WO1999037327A1

WO1999037327A1 - A virus vaccine of enhanced immunogenicity against aujeszky's disease

Info

Publication number: WO1999037327A1
Application number: PCT/EP1999/000745
Authority: WO
Inventors: Judit Erdei; Maria Kelemen; Béla KÖVES; Béla LOMNICZI; Ferenc MISÁK; Jozsef Nagy; Enikó WEHMAN
Original assignee: Sanofi Sante Nutrition Animale
Priority date: 1998-01-26
Filing date: 1999-01-26
Publication date: 1999-07-29
Also published as: AU2521899A

Abstract

The present invention concerns a vaccine against Aujeszky's disease containing an avirulent live virus or an inactivated virus, wherein said virus is an Aujeszky's disease virus producing large plaques in cell culture and expressing the glycoprotein gC, in association with a pharmaceutical acceptable carrier.

Description

"A virus vaccine of enhanced immunogenicity against Aujeszky's disease"

The subject-matter of the invention is a veterinary live or inactivated virus vaccine of enhanced immunogenicity against Aujeszky's disease the method for its production, and a particular virus strain forming the basis of the vaccine

Aujeszky's disease was named after the scientist first describing it, the Hungarian Aladar Aujeszky (Zbl Bakt Oπg 1/32, 353-357 1903) Aujeszky's disease is an infectious disease caused by a virus io belonging to the group of herpesviruses Numerous mammalian species except the horse are susceptible to the causative virus which is spread by pigs

Infection often occurs in epizootic form in domestic animals Its symptoms include encephalitis, paraesthesia, and pruritus, which signs l ^ prompted the former name of the disease (pseudorabies)

In pigs, paraesthesia is absent and the clinical picture is dominated by signs related to encephalitis, such as intense excitement compulsive movements, and convulsions extending to the whole body After the onset of clinical signs the animals, especially the baby piglets, usually

:iι die The rate of piglet mortality caused by Aujeszky's disease decreases with the advancement of age, however the respiratory disease the nervous signs and the complications occurring in older pigs markedly impair the profitability of pig production Virus-infected animals may carry the pathogens for a long time and, when exposed to stress such as farrowing,

: may spread the disease through virus shedding Infected sows often abort their fetuses Infected pigs also play an important role in spreading Aujeszky's disease to ruminant animals (cattle and sheep) Both the virus and the colostral antibodies may be excreted by infected sows via milk

According to the present state of knowledge, the disease is

'<» incurable The only possible approach of control is prevention aimed at protecting individual animals and stocks Prevention is based upon prophylactic inoculations and vaccination A hyperimmune serum was developed for the preventive treatment of baby piglets for the control of Aujeszky's disease (Acta Vet. Acad. Sci. Hung. 7, 423-427, 1957).

When used at the appropriate time, the hyperimmune serum reduces the losses. However, its disadvantage is that it is rapidly excreted from the organism (in 10-14 days), thus its protective effect decreases. In addition, the high cost of the hyperimmune serum makes it unfeasible for use in growing pigs and in herds of adult animals.

In order to eliminate the drawbacks of hyperimmune serum, vaccines containing a live virus strain of attenuated virulence or a variant thereof were developed.

The Hungarian patent specification no. 149.286 presents a method for the production of an immunogenic Aujeszky's disease virus (ADV) variant of reduced virulence and for the production of a vaccine prepared from that variant.

According to this method, Aujeszky's disease virus is grown in mammalian cell cultures, especially in pig kidney epithelial cell cultures, by Dulbecco's plaque methods. The virus strain thus obtained was named variant "K" (Magyar Allatorvosok Lapja lδ, 42-45, 1961 ).

Preferably such a virus is used for selecting variant "K" which has been grown through 20-30 passages at a temperature between 25 and

40 °C. Virus variant "K", which was deposited at the Hungarian National Institute of Public Health under no. 601 121 1 , was processed into a vaccine containing live virus.

Adorjan Bartha subsequently replaced the virus variant of reduced virulence contained by the live virus vaccine by the trypsin- and heat-resistant virus variant deposited at the Hungarian National Institute of Public Health under no 00166.

It is a disadvantage of live virus vaccines that they do not confer potent immunity on newborn piglets possessing maternal antibodies up to 4-6 weeks of age, and that they are not entirely safe for dogs, minks and cats. After the use of vaccines containing either live virus or inactivated virus, the conventional serological methods used for checking the animals' immune status (especially the virus neutralisation test) cannot distinguish between antibodies induced by natural infection of the animals and those elicited by preventive vaccination; thus, it cannot be determined whether the animal possesses natural or vaccination-induced immunity. A further disadvantage of the hitherto known vaccines is that while they confer protection on the animals, a certain proportion of the animals may become virus carriers. While virus carriers do not become affected, they may infect their surroundings and thus also the animal stocks living there; this means that such vaccines do not prevent the spread of the wild virus (Current Topics in Veterinary Medicine and Animal Science 49, 209-213, 1988).

At present, numerous avirulent, live Aujeszky's disease vaccines lacking pathogenicity to pigs but possessing immunising ability against the disease are on the market. The avirulent strains forming the basis of these vaccines share the property that one of the genes responsible for the virulence of the virus strain has lost its function. ADV has multiple genes (absolute virulence genes) the loss of function of even one of which causes the virus to lose its pathogenicity. In the vaccine strains already used at present, one of the two main virulence genes are defective: either the gene designated UL21 or the thymidine kinase (TK) gene (Fig. 2).

The loss of virulence of the vaccine strain designated K/61 is caused by the mutations occurring on the UL21 gene (Virology 212, 466- 473, 1995).

No other vaccine strain having an UL21 gene defect is known_. The vaccine containing the K/61 strain has been on the market since the mid-1960s, and it has proved to be reliable in the respect that no increase in its virulence has been observed even after 30 years of use.

The virus strain designated K/61 is otherwise a naturally occurring mutant which, besides being avirulent, produces small plaques in cell culture (the Hungarian for "small" is "kicsiJ hence the abbreviation: K). It was precisely on that basis, i e by plaque selection, that the strain was found (Bartha, 1961 , HU 149 286)

On both pig kidney and chicken cells it forms much smaller plaques than the virulent virus (e g the parent strain) or any other vaccine strain (Arch g Virusforsch 1974, 44, 205-214) The detrimental effect of small-plaque type can be confirmed also in pigs in vivo When inoculated intranasally to some weeks old piglets, the individual vaccine strains grow to different titres This can be followed precisely by taking nasal swab samples on a daily basis It can be established that strain K/61 has much poorer replicating ability in the nasal mucosa of piglets than do vaccines containing a thymidine kinase gene negative, large-plaque virus strain While the thymidine kinase gene negative strains replicate for 10-12 days, yielding several million infective viπons at the time of virus peaks, strain K/61 has a virus yield corresponding to about 1 % of the former and, correspondingly, its persistence in the nasal cavity is some days shorter

The group of TK-negative vaccines, i e those having a defective TK gene, include several virus strains one of them, the Tatarov strain (designated MK-25), was produced from virulent virus by passage in cell culture in the presence of mutagenic chemicals, e g bromodeoxyuπdine (Zentralbl Vet Med B 15, 857-863) As a result of mutations (nucleotide exchanges) occurring in the TK gene during that procedure the TK gene will lose its ability to function While this does not affect the replicating ability of the virus in the cells of tissues except those of the nervous system, the virus will be unable to invade the nervous system and, thus, will lose its virulence Recently the TK gene is rendered inoperative by genetic manipulation, when the entire gene or a portion thereof is deleted Attenuated virus strains obtained in this way form the basis of numerous live virus vaccines (Suvaxyn 783, Omnivac, Tolvid, Marker-vac) Thus, in both groups either one or the other so-called absolute virulence gene has become inoperative or has been artificially deleted

The authors of the present invention have constructed avirulent Bartha's K/61 virus strain variants that produce large plaques and express the glycoprotein gC (gC⁺) (previously known as glycoprotein III), said virus strain variants being useful as vaccines as they exhibit enhanced immunogenicity and excellent protection of animals against virulent Aujeszky virus. A subject of the invention is thus a vaccine against Aujeszky's disease containing an avirulent live virus or an inactivated virus, wherein said virus is a Aujeszky's disease virus producing large plaques in cell culture and expressing the glycoprotein gC, in association with a pharmaceutically acceptable carrier. Said virus is preferably a Bartha's K/61 virus strain variant.

"Bartha's K/61 virus strain variants" refer to naturally-occuring variants or to genetically engineered strains obtained from the known Bartha K 61 strain.

The virus used in the vaccine of the invention can be alive or can be inactivated by any method well-known by one skilled in the art.

The live strains may be rendered avirulent by deleting TK, UL21.

Furthermore, using strains that lack the gE gene (previously known as gl) is particularly advantageous because it further allows the distinction between immunized animals, that do not produce gE-specific antibodies, and infected animals, that produce gE-specific antibodies.

The virus strains that compose the vaccines of the present invention may advantageously possess a detectable marker, such as a genetic marker detectable by analysis with DNA restriction enzymes. Such a detectable marker allows any one skilled in the art to identify the vaccine strain of the invention, at least to solve the following problem :

The single immunising dose of vaccines containing live Aujeszky's disease virus is at least 10⁵ infective particles. Over the years, billions of vaccine virus particles have been released into the environment from the Hungarian pig herds alone. It would be a basic requirement that every manufacturer could unmistakably recognise their own vaccine, so that they can ward off any criticism concerning that vaccine. In the past few decades, the view has repeatedly arisen that the symptomless forms of Aujeszky's disease virus infection are in fact caused by independently spreading vaccine strains, or that vaccine strains are responsible for causing disease in day-old piglets (Arch. Virol. 124, 225-234, 1992).

As shown in example 1 , the extra SamHI cleavage site occurring in BamHI fragment no. 2 can be advantageously selected as a structural marker capable of strain recognition. That cleavage site does not occur in Hungarian strains at that site, it can be easily and unmistakably identified on the gel during the electrophoresis used for the analysis, and it is in the immediate vicinity of the gC gene occupying the right end of fragment 2; therefore, if the marker can be found in any virus progeny of Bartha type, it is very likely that the DNA fragment carrying the gC gene next to the marker has also been transferred into the DNA of strain K/61 . Namely, the marker selected for labelling the vaccine strain also served as a marker of the DNA segment carrying the gC, which below proved to be a major help during the search for the progeny virus. In the examples, the correctness of the outlined approach has been proved by the ratio of the recombinants inasmuch as the recombinant (10a) randomly selected from the dual structural mutants (gE deletion at map units 0.85-0.88 and occurrence of a SamHI cleavage site between map units 0.32 and 0.34) has proved to be also gC positive when tested with a gC-specific monoclonal antibody (designated b68-C3 and recognising gC).

A subject of the present invention is more particularly a modified K/61 NM virus strain deposited under n° NBIMCC 3551 , on December 20, 1997.

This avirulent strain exhibits the following properties : it possesses the gC gene ; it forms large plaques in cell culture ; it possesses a marker gene enabling individual strain recognition.

This strain was obtained by recombination, i.e. by co-culturing donor and the recipient strain in a susceptible cell culture, and selecting 7

those recombinants of the arising recombinant progeny which carry the desired properties.

It is also possible to obtain an equivalent strain by genetic engineering procedures, wherein a gene fragment of precisely known function is deleted from the DNA of a virulent virus donor strain, incorporated into a bacterial plasmid vector, and transferred into a recipient.

The produced strains may be stored at a temperature of -65/- 75°C if desired or lyophilized at a temperature between -20°C and 4°C.

The new mutant virus strain (K/61 NM) NBIMCC 3551 has more particularly the following properties:

1. It is UL21 negative like the parent strain designated K/61. This actually means that the mutant having new properties is similarly avirulent as the parent strain K/61. This was checked by testing the virulence of the mutant grown in cell culture by the intracerebral (i.e.) inoculation of day-old chickens. The chickens remain healthy even after inoculation with 10⁵ plaque-forming units (PFU) of infective particles, while the virulent virus kills them already in a dose as low as 10² particles.

2. The K/61 NM virus strain forming the basis of the invention is gE negative, which means that its DNA fragment containing the glycoprotein E gene is missing (has been deleted). This can be checked and proved by DNA analysis with restriction enzymes (RE) SamHI and Kpn\ (Figure 1 ). A 2.7 million dalton (md) DNA fragment has been deleted from this gE-negative mutant between genome map units 0.85 and 0.88 (Figure 2).

3. The K/61 NM virus strain forming the basis of the invention is a large-plaque variant, which can be determined by plaque production on chicken embryo fibroblast (CEF) cell cultures. After cultivation for 3 days, the original K/61 parent strain produces plaques smaller than 1 mm, while the large-plaque strain K/61 NM gives rise to plaques measuring 2 mm or more in diameter. 8

4. The K/61 NM virus strain forming the basis of the invention possesses an RE cleavage site marker which is absent from the parent strain designated K/61. This can be demonstrated by RE analysis (Figure 1 ).

5. The gC function of the virus strain designated K/61 NM has been restored: while the original K/61 defective in gC does not react with a monoclonal antibody designated b69-C3, that monoclonal antibody reacts with K/61 NM in the same way as with any other gC-positive virus strains.

The virus strain n° NBIMCC 3551 is useful as a vaccine vector, together with a diluent or carrier.

The present invention thus provides a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector as previously defined.

The present invention also provides a method for preparing a vaccine as defined above wherein a therapeutically or prophylactically effective amount of an avirulent live or inactivated Aujeszky's disease virus producing large plaques in cell culture and expressing the glycoprotein gC is associated with a pharmaceutically acceptable carrier.

Another subject of the invention is a method for inducing an immune response against the Aujeszky virus in an animal (e.g. pigs, cattle or sheep) which involves administering to the animal an immunogenically effective amount of a vaccine vector of the invention to elicit an immune response, e.g. a protective or therapeutic immune response to the Aujeszky virus.

In vaccination methods for treating or preventing infection in a mammal, a vaccine vector of the invention can be administered by any conventional route in use in the vaccine field, particularly, to a mucosal (e.g_. ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. The intranasal route or the intradermal, subcutaneous or intramuscular route are four of the preferred routes. The administration can be achieved in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by the skilled artisans such as the route of administration or the condition of the animal to be vaccinated (weight, age and the like).

Vaccines of the invention can comprise adjuvants which are well-known by one skilled in the art and depend upon the route of administration. Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labiletoxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof.

Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.

For enhancing immunogenicity the virus can also be homogenized with an oil adjuvant, e.g mineral oil derivative such as paraffin oil, and/or surfactants e.g mannide monooleate, sorbitan oleate and ethoxylated sorbitan oleate.

Any pharmaceutical composition of the invention can be manufactured in a conventional manner. In particular, it can be formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier can be selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field.

In a preferred embodiment, the virus is mixed with the protective colloids generally used in vaccine production, lyophilized after distribution into sterile ampoules in suitable volumes, then the ampoules are sealed under vacuum or in nitrogen gas atmosphere. In another 10

embodiment, the virus is inactivated, e g with ethylene imine, prior to lyophilization

The present invention is illustrated by the following examples and figures, without restricting the scope of protection to them

LEGENDS OF THE FIGURES

Figure 1 represents a restriction enzyme fragment distribution of the DNA of virus strains K/61 and K/61 NM on agarose gel Fragments designated Bam 7 and Kpn I are fragments of normal size Fragments designated Bam T and Kpn I' are deletion fragments Fragments designated Bam 2a and 2b are markers

Figure 2 represents the physical map of the restriction fragments of the DNA of strain K/61 NM

Figure 3 shows gene defects on the genome of strains K/61 and K/61 NM

Figure 4 is a diagram showing antibody levels in piglets inoculated with strain K/61 and strain K/61 NM

EXAMPLES

Two parent strains were used for the recombination One was the recipient strain designated K/61 , deposited under no NBIMCC 3550, on December 20, 1997 while the other was the donor strain designated Sp-12, deposited under no NBIMCC 3552, on December 20, 1997 The new virus strain variant, which was designated as K/61 NM [N = large plaque variant ("nagy" means "large" in Hungarian), M = recognisable marker], was deposited at the National Bank for Industrial Microorganisms and Cell Cultures in Bulgaria (Sofia 1113, 125 "Tsaπgradsko shaussee" blvd , bl 2, fl 5) under no NBIMCC 3551 , on December 20, 1997 11

EXAMPLE 1 : Recombination

A PK-15 (permanent pig kidney) cell culture containing approx 2-4 x 10⁶ cells is simultaneously inoculated with virus fluid grown on the same type of cells and containing approx 10⁷ PFU (plaque-forming units) of parent strain K/61 (deposit no NBIMCC 3550) and with approximately the same dose of virulent Aujeszky's disease virus strain Sp-12 (deposit no NBIMCC 3552) The virulent strain designated Sp-12 has a SamHI cleavage site distribution which has not occurred in any of the more than 500 field isolates of Aujeszky's disease virus analysed in Hungary in the past decades This rare marker can be recognised by the presence of an extra SamHI cleavage site on SamHI fragment 2, which cleaves that 12 md (megadalton) fragment into two subfragments of unequal size, a 6 5 md (2a) and a 5 5 md (2b) subfragment The DNA possessing that new cleavage site can easily be recognised after digestion with SamHI, as the two new subfragments (Bam 2a and 2b) are located at a very conspicuous site, above and below fragment 4, after electrophoretic separation [Med Vet (Spain) 7, 27-33, 1990] Hereinafter that structure is referred to as Bam 2a+2b PK-15 cell culture is inoculated with a mixture of strains K/61 and Sp-12 for 1 hour at a temperature of 37 °C, washed three times, covered with MEM (Sigma) medium containing 1 % calf serum, and the virus was grown in a thermostat of 37 °C temperature for two days After two days, the cell layer was completely destroyed and the supernatant fluid contained high titres (>10⁷ PFU/ml) of Aujeszky's disease virus

That virus population consists of three different types of virus (a) one of the parent strains, i e strain designated K/61 , (b) the other parent strain, virulent virus designated Sp-12, and (c) their genetic recombinants which include both virulent and avirulent recombinants As the latter mentioned constitutes the basis of the vaccine according to the invention, it has to be separated from the other two As the ratio of recombinants, and within that especially the ratio of recombinants possessing the desired property, is unpredictable, dozens of large plaques 12

have to be picked out and analyzed for DNA structure The plaques were picked off with a Pasteur pipette and propagated in PK-15 cell culture at 37 °C for 48 hours (each in one Petπ dish 8 cm in diameter) After harvesting the virus the DNA was purified, digested with the restriction enzyme SamHI, and analyzed by electrophoresis (J Virol 49, 970-979, 1984) From each plaque, 1 ml virus sample was preserved for virulence test The virulence of each large-plaque virus exhibiting the double structural marker of the DNA (deletion of the gE gene and presence of the Sam 2a+2b marker) was tested by intracerebral inoculation of day-old chickens Only a certain proportion of mutants possessing the above DNA structure is avirulent, the majority of them are virulent Day-old chickens were inoculated with 0 05 ml of virus into a region close to one hemisphere of the cerebrum (the inoculum contained approx 10⁵ PFU live virus), then the chickens were kept under observation for 14 days According to earlier experience (J Virol 52, 198-205, 1984), the result of the test performed by the intracerebral inoculation of day-old chickens is parallel to that of the much more expensive virulence test on piglets The plaque progeny showing the desired DNA structure and not causing disease in chickens after intracerebral inoculation were subjected to further three plaque purification cycles carried out by the known method, and then they were stored at a temperature of -70 °C After plaque purification (aimed at removing contaminating plaques, if any), the analysis of DNA structure with RE and the virulence test in day-old chickens were repeated, then the recombinant designated 10a was selected from the possible recombinant candidates for further study The strain designated 10a constitutes the basis of the present invention, and it has been deposited with the designation K/61 NM and under no NBIMCC 3551

EXAMPLE 2 : Production of the vaccines

a The virus strain designated K 61 NM, which has been tested for sterility and for the presence of extraneous viruses, is distributed into suitable aliquots and stored either at -65 to -75 °C, in liquid nitrogen or in lyophi zed form at a temperature of +4 °C or -20 °C 13

b Using PK-15 or other suitable permanent cell lines (e g VERO, RK-13, ST, MDBK, etc ) or primary or secondary chicken embryo fibroblast, calf testicle, calf kidney etc cell cultures, we prepare stationary or roller cultures in the manner usual in vaccine production, e g by the use of MEM-H (Sigma) culture medium and 5-10 % v/v calf serum and incubation at 37 °C

c When they reach 80-100% confluency, the cultures prepared according to point (b) are inoculated with the appropriate dilution of virus strain K/61 NM, then the virus-containing supernatant fluid is collected under sterile conditions when a 80-90% CPE has occurred d The virus suspension according to point (c) is mixed with the protective colloids generally used in vaccine production, lyophilized after distribution into sterile ampoules in suitable volumes, then the ampoules are sealed under vacuum or in nitrogen gas atmosphere e The vaccine produced according to point (d) is resuspended in sterile physiological saline or in oil emulsion immediately before use f The virus suspension according to point (c) is inactivated preferably with ethylene imine of 200 μg/ml concentration at 37°C for 24 hours The ethylene imine is cychzed from bromo ethylene amine hydrobromide immediately before use At the end of inactivation ethylene imine in excess is neutralized by adding sodium thiosulphate solution to the treated virus The pH value of the suspension is adjusted to 7 2-7 4 by adding a suitable buffer e g (glycme-HCI) Inactivation of the virus suspension is controlled by inoculating cell-cultures and/or susceptible rabbits

g For enhancing immunogenicity the inactivated virus suspension according to point (f) is homogenized after the addition of an oil adjuvant The volume proportion of the inactivated antigen is 50-80% v/v The composition of the oil adjuvant in terms of volume proportions mineral oil derivative, preferably paraffin oil 55-57% v/v, at least two 14

surfactants, preferably mannide monooleate (ICI) and/or sorbitan oleate and ethoxylated sorbitan oleate altogether 45-25% v/v Homogenization results in "oil in water"-type emulsion disappearing in a short time (two weeks) after inoculation without leaving a trace at the site of injection

EXAMPLE 3 : Virulence test of the live vaccine

Five 4-week-old (negative) piglets free from antibodies to Aujeszky's disease virus were inoculated intranasally with the vaccine produced as described in example 2 One ml fluid containing 10⁷ PFU of virus per nostril was inoculated deep into the nasal cavity (The virulent virus causes lethal disease in negative piglets of that age even in a dose corresponding to 1/10⁵ part of the above dose ) The intranasally inoculated piglets were kept under observation for 14 days, during which period their body temperature was measured on days 1-10 and the animals were weighed on every second day During the period of observation, no body temperature values exceeding 40 °C (i e deviating from normal) were recorded, the piglets did not show any clinical signs other than their normal behaviour, their body weight gain showed an increasing tendency corresponding to the breed (Hungarian Large White * Danish Landrace), and no stagnation of body weight (because of a possible inappetence) or weight loss could be observed

Thus, the highly susceptible 4-week-old piglets survive intranasal inoculation with a very high virus dose without showing any clinical signs of disease, indicating that virus strain K/61 NM is as safe as its "parent" virus strain K/61 , despite the fact that through recombination with a virulent virus (Sp-12) its DNA has taken over DNA fragments from the genome of Sp-12 at least at the following two sites a around the cleavage site resulting in the Bam 2a+2b marker (this is between map units 0 32 and 0 34) in the central region of SamHI fragment 2, and 15

b. The recombination event introducing the Bam 2a+2b cleavage site into strain K/61 has restored the gC function as well. A monoclonal antibody (designated B69-C3) is known to exist which recognises, and binds to, every Aujeszky's disease virus strain having an intact gC function, e.g. the virulent strains. That monoclonal antibody does not react with the original K/61 strain while it binds to strain K/61 NM. According to data of the literature, this means that strain K/61 NM has become gC positive.

EXAMPLE 4 : Efficacy test of the live vaccine produced from strain K/61 NM in pigs

Two experiments were carried out to study the efficacy of the genetically improved live strain (K/61 NM). a. Study of the replicating ability of strain K/61 NM in the nasal mucosa of piglets (young pigs), in order to detect an increased growth potential as compared to the original strain K/61 . Namely, if virus replication does not increase in a detectable manner in some tissue of the pig where it is easily measurable, no increase can be expected in the immunogenic potential either. Since from the nasal cavity of piglets intranasally inoculated with an extremely large dose (as described in Example no. 3) a large quantity of virus can be reisolated already because of the large dose of inoculum used, the authors of the invention studied the growth potential of strain K/61 NM in the nasal cavity after using an inoculum of lower dose level (10⁴ PFU/nostril). The piglets were inoculated with a dose of 10⁴ PFU/nostril, then nasal swab samples were taken daily from both nasal cavities and the virus content of the nasal secretion was precisely measured by plaque titration. Strain K/61 NM was found to be detectable for 8-10 days, and that the peak titres measurable on day 5-6 corresponded to 10⁵— 10⁶ PFU/ml (nasal secretion). In similar experiments performed with strain K 61 these values corresponded to maximum 10⁴-10⁵, indicating that the growth potential of strain K/61 NM in the nasal cavity had increased without any rise in its virulence. If the 16

growth potential of the virus measurable in pigs has increased, this is bound to be accompanied by enhanced immunogenicity of the vaccine strain b Induction of elevated antibody titres in the blood of pigs Six week-old piglets free from specific antibodies to

Aujeszky's disease virus (6 animals per group) were immunized intramuscularly with different, decreasing doses (10⁶, 10⁵ and 10⁴ infective particles) of the original K/61 strain and of the genetically improved new virus according to the invention (strain K/61 NM) Blood samples were taken every 10 days and their antibody levels were determined by the ADV-ELISA tests serving for the detection of Aujeszky's disease virus specific antibodies The antibody response induced by the two vaccine types is presented in Figure 4 (Since no difference was found between the values of groups inoculated with the 10⁵ or the 10⁶ dose for any strain, their geometric means were aggregated and are shown in Figure 4) In summary, it can be established that on days 10, 20, 30 and 45 the antibody titres (geometric means per group) of piglets inoculated with the original K/61 strain varied within the range of 1 50-1 300 irrespective of the virus dose administered, while the values of antibody levels induced by strain K/61 NM according to the invention were between 1 400 and 1 500, also irrespective of the virus dose inoculated These experiments clearly demonstrate the enhanced antibody-inducing ability, i e increased efficacy, of the virus strain designated K/61 NM

EXAMPLE 5 : Efficacy test of the inactivated vaccine produced from strain K/61 NM in pigs

Six 6-week-old piglets free from specific antibodies to Aujeszky's disease virus were immunized intramuscularly with the inactivated vaccine which was prepared according to example 2, points f and g Six piglets of the same age and origin were inoculated twice at an interval of 21 days with an inactivated vaccine prepared from the gE negative LK 17

strain (control vaccine). Two weeks after the second injection blood samples were taken and the antibody levels were determined by the ADV-ELISA tests serving for the detection of the antibodies specific to the Aujeszky's disease virus

The geometric mean titer (GMT) induced by two injection of a vaccines prepared from strain K/61 NM was 1 4000 while GMT induced by the control vaccine, which had been evaluated by comparison with other vaccines as a good vaccine, was 1 :2000

BUDAPEST TREATY ON THE INTERNA1 IONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM RECEIPT IN THE CASE OF AN

Dr. Maria Kelemen ORIGINAL DEPOSIT

PHYLAXIA - SANOFI , , , ,. _ . _ , . .,

. . . . ₀. - i . _τr. issued pursuant to the Rule 7J by the u H - , 1 10Y7 Budape ⁹st, M ^Naic^tir^ooⁿo^ar^lg ^Ban^aiⁿs^km ^fs^or a ^,nⁿd^du C^se^Mll ^a C^lultures,

Szallas u. 5. _{Sofja Bu}|_garja

HUNGARY

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: NBIMCC No 3550

Aujeszky's disease virus, Ay virus K/61

II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by: E3 a scientific description □ a proposed taxonomic designation

(Mark with a cross where applicable)

III. RECEIPT AND ACCEPTANCE

NBIMCC accepts the microorganism identified under I above, which was received by it on 20.12.1997. (date of the original deposit)¹

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I, above was received by the NBIMCC on (date of the original deposit) and a request to convert the original deposit to a under the Budapest Treaty was received by it on (date of receipt of request for conversion)

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: NATIONAL BANK OF INDUSTRIAL MICROORGANISMS AND CELL CULTURES

Signature (s) of person (s) having the power to represent the International Depositary Authority or of authorized official (s): Λ

Address: 1113 Sofia, 125 Tsarigradsko chausse blvd., bl. 2, P.O.Box 239 ^ γγιι^y

Date: 30.01.1998.

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired.

Form BP/4 19

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

TO: VIABILITY STATEMENT,

Dr. Maria Kelemen

PHYLAXIA - SANOFI issued pursuant to Rule 10.2 by the Veterinary Biologicals Co. LTD. National Bank for Industrial H - 1107 Budapest, Microorganisms and Cell Cultures, Szallas u. 5. Sofia, Bulgaria HUNGARY

DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM

NBIMCC No 3550

Name: Dr. Maria Kelemen

Address:PHYLAXIA - SANOFI

Veterinary Biologicals Co. LTD. Date of the deposit or of the transfer: 20.12.1997.

H - 1107 Budapest, Szallas u. 5. HUNGARY

111. VIABILITY STATEMENT

The viability of the microorganism, identified under II, above was tested on 21.01.1998¹. On that date, the said microorganism was: viable

D no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED '

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: Signature (s) of person (s) having the power to represent the International Depositary

NATIONAL BANK FOR INDUSTRIAL MICROORGANISMS Authority or of authorized official AND CELL CULTURES

Address:

1113 Sofia, 125 Tsarigradsko chausse blvd., Date: 30.01.1998. bl. 2, P.O.Box 239

¹ 1n the cases referred to in Rule 10.2 (a) (ii) and (iii), refer to the most recent viability test. ² Fill in if the information has been requested and if the results of the test were negative.

Form BP/9 BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

TO: RECEIPT IN THE CASE OF AN

Dr. Maria Kelemen ORIGINAL DEPOSIT

PHYLAXIA - SANOFI issued pursuant to the Rule 7J by the Veterinary Biologicals Co. LTD. National Bank for Industrial H - 1 107 Budapest, Microorganisms and Cell Cultures, Szallas u. 5.

Sofia, Bulgaria HUNGARY

IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: NBIMCC No 3551

Aujeszky's disease virus, Ay virus K 61 NM

II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by: [5_1 a scientific description □ a proposed taxonomic designation

(Mark with a cross where applicable)

III. RECEIPT AND ACCEPTANCE

IV. RECEIPT OF REQUEST FOR CONVERSION

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: NATIONAL BANK OF INDUSTRIAL MICROORGANISMS AND CELL CULTURES

Signature (s) of person (s) having the power to represent the International Depositary Authority or of authorized official (s): ~ . r —

Address: 1113 Sofia, 125 Tsarigradsko chausse blvd., bl. 2, P.O.Box 239

Date: 30.01.1998.

Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired. - -

Form BP/4 21

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

TO: VIABILITY STATEMENT,

Dr. Maria Kelemen

DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM

NBIMCC No 3551

Name: Dr. Maria Kelemen

Address:PHYLAXIA - SANOFI

H - 1107 Budapest, Szallas u. 5. HUNGARY

VIABILITY STATEMENT

D no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED

V. INTERNATIONAL DEPOSITARY AUTHORITY

NATIONAL BANK FOR INDUSTRIAL MICROORGANISMS Authority or of authorized official (s): AND CELL CULTURES

Qγfii< >

Address:

¹ In the cases referred to in Rule 10.2 (a) (ii) and (iii), refer to the most recent viability test. Fill in if the information has been requested and if the results of the test were negative.

Form BP/9 BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM RECEIPT IN THE CASE OF AN

Dr. Maria Kelemen ORIGINAL DEPOSIT

PHYLAXIA - SANOFI _{jssued pursuant to tne Ru)e ηΛ by tne} u π 1 ^B _/! ^l0g,C ₊ ^{alS Co}- ^TD' National Bank for Industrial

H - 1 107 Budapest, Microorganisms and Cell Cultures,

Szallas u. 5. _Sofia Bulgaria

HUNGARY

IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: NBIMCC No 3552

Aujeszky's disease virus, Ay virus Sp-12

II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by: [3 a scientific description □ a proposed taxonomic designation

(Mark with a cross where applicable)

RECEIPT AND ACCEPTANCE

IV. RECEIPT OF REQUEST FOR CONVERSION

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: NATIONAL BANK OF INDUSTRIAL MICROORGANISMS AND CELL CULTURES

Signature (s) of person (s) having the power to represent the International Depositary Authority or of authorized official (s): Λ _^JI r — ^~

Address: 1113 Sofia, 125 Tsarigradsko chausse blvd., bl. 2, P.O.Box 239

Date: 30.01.1998.

Form BP/4 23

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

TO: VIABILITY STATEMENT,

Dr. Maria Kelemen

DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM

NBIMCC No 3552

Name: Dr. Maria Kelemen

Address:PHYLAXIA - SANOFI

H - 1107 Budapest, Szallas u. 5. HUNGARY

VIABILITY STATEMENT

The viability of the microorganism, identified under II, above was tested on 21.01.1998¹. On that date, the said microorganism was:

IS viable D no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED '

V. INTERNATIONAL DEPOSITARY AUTHORITY

NATIONAL BANK FOR INDUSTRIAL MICROORGANISMS Authority or of authorized AND CELL CULTURES

IICA

Address:

1113 Sofia, 125 Tsangradsko chausse blvd. Date: 30.01.1998. bl. 2, P.O.Box 239

¹ In the cases referred to in Rule 10.2 (a) (ii) and (iii), refer to the most recent viability test.

² Fill in if the information has been requested and if the results of the test were negative.

Form BP/9

Claims

1. A vaccine against Aujeszky's disease containing an avirulent live virus or an inactivated virus, wherein said virus is a Aujeszky's disease virus producing large plaques in cell culture and expressing the glycoprotein gC, in association with a pharmaceutically acceptable carrier.

2. A vaccine according to claim 1 , wherein said virus is a Bartha's K/61 virus strain variant.

3. A vaccine according to any of claims 1 or 2 wherein said virus further possesses a marker gene enabling individual strain recognition.

4. A vaccine according to claim 3, wherein said virus is a virus of the K/61 NM strain, deposited under n┬░ NBIMCC 3551 on December 20,

1997.

5. A vaccine according to any of claims 1 to 4 wherein said virus is a live virus.

6. A vaccine according to any of claims 1 to 4 wherein said virus is inactivated.

7. A method for inducing an immune response against the Aujeszky virus in an animal which involves administering to the animal an immunogenically effective amount of a vaccine according to any of claims 1 to 6.

8. A recombinant virus of the K/61 NM strain, deposited under n┬░ NBIMCC 3551 on December 20, 1997.

9. A method for preparing a vaccine according to any of claims

1 to 6 wherein a therapeutically or prophylactically effective amount of an avirulent live or inactivated Aujeszky's disease virus producing large plaques in cell culture and expressing the glycoprotein gC is associated with a pharmaceutically acceptable carrier.

10. A method according to claim 9 wherein said virus is further associated with an adjuvant enhancing immunogenicity.