolation of the strain, for its subsequent passage and for the production of the vaccine, eliminates the risk of latent viruses being transmitted through the vaccine can be transferred to the vaccinated animal. By using a virus vaccine to immunize the dogs which has been grown in cell cultures derived from the same species, the possibility of allergic or hypersensitive reactions occurring in the vaccinated animals is also eliminated. External tests show that the use of this vaccine in a closed group of dogs previously susceptible to tracheobronehitis can lead to a 64% decrease in mortality.

According to the invention, a strain of the SV, - is isolated Canine virus and subjects it to at least five serial passages in MDCK cell cultures to produce a non-pathogenic to produce live virus vaccine, which when administered naturally or parenterally for Immunity leads. With regard to the antigenic character, it is indistinguishable from the pathogenic SV, - similar dog parain- +) from warm

509832/0963

fluenza virus (Penn / 343/67) »described by PJ Rosenberg, PS Mef, JB Todd and JS Rief in" Studies of Canine Respiratory Viruses. I. Experimental Infection of Dogs with an SV, - like Canine Parainfluenza Agent ", Im. J. of Epidemiology 94 (1971), 147, or from isolates which were made by the inventor or other researchers (M. Appel, PHPickerill, M, Menegus, DH Percy, IM Parsonon and Β.Ξ. Sheffy, “Current Status of Canine Respiratory Disease ", 20th Gaines Vet. Symposium, Manhattan, Kansas College Vet. Med., KSU Hewer Knowledge About Dogs, Oct. 22, 1970, pp. 15-23) were obtained from other epizootic diseased animals. A vaccine strain in the state of the fifth Passage (Penn / 103/70) was deposited with the American Type Culture Collection (ATCC) and was given the designation - ¹¹ ATCC VR 666 ". The trunk is available to everyone. It was originally isolated in MDCK cells and brought to high titer values by subsequent passage in these cells. The vaccine produced from this strain was found to be safe and provides resistance to natural irritation in external conditions.

The canine parainfluenza virus is bred for the vaccine by subjecting the original isolate several times to a passage in MDCK cells, which uses the infected supernatant obtained from the previous passage as the germinal substance. ^{The inoculated cultures are incubated stationary at 36 °} C. for 6 to 7 days for each passage.

The propagation of the MDCK cells can after any the standard methods explained in the literature. Using one of the recommended standard methods of breeding MDCK cultures use a tissue culture system Eagle Minimal Essence Medium in Earle Buffered Saline, where one gets 10 jSiges Petal calf serum, 100 penicillin units

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and add 100 mcg streptomycin / ml. To maintain the cultures, preference is given to using 2% calf serum before inoculation MDCK cell layers with medium 199, the the aforementioned antibiotics have been added to be washed. After the virus inoculation, the cultures should be in the same Medium with the addition of stabilizers in the form of SPG and 0.5 ^ gelatin, are stored.

To prepare the vaccine, use is made of the viruses in the supernatant to which the stabilizers (SPG and 0.5 % gelatin) have been added, as well as the intracellular viruses released by several rapid freeze-thaw cycles . centrifuged at 3000 rpm, the clear supernatant obtained constitutes the vaccine final product, which must be stored until use at -75 to -85 ⁰ C This final product shows when tested on guinea pig erythrocytes, a HA titer of 1..: 128 to 1: 256 and with titration in MDCK cell cultures a TCD ₁ - _A - ¥ ert of 1O ⁶ * ⁷ " ⁷ ' ⁷ .

In view of the instability of the vaccine at temperatures above -70 ⁰ C a Gefriertrocknungsmethöde is provided which permits the long-term preservation of the vaccine at refrigerator temperatures. In the manufacture of the lyophilized product, one less freeze-thaw cycle is required prior to clarification than is the case with the aqueous product. After resuspension in a suitable diluent, such as water (pH 7.0) *, the HA and infection titers of the freeze-dried product correspond to the relevant titers of the aqueous product.

5G8832 / 03S3

! EABBI ₁ IiE VIII

(ά)
In dogs given PI-103 vaccine ^v 'rhinopharyngeal

and the results obtained with dogs who come into contact with them

Vaccinated dogs (Each including
number with clinical
Reaction ("*>) with virus
drop out
manure with before
existing
Antibodies with serum wall
lung (0.5 nil in each nostril
administered and 1 ml
sprayed in the throat)
Contact animals 9 0 9 (c) 0 9 509832 (with the vaccinated Hun
on the 4th day after the '
Vaccination united)
Comparison animals 8th 0 8 (°) 0 Area =
1: 256 ( ^d )
8th ο
co
m 2 0 0 0 Area =
1: 256 W.
0 -. 1: 128
I. = 1: 64-

(isolated from those who have been vaccinated and those who come into contact with them Dogs)

(a) The vaccine contains 10 ⁶ ' ⁷ ₅₀

(b) Pieber, rabbits, conjunctivitis, or spontaneous cough.

(c) Start 2 days after vaccination or after contact with the vaccinated Dogs and lasts about 1 week.

(d) Serum neutralizing antibody titre. -

cn ό

O CD CD

- 15 -

The following are the preferred embodiments of the present invention explained.

The aqueous end product is produced in two stages. First, to a seed virus for the vaccine is prepared by mixing four times transmits the isolate in MDCK rows or subjecting a passage while a 6- Ms 7-day incubation period at 36 ⁰ C applies (in such an incubation, the maximum Infektionstlter be achieved ). Each yield, especially those of as a seed virus to be used for the vaccine production passage is mixed with stabilizers, before being divided into small aliquots until use at below -70 ⁰ C to freeze. The stabilizer (SPG) is used in a final concentration

resulting amount according to the method of Bovaraick et al. ""The Influence of Certain Salts, Amino Acids, Sugars and Proteins on a Stability of Rickettsiae," J. Bact. 89 (1950), 509 to 522, while adding sterile gelatin with a view to a final concentration of 0.5%. For reasons of expediency, the yields of the initial series passages can be frozen and transferred again at a later point in time in order to generate further germ virus stocks for vaccine production. These germ stores, which represent the liquid supernatant obtained from the infected cells, generally have a hemoagglutination titer (HA titer) with guinea pig erythrocytes of 1:64 and an infection titer (TCD _cn ) of 10 ^b ''''

In the second stage of vaccine production, virus replication is allowed to take place in MDCK cells, which are in 95 ml cells or roll tubes with the help of a germ or vaccine virus, which is subjected to at least four to five passages was to be bred. Before inoculation with the virus the growth medium is removed and the cells are washed twice

509832/0983

1 submitted later]

with medium 199, which contains 100 ax penicillin and 50 / Ug streptomycin per ml. The germ virus is then given in the form of a 100-fold dilution in medium 199, which contains the antibiotics as well as 0.1 % SPG and 0.5 % gelatin, in sufficient amounts to cover the cell monolayers in the small flasks or tubes. The cultures are then returned to the incubator for 1 hour. The rolled tubes returned to the rotating drum do not need to be shaken afterwards, while the vials incubated in a stationary position have to be shaken every 15 minutes. After the one hour has elapsed, the inoculated cultures are mixed with volumes of medium 199 containing the antibiotics and stabilizers suitable ^{for the respective container 1. The inoculation cultures are incubated for 6 to 7 days.} The pH is monitored and excess acidity is eliminated by adding a small amount of fresh medium while maintaining a pH of 7 to 7.2. However, such an addition is seldom necessary.

After the incubation has ended, sterile gelatin corresponding to a final concentration of 0.5 % and SPG is added to each vessel. At

closing the cultures are immediately frozen and stored at -70 to -85 ⁰ C stored for convenience, to preparation of the final product vaccine at these temperatures.

The final vaccine is made by giving the frozen cultures a total of 1 or 2 freeze-thaw cycles subjected to release intracellular virus. The liquid cell suspensions obtained from the individual containers can be used unite. The whole is to be centrifuged for 10 minutes at 3000 rpm. The clarified liquid supernatant represents the aqueous end product. This should be done immediately

509832/0963

Filled in the desired volumes in ⁺ bottles and, after carefully closing them, immediately stored at -75 to -85 ° C. When tested with guinea pig erythrocytes, the end product has an HA titer of 1: 128 to 1: 256 and when titrated in MDCK cell cultures

Value of 1Cr ''"'' ⁷ per ml.

Since the vaccine is relatively unstable if it is stored at temperatures above -7 ° C., a freeze- drying process was developed which allows the vaccine to be properly preserved. Table IX illustrates the instability of the aqueous product when stored at -2O ⁰ C, the temperature of a conventional freezing apparatus. Freeze-drying does not appear to have an adverse effect on infectivity. Experiments demonstrate that the freeze-dried product can be stored for at least 8 months at 4 ⁰ O without a loss of infectivity of suffering (see. Table X). After resuspension, the stability of the infectious capacity is the same as that of the aqueous vaccine (cf. Table XI). Freeze-drying uses a single cycle of freezing and thawing cells and fluid prior to centrifugation. Centrifugation and clarification are carried out as before; the supernatant should be freeze-dried immediately in the form of 2 ml to 10 ml volumes. The lyophilized product can be stored ^{at 4 ° C. until use.} The resuspended product should be used immediately.

+) matching

509832/0963

TABLE IX

Stability of stored at different temperatures ATCC VR 666 vaccine

• storage temperature Storage time TCD50 per 0.2 ml Change of the dekadi
logarithm I. cn -75 ⁰ C 5 days 10 ⁷ O ω O
co 7 month 10 ⁷ O I. 832, -25 ⁰ C 1 month 10 ⁵ , 5 -1.5 ^ *.
O 2 1/2 months 10 * , 5 -2.5 to
σ> 4 months <10 ¹ - 10 ⁴ -3 to -7 2505066 ο 5 months <10 ¹ - 10 ⁴ , -3 to -7 7 month <1P ¹ - 10 ⁴ -3 to -7

2505068

TABLE X

Stability of freeze-dried ATCC TE 666 vaccine at 4 ^° C

vaccine La & ex-ungbdauei * 503) 50
per ml modification original liquid
speed 0 10 ^{6 7} 0 lyophilic at 4 ° C
keep ■ 1 week 10 ^{6 7} O ti 1 month 10 ^{6 7} 0 η 5 months 10 ⁶ ' ⁷ 0 It 8 months 0

509832/0963

TABLE XI

Instability of freeze-dried vaccine after. Resuspension and stored at 4 ⁰ C

Storage daiier mitigation of the

TCD5O *
(Logarithm)

Resuspended <1 day O

freeze-dried

Vaccine stored at 4 C for 2 to 14 days - 1

1 month '' -2

2 months - 3

* The changes are the same as in fresh yields of this virus which were stored ^{at 4 ° C.}

509832/0963

Testing the vaccine example 1

In the experimental animal facility of the ^ Flow Laboratories, an extensive series of experiments was carried out in a large kennel, which is specially equipped to / for hybrid dogs that have undergone suitable treatment for research purposes. deliver. The animals in this kennel were constantly afflicted by diseases of the respiratory organs. The kennel always contained a colony of 500 to 800 dogs, with 60 to 100 new animals being added weekly. All dogs that were added were immediately vaccinated against distemper, hepatitis, leptospirosis and rabies, except that they were freed from worms, shaved, bathed etc. Of the entire colony, an estimated $ 10 per week died due to respiratory disease. This mortality means that more than 50 % of the dogs admitted to the kennel weekly do not survive their 6- to 8-week stay.

In the first attempt, two groups of newly admitted hybrid dogs (31 animals) were separated from the rest of the colony and administered the aqueous vaccine, which was stored at ~ 75 ° O and contained 10% TCD, -q / ib1. The. Dogs were administered 1 ml doses in both the right and into the left Nackenamskel. Seven to ten, fag © naoh aev vaccination, all dogs except for the animal were resumed in a hurry "general colony. At the SPagea-0, % 7, 10 20 Ehinopharyngeal Abstrlelie for the Viri & laoli were removed. Blood samples for the Antibodies & Xysie threw @m_ administration day and 4 weeks after the vaccination sMgemnmme All animals were at least 6 weeks long a © obac? M @ t | # 1 © four E ^ sM, © trjuräesi left in isolation

509832 / OSS3

antibody persistence was retained for 15 months.

All here remained undamaged, even natural ones Irritation. With one exception (from which viruses were only obtained on day 7), none of the animals excreted viruses (See Table XII), Although none of the dogs had detectable circulating antibodies at the start of the study, 29 of the 31 animals showed 4 weeks after vaccination El'öe serodonversion with a duro-average fclien H-T-titer of 1:55 · Those appearing later than the H-I antibodies neutralizing antibodies had an average titer of 1:78 at this point in time. (see Table XIII). Neutralizing antibodies developed in all four dogs kept in isolation for 15 months high titers persisted. On the other hand, the H-I antibodies in these dogs decreased even though they did after 15 months were still present, starting from ¥ ert after one month by 2 to 4 times (see Table XIV).

example

Using the same equipment as in Example 1, a total of 38 dogs with a 10 '' vaccine containing vaccines. Of these dogs, twenty were intramuscularly and the remaining eighteen were subcutaneously im

Vaccinated. G-eschwiMigkeit "jnd O-rad of SeroumwandliHig are for" about the same as both Verafefolgungswege "© at the first attempt (Ss ^ elle Xf) ₈ Bie vaccine is soiEit assülieinend feei intraiansfealärer or sublcutaner YeT- abreieliimg equally well to stimulate antibody sirkuliersnder

TABLE XII

Disease and Yirus Shedding in Dogs That Were "at -75 C stored ATCC YR 666 yakzine was administered

Dose and Yerab number Number of virus-shedding number consequently of the sick. _{Days NACIL} ^ _ex vaccination:

O 3rd day 7th day 10th day 20th day

cn 2x10 'TCD50 dogs dogs ο intramuscular (O * co 31 0 O CO O) CJ

.0

Total number of those leaving animals

1/31

Dose and route of administration

2x10 'TCD50 intramuscular

TABLE ΣΙΙΙ

Antibody reactions. of dogs compared to -75 ⁰ C
stored ATCC VR 666 vaccine

number

the

dogs

H-I liter

converting number

Average

average

29/31 ($ 93) Counter hands! Egg ter

converting mean average value worth cut

(D)

<8/64 <8/78

(a) Pre-vaccination titre / post-vaccination titre

(b) the positively reacting animals

(c) two dogs not tested

cn co co

TABEI & E XIY

Persistence of antibodies in dogs which stored at -75 ⁰ C ATCC YR 666 vaccine was administered

Dose and yer- dog 1 day H-I titer 2 mo 6 mo ■ 9 mo • 15 mo Ni sutralj ide titer 9 mo 15th Mon- Mg delivery route No. 1 mo nate nate nate nate O day 1 mo 6 mo nate nate 56 ö nat nat nate 64 cn
ο <8 64 32 32 32 256 28 co
CS 2 χ 10 ⁷ 4655 128 <8 256 256 128 c *> <8 64 32 64. 64 128 IO
"%" T0D50 4662 <8 128 32 32 '32 <8 32 256 64 2 O
co Intramuscular 4679 <8 64 32 32 64 64 <8 32 64 128 ι ' co 4684 <8 64 32 <8 <16 128 1 4656 16 <8 64 ' cn CD 90S CD Lsierei 2 mo nate 128 128 / 16 '64 •

TABLE XV

H-I antibody responses in dogs to -75 ° C ATCO VR 666 vaccine stored frozen and administered in various ways

Dose and route of administration

2 χ 1O ⁶ ' ⁷ TCD50 intramuscular

2 χ 1O ⁶ ' ⁷ subcutaneously

Total number of sero-converting dogs showing number of dogs

20th

18th

17 (85

17 (94

Titer before Titer later 512 I.
σ \
I. G.M. area G.M. * area 512 <β <8 64 * 16 to <8 <8 51 8 to

* of the positively reacting animals cn

cn ο

DG CD

example

This test attempted to reduce the loss of infectious capacity due to high storage temperatures; to be related to immunization capacity. Ueun dogs were intramuscularly mounted with stored at -75 ⁰ C aqueous vaccines nine dogs with the same, -20 ⁰ C stored at 'vaccine batch and ten dogs with the same, at 4 ⁰ C vaccine batch vaccinated. Table XVI shows that there is a direct relationship between the level of antibody response and the amount of live virus remaining in the vaccine. The highest titers were obtained with the samples stored at -75 ° C the preparation with the lowest stored at 4 ⁰ C material.

A similar analysis was carried out with freeze-dried vaccines in order to determine their ability to immunize against them of aqueous vaccines stored at -75 ° C to compare. 29 dogs were intramuscularly 2 ml of a resuspended

Preparation with'einem content of 10 'TCDp-q / πιΙ administered; it was found that the antibody reactions; those are very similar to those caused by the samples stored at -75 ⁰ C vaccine (see Table XVII.). .

example

From mid Pebruar 1970, all new entries of mongrel dogs colony in the above-described ¥ else were immunized intramuscularly with 2 ml of the ATCC VR-666 vaccine that had been stored at -75 ⁰ C, ^ inoculated. Since it was known that antibodies against this virus were detectable as early as the 8th day after vaccination, the freshly vaccinated dogs were kept isolated for 7 to 10 days before union with the general colony. This course of action was

509832/0963

maintained until nearly 100 % of the colony was vaccinated. Thereafter, newcomers were reunited with the general colony without the 7 to 10 day segregation.

S098 3 2/096

TABLE XVI

HI antibody response from dogs versus 5 months
ATCC VR 666 vaccine stored at different temperatures

Camp- TCD50 Dose and admin number converting (100 i \ H-I titer By- I.
ro temp. . per ml delivery route the Number of funds ■ cut VO en üunae (89 *) value I. ο (90 #) co 9 <8/32 00
GO -75 ⁰ C 10 ⁶ 2 ml intramuscularly 9 ) <8/52 * to
"« Ν noisy 8th <8/16 O
CD -20 ⁰ C 1O ³ ' ⁵ Il 9 9 <8 / i6 <β / 15. CD
CO +4 ⁰ C 10 ^{1 0} η 10 <8/8

* Titer before vaccination / titer after vaccination

CD CD CD

TABLE XVII

Canine Antibody Responses to Freeze-Dried ATOO VR 666 Vaccine

Dose and route of administration

2 10 ⁶ ' ⁷ TCD50 intramuscular

number

the

dogs

29

H-I-TIter

Neutralizing titers

Converting Mean Average Average Number

converting number

Medium average

(90 50 <8/64 ^ <8/48 20 (70 50 ^ <8 / i6

<8/83

(a) Pre-vaccination titer / post-vaccination titer

(b) the positively reacting animals

(c) the H-I antibodies appear before the neutralizing antibodies. These sera are taken before the neutralizing antibodies reach their maximum.

The mortality shown in Table XVIII during the 5-month period before the start of the vaccination shows that (“calculated on a 4-week basis) an average of 5» 5 "to $ 10.8> of the respective colony succumbed to a respiratory organ disease. As is normal for an alternating colony of this type, weekly mortality varies considerably. In a few weeks it reached values of 14 to 16 io thereafter, presumably as a result of the decrease in the number of remaining potential recipients. When new recipients were admitted, this decrease was always followed by a marked increase in weekly mortality.

The effects of the vaccinations only became apparent after the majority of the dogs in the colony had been vaccinated. For the week ending March 24, 1971, 50 # of the dogs present had been vaccinated. The total colony mortality that week was $ 4. As the percentage of dogs vaccinated increased, the mortality steadily decreased. When 70 to 100 # of the dogs in the kennel were vaccinated, the weekly mortality was $ 2. No vaccine was available from April 5 to May 8, 1971; new, unvaccinated dogs were admitted, while vaccinated .. dogs were sold. During this period, the mortality increased from 2.1 # (1 Wocher before) to 4.7 f> \ With the resumption of vaccinations, the mortality fell again immediately. Before the former value was reached again of about $ 2>, inadvertently stored at -20 ⁰ C aqueous vaccine was administered. This fallacy was discovered when it was discovered that more dogs were dying of respiratory disease and the weekly mortality rate was increasing. Resumption of vaccination with an effective vaccine gradually reduced mortality to permanent values of 0.4 to 1 $ .
+) and

509832/0983

TABLE ZVIII

co
m co

Weekly mortality in a bastard dog kennel during a Comparison period and a period of vaccination with frozen ATCC VR 666

period

16 September
14.10.
12/10/70

7. 1.

4. 2.

-13.10.70

- 11.11.70

- 6. 1.71

- 3. 2.71

- 17.271

Total number of dogs at risk

2894 3015 2328 2975 1245

Animals that have died or have been euthanized due to illness

313 203 196 162 101

Vaccination status

0
0
0
0
0

Start of vaccinations in the week after

10. 2. - 10. 3.71 2268

March 11th - 7th 4.71 2074

8.4

- 5.71

6th 5. - 2. 6.71 3. 6th - 30. 6.71 1. 7th - 28. 7.71 29 7th - 25th 8.71 26th 8th. - 22. 9.71 23 9. - 6. 10.71

2676

2930 2429

2492 2589

2278 1049

10.2 .;

of 601 dogs vaccinated.
<10 - 40 p

gradually to 100 #
elevated

average weekly mortality per total colony

10.8 6.7 8.3 5.5 8.1

i
j

7.3

no vaccination, gradually decreased to $ 40

- 80 ^

-20 ° C-Val: zine with a low titer in this one
Period used

$>
%
$
%

4.7

4.5 6.9

3.4

1.1 1.1 0.4

The overall mortality during the comparison and vaccination periods is shown in Tables XIX and XX. The weekly mortality for the reference period "is $ 7.4. Even including the period during which no vaccine was available, the mortality during the vaccination period is $ 3.92>. If one takes into account only the period during which d ' If the vaccine was available, the mortality decreased to $ 3.39. In the Chi ² test, ρ is less than 0.001 * Table XXI shows that the above values can be maintained when using freeze-dried vaccine.

The suitability of the aforementioned vaccine for the prevention of iracheobronehitis can best be demonstrated by testing the mortalities of dogs which were admitted to the kennel before or after the start of vaccination with the ATCC VE 666 vaccine ^ From 1980 of September 1970 1141 dogs admitted up to March 1971 »died or were euthanized due to a severe respiratory disease. After the vaccinations were started, of the 2391 dogs admitted over the next 9 1/2 months, only 626 died or were euthanized for the same reason. Instead of a mortality of $ 58 6, only $ 26> of the dogs succumbed to a respiratory organ disease during the vaccination period. Then in 1972 mortality continued to decline; 79 of every 100 dogs that were added survived, which corresponds to a mortality decrease of 64 i> .

The loss of $ 20 of all newly admitted dogs as a result respiratory disease is still a problem. Several other pathogens can cause symptoms similar to how Cause tracheobronchitis. To a certain extent, the aforementioned mortality can be explained by the fact that some animals +) (see Tables XXII and XXITI) '.

509832/0963

which are pre-infected, and indeed not only with the canine parainfluenza virus, but presumably also with distemper, Ich- and Ich-like viruses, and probably also with bacteria. No vaccine is "able to prevent the full progression of these diseases." By giving the aforementioned canine Parainfluenza virus vaccine to all newly arrived dogs

administered, however, was clearly the transmission of a! Main causative agent of respiratory organ infections on other inaassen of the kennel. The mentioned canine parainfect luenza virus vaccine is when described in the Manufactured and administered in a manner that is undoubtedly risk-free and immunogenic, and enables effective Reduction of the mortality that occurs with outbreaks of respiratory diseases in dog kennels.

509832/0963

TABLE XIX

Average weekly mortality per total colony in
Periods without or with vaccination (with frozen ATCC YR 666)

Period vaccination died survived ' ^a ' total ^ 'mortality ^ ⁰ ·

16.9.70 - 11.271 no '885 10 961 11 846 $ 7.47>
20. ' 5:? 1.- Ι'Λ'Ά] ' ^{äa 559 15 952 1β 511.3.39} *

to ■

^ (a) Total number of dogs surviving each week.

^ (b) Total number of dogs at risk per week.

& dead dogs

cn ( _c ) mortality =

■ Total number of dogs / weeks in danger

(d) P = <0.001

TABLE XX

Average weekly mortality per total colony in periods with and without vaccination (with frozen ATCC VE 666)

period

Vaccination died survived

total

mortality

16.9.70 - 11.2.71 10.2.71 . - 6.10.71

no

3a

885 792

10961 19434

(a) Total number of dogs surviving each week.

(b) Total number of dogs at risk per week.

dead dogs

(c) mortality =

11846
20226

7.47 *
£ 3.92

(e)

Total number of dogs / weeks of endangerment

(d) Including the periods from 7.4. - May 19, when there was no vaccination and the percentage of vaccinated dogs in the colony fell to 45, and from May 27 to June 30, when vaccines with a low titer stored ^{at --2O 0 were used;} there was some increase in mortality during these periods.

(e) P = ^ 0.001

V »

CD CD CD

TABLE XXI

Weekly mortality after starting freeze-dried vaccination Preparation*

week 21. 2.73 Total colony recorded
dogs dies or
due to illness
euthanized Mortality (#)
per total
colony I.
Previous year 15th 2. - 28. 2.73 222 40 4th 1.8 I. 22nd 7th ■ 3.73 243 48 3 1.2 29 2. - H. 3.73 253 45 8th 3.1 8th. 3.- 21. 3.73 255 44 O O 15th 3.- 273 47 · 2 0.8 TOTAL 1246 224 17th 1.3

* Starting on 02/12/73, the new dogs were given intramuscular 2 χ 10
of the preparation administered.

6.7

TCD

50

ro cn ό

! TABLE XXII

Influence of the use of ATCC VR 666 vaccine on the Mortality of dogs admitted to the hybrid colony

cn ο co oo approx

period 10. 3.71 recorded
dogs vaccinated! eggs 16.9.70- 1980 none "until 02/10/71 7th 4.71 <10 to <40 i> between
the 10.2. and 10.3. 3/11/71 - 5. 5.71 346 100 $> vaccinated 8.4.71 - 30th 6.71 486 no vaccination; number
gradually sinks to
40 # from 6.5.71 - 751 Vaccination resumed
taken, but at all
neuter · vaccine ver
applies, which is at -20 C
stored "

i.7.71 - 6.10.71

808

ft vaccinated

dead or euthanized mortality fi

animals

1141

55
125

, 299

147

57.6

15.8
25.7

39.6

18.1

CD CD CD

TABLE XXIII

Mortality "in dogs that were in the hybrid dog kennel during periods without I) ZW. With vaccination be included

comparison

16.9.70 - 10.5.71

vaccinated

3/11/71 - 12/31/71

vaccinated

1.1.71 - 31.12.71

Admitted dead or euthanized dogs

1980

2391

3724

1141 626

1014

mortality

57.6

26.1

27.2

'vaccinated
1.1.72 - 31.12.72

2339

507

21.2

NOTE ·: The comparison period includes the period from 10.2. - 10.3.71, while Aem less than 40 years of the dogs were vaccinated. During certain periods after the start of the vaccinations with PI 103 no vaccine was available and it was inadvertently used vaccine which has been inactivated by storage at -2O ⁰ C. Morbidity and mortality increased during these time periods regardless of season, but decreased again as soon as the vaccine with good infectivity was reused (Table XVII). ,

509832/0963

Example 5

This test was carried out in another kennel of the experimental animal facility of the plow laboratories, in which beagles were bred. There were in the kennel a total of about 500 dogs. In particular, the 5 to 8 week old young dogs developed severe symptoms Respiratory disease. More than $ 50 of the young fell ill, including those affected by the disease Animals perished. As with the first colony, one of the suggested ones was obtained from the sick young dogs Parainfluenza pathogen similar to vaccine strain. It was set up a vaccination plan for young dogs.

As a result of a decision by the Flow Laboratories to dissolve the sniffer dog colony within two months, the options for testing the vaccine were severely restricted. Between 19 February and 10 April 1971, 230 young dogs between the ages of 5 and 7 years were vaccinated. They were given 1 ml of vaccine, evenly distributed and indexed into the neck muscles. The usual mortality from respiratory disease in this age group suggests that 50 to 60 $> of dogs would have become sick and perish if they had not been vaccinated. In the vaccinated group, the mortality was $ 30.

The reliability of this test was greatly reduced by the shortened test period. From some of the vaccinated Dogs that died and other non-vaccinated dogs young dogs suffering from a respiratory disease or were about to die due to this disease, parainfluenza viruses have been isolated. Many of the vaccinated Young dogs that have died may have been naturally infected prior to vaccination. If-

509832/0963

More extensive and better controlled investigations are required, as those already available show Values that the described canine parainfluenza traeheobronchitis vaccine has practical applicability for colonies of the type mentioned.

509832/0963

Claims (1)

PATENT CLAIMS 1. A process for preparing an aqueous canine tracheobronchitis vaccine, characterized in that an SVC-like canine tracheobronchitis virus strain of a series passage in one of! Impurities free, contiguous ⁺ Warmblood kidney cell culture is subjected. 2. The method according to claim 1, characterized in that one in an additional First stage isolate the virus strain in the cell culture. 5. The method according to claim 1, characterized that one increases the virus yield in further stages by using the 'intracellular Virus is released by rapid freeze-thaw cycles of the cell culture and the resulting liquid cell suspension Centrifuged at low speed to produce the aqueous vaccine in the form of a clarified supernatant. 4- The method according to claim 1, characterized in that one (a) in further stages SPG SPG and gelatin added to achieve a final concentration of 0.5 # gelatin, around the HA and infection titer from the + (continuous-line) 509832/0963 1 NAUTRICHI virus yields obtained in cell culture to stabilize and (b) the medium during the propagation of the virus-containing Cell culture SPG and gelatin added to achieve a final concentration of 0.5 % gelatin to prevent inactivation during propagation. 5. The method according to claim 1, characterized in that the virus strain at least five times a serial passage - subjected to Darby canine kidney cells in Madin. 6. The method according to claim 1, characterized that the virus strain Penn / 103/70, ATCC VR 666 is. 7. The method according to claim 5, characterized in that each passage 6 incubated to 7 forecast at 36 ⁰ C. 8. The method according to claim 3, characterized in that one uses to release the virus particles from the cells 1 ■ or 2 freeze-thaw cycles. 9. Process for the preparation of an aqueous canine tracheobronchitis vaccine, consisting of the following levels: (1) Isolation first and. then at least five times serial passage of the canine tracheobronchitis virus strain having the designation ATCC VR 666 in a Madin Darby Canine Kidney cell culture, wherein each passage is incubated for 6 to 7 days at 36 ⁰ C, 509832/0963 (2) Stabilization of the HA titer and infection titer by adding SPG and gelatin corresponding to one Final concentration of 0.5 1 ° gelatin for the recovery of the virus strain and SPG . - and of gelatine corresponding to a final concentration of 0.5 % gelatine for the propagation of the virus strain, (3) Increase in the yield of virus by releasing the intracellular virus with the aid of 1 or 2 batches Cell culture and freeze-thaw cycles (A) Centrifugation of the resulting liquid cell suspension at low speed to produce the aqueous vaccine in the form of a clarified supernatant. 10. A process for the preparation of a freeze-dried canine tracheobronchitis vaccine, consisting of the following stages: (1) Series passage - "" _ ■ of an SV ^ -like canine tracheobronehitis virus strain in a contiguous warm-blooded animal free of foreign matter Do not do it, (2) Addition of SPG ' and of gelatin corresponding to a final concentration of 0.5 # gelatin (upon recovery of the virus strain from the cell culture) as well as from SPG and of gelatin corresponding to a final concentration of $ 0.5> gelatin (during the propagation of the virus strain), (3) Increasing the virus yield by releasing the intracellular virus with the aid of rapid freeze-thaw cycles the cell culture, 509832/6963 (4) Centrifugation of the resulting liquid cell suspension to produce a clarified, yirus-containing one Overhangs, (5) freeze-drying the supernatant, (6) Store the freeze-dried product at ⁴⁰ G until use, and (7) Resuspending the product in an appropriate ¹ resuspending medium to produce the freeze-dried vaccine. 11. The method according to claim 10, characterized in that one -more First, isolate the virus strain in the contiguous warmblood er-merenzellkultur series. 12. The method according to .Anspruch 11, characterized in that the virus strain first isolated and then at least five times of an iSeries passage. in a Madin Darby canine kidney cell culture subject. 13. The method according to claim 10, characterized that the virus strain is Penn / 103/70 ATGC VE 666. 14. The method according to claim 12, characterized in that that each passage is incubated for 6 "to 7 days at 36 ° C. 15. The method according to claim 10, characterized in that there is a single Applies freeze-thaw cycle to release virus strain from cell culture. S09832 / 0963 1 SUBSCRIPTIONJ 16. Process for the preparation of a freeze-dried Canine tracheobronchitis vaccine, ordered from the following stages: (1) First, isolation and then at least five times serial passage of the canine tracheobronchitis virus strain having the designation ATCC VR 666 in a Madin Darby Canine Kidney cell culture, wherein each passage is incubated for 6 to 7 days at 36 ⁰ C, (2) Addition of SPG. and gelatine corresponding to a final concentration of 0.5 Ί » gelatine (when extracting the virus strain from the cell culture) as well as SPG . and of gelatin corresponding to a final concentration of $ 0.5> gelatin (during the propagation of the virus strain), (3) Increasing the virus yield by releasing the intracellular virus using a rapid freeze-thaw cycle the cell culture, (A) centrifuging the liquid cell suspension obtained to produce a clarified, virus-containing supernatant, (5) freeze-drying the supernatant, (6) Store the freeze-dried product at 4 ° C until use and (7) Resuspension of the product in a suitable one Resuspension medium to create the freeze-dried vaccine. 17. Method for stabilizing the HA-Iiter and the infection-titer a virus-containing cell culture, d a - 509832/0963 characterized by that one incorporated into this SPG and gelatin. 18. The method according to claim 17, characterized indicates that SPG to achieve a final concentration of 1 ° SPG and gelatine to achieve a final concentration of 0.5 $ gelatin for obtaining the virus from the virus-containing cell cultures and SPG to achieve a final concentration of 0.1 io SPG and Gelatin to achieve a final concentration of 0.5 %> gelatin added for the propagation of the virus-containing cell culture. 19. A method for preserving the infectious capacity of a virus yield in freeze-drying, characterized in that one SPG to achieve a final concentration of 1 # SPG and gelatin to achieve a final concentration of 0.5 # gelatin added. 20. Traeheobronchitis vaccine produced by a process according to claim 1 to 19 509832/0963