ES2388909T3

Porcine reproductive and respiratory syndrome vaccine, based on isolate JA-142

Abstract

A porcine respiratory and reproductive syndrome virus (SRRP) comprising a sequence represented by SEQ ID NO: 1 or by a sequence having at least 95% sequence identity with SEQ ID NO: 1.

Term

Term ended

Projected expiry passed 21 April 2020, 6.4 years ago.

21 claims: 12 independent, 9 dependent

1
ES 2 388 909 T3 REIVINDICACIONES 1. Un virus del síndrome respiratorio y reproductivo porcino (SRRP) que comprende una secuencia representada por SEC ID N°:1 o por una secuencia que tiene al menos el 95 % de identidad de secuencia con SEC ID N°:1.
2
El virus de la reivindicación 1, comprendiendo el virus la secuencia representada por SEC ID N°:1.
3
Un virus del SRRP que tiene el número de acceso ATCC VR-2638.
4
El virus de la reivindicación 1, comprendiendo el virus la secuencia representada por SEC ID N°:2.
5
El virus de una cualquiera de las reivindicaciones 1 a 3, atenuado por un procedimiento para atenuar un virus que incluye las etapas de replicar sucesivamente dicho virus mediante inoculación y replicación del virus en cultivos celulares individuales respectivos hasta lograr la atenuación, retirar las muestras que contienen virus de al menos determinados de dichos cultivos celulares mientras el virus se está replicando a una velocidad logarítmica y antes de la inducción de efectos citopáticos en dichos determinados cultivos celulares e inocular el siguiente pase de cultivo celular respectivo con dichas muestras.
6
El virus de la reivindicación 5, produciéndose dicha etapa de retirada aproximadamente 24 horas después de la inoculación de dichos cultivos celulares.
7
El virus de la reivindicación 5, comprendiendo dicho cultivo celular una monocapa de células que tiene una confluencia superior a aproximadamente el 70 %.
8
El virus de la reivindicación 5, que además comprende la etapa de analizar periódicamente dicho virus progenie para evaluar la atenuación.
9
El virus de la reivindicación 5, que además comprende las etapas de conservar dichos determinados cultivos celulares después de dicha etapa de retirada y observar si la inducción de efectos citopáticos tiene lugar en dichos cultivos celulares conservados.
10
El virus de la reivindicación 5, comprendiendo dicha inoculación el número más pequeño de virus que da como resultado en última instancia ECP.
11
El virus de una cualquiera de las reivindicaciones precedentes, habiéndose pasado dicho virus un mínimo de 200 veces en cultivo celular.
12
El virus de una cualquiera de las reivindicaciones precedentes, estando dicho virus atenuado.
13
El virus de una cualquiera de las reivindicaciones precedentes, siendo dicho virus avirulento.
14
El virus de una cualquiera de las reivindicaciones precedentes, siendo dicho virus capaz de generar respuestas de anticuerpos en ganado porcino.
15
El virus de la reivindicación 14, siendo dicha respuesta de anticuerpos específica para cepas de virus del síndrome respiratorio y reproductivo porcino.
16
El virus del SRRP atenuado de una cualquiera de las reivindicaciones 5 a 10, obtenido mediante atenuación de un virus que tiene una secuencia de ácido nucleido representada por SEC ID N°:2.
17
Una vacuna que comprende el virus según una cualquiera de las reivindicaciones precedentes.
18
La vacuna de la reivindicación 17, que además comprende un agente vehículo farmacológicamente compatible.
19
Una secuencia de ácido nucleico aislada que comprende al menos el 95 % de identidad de secuencia con SEC ID N°:1.
20
Una secuencia de ácido nucleico aislada que comprende la secuencia tal como se muestra en SEC ID N°:1.
21
La vacuna tal como se define en la reivindicación 17 ó 18 para su uso en la inmunización de ganado porcino contra el síndrome respiratorio y reproductivo porcino, en la que dicha inmunización comprende administrar dicha vacuna a dicho ganado porcino.

Independent claims21

666 paragraphs in 26 sections, as filed

ES 2 388 909 T3

DESCRIPTION

Isolated JA-142-based Porcine Reproductive and Respiratory Syndrome Virus Vaccine

Background of the invention

Field of Invention

The present invention relates, in general, to attenuated avirulent atypical porcine respiratory and reproductive syndrome (PRRS) virus (PRRSV) and the corresponding live virus vaccines for administration to pigs to confer effective immunity in pigs against VSRRP.

Description of the prior art

SRRP emerged in the late 1980s as a major viral disease of pigs. PRRSV causes severe reproductive failure in pregnant sows, manifested in the form of premature farrowing, increased numbers of stillbirths, mummified and weak born pigs, decreased farrowing rate, and delayed return to estrus. Furthermore, the respiratory system of PRRSV-infected pigs is adversely affected, as demonstrated by lesions appearing in the lungs of infected pigs. To combat the problems associated with RSVR infection, vaccines have been developed that confer immunity to then existing PRRSV strains.

Outbreaks of an exceptionally severe form of SRRP, hereinafter referred to as "atypical SRRP," were first recognized in North America in the latter part of 1996. They differed from “typical SRRP” epidemics in that: 1) the clinical signs were longer as well as more severe; 2) the incidence of abortions was higher, especially during the first part and the middle of the gestation; 3) there was a higher incidence of mortality in gilts and mutiparous sows; 4) PRRSV was isolated less frequently from aborted fetuses, stillborn pigs, and live-born pigs — perhaps because abortions were more often the result of acute maternal disease rather than transplacental infection; 5) lung lesions in affected young pigs were more extensive; and 6) commercially available vaccines provided little or no protection. Taken together, these observations indicated the emergence of more virulent and antigenically different strains of PRRSV and the need for a new generation of PRRS vaccines.

The most frequently used procedure for producing live attenuated virus vaccines is to serially passage the virus on a substrate (usually a cell culture) other than the natural host (S) until it is sufficiently attenuated (i.e., its virulence is reduced or ability to produce disease) for use as a vaccine. For the first passage, a cell culture is infected with the selected inoculum. After obtaining clear evidence of virus replication (eg, virus-induced cytopathic effects [CPE] on infected cells), an aliquot of the cell culture medium, or infected cells, or both, from the first passage is used to infect a second cell culture. The procedure is repeated until one or more critical mutations in the viral genome cause sufficient attenuation so that the virus can be used safely as a vaccine. The degree of attenuation is usually determined empirically by exposing the natural host (S) to progressively higher passage levels of the virus.

The above procedure is basically valid and has been used successfully for the development of numerous vaccines for human and veterinary use. However, it is relatively ineffective because the log phase of virus replication, during which mutations are most likely to occur, is often completed long before evidence of viral replication becomes visibly evident.

Murtaugh et al. (1995) Archives Virol. 40, 1451-1460 compares the sequences encoding structural proteins of the VR-2332 and Lelystad viral strains of the SRRP virus. Comparative sequence analysis indicates that there were degrees of amino acid identity with the Lelystad virus open reading frames ranging from 55% in ORF5 to 79% in ORF6.

Mengelin et al. (1999) AJV 60 (3), 334-340 describes the identification and clinical evaluation of wild vaccine-related strains suspected of porcine reproductive and respiratory syndrome virus. It was concluded that in pig herds, the vaccine strain may have persisted and mutated to a less attenuated form.

EP 0835929 A1 describes a new attenuated strain (CNCM Institut Pasteur 1-1642) of SRRP virus, together with an attenuation replication procedure thereof using a clone obtained from monkey liver cells MA-104 ( CNCM Institut Pasteur 1-1643).

US 5,476,778 discloses a method of attenuation of porcine respiratory syndrome and infertility virus which comprises passing the virus through a simian cell line under well defined conditions.

ES 2 388 909 T3

US 5,587,164 discloses a method for culturing SRRP virus in tissue culture that is susceptible to infection to replicate the virus in an amount sufficient to protect animals against SRRP or can be used to diagnose SRRP or identify the molecular structure of PRRS virus to develop recombinant products, said procedure comprising inoculating the viruses into tissue tissue and harvesting the replicated virus

Therefore, there is an undoubted need in the art for a vaccine that confers effective immunity against PRRSV strains, including recently discovered atypical RSVR strains. There is also a need in the art for a method of preparing such a vaccine. Ultimately, what is needed is a virus passage procedure that attenuates the virus more efficiently than previously thought possible, with the resulting attenuated virus generating PRRSV-specific antibodies in pigs, thereby conferring immunity. effective against subsequent infection by PRRSV.

Summary of the invention

The present invention relates to the following embodiments (1) to (21):

(1) A porcine reproductive and respiratory syndrome virus (SRRP) comprising a sequence represented by SEQ ID NO: 1 or by a sequence having at least 95% sequence identity with SEQ ID NO: 1.

(2) The virus of embodiment (1), the virus comprising the sequence represented by SEQ ID NO: 1.

(3) An SRRP virus having the ATCC accession number VR-2638.

(4) The virus of embodiment (1), the virus comprising the sequence represented by SEQ ID NO: 2.

(5) The virus of any one of embodiments (1) to (3), attenuated by a method for attenuating a virus that includes the steps of successively replicating said virus by inoculation and replication of the virus in respective individual cell cultures until achieving the attenuation, withdrawing the samples containing virus from at least certain of said cell cultures while the virus is replicating at a logarithmic rate and before the induction of cytopathic effects in said certain cell cultures and inoculating the next respective cell culture passage with said samples.

(6) The virus of embodiment (5), said withdrawal step occurring approximately 24 hours after inoculation of said cell cultures.

(7) The virus of embodiment (5), said cell culture comprising a monolayer of cells having a confluence of greater than about 70%.

(8) The virus of embodiment (5), further comprising the step of periodically analyzing said progeny virus to assess attenuation.

(9) The virus of embodiment (5), which further comprises the steps of preserving said certain cell cultures after said withdrawal step and observing whether the induction of cytopathic effects takes place in said conserved cell cultures.

(10) The virus of embodiment (5), said inoculation comprising the smallest number of viruses that ultimately results in CPE.

(11) The virus of any one of the preceding embodiments, said virus having been passed a minimum of 200 times in cell culture.

(12) The virus of any one of the preceding embodiments, said virus being attenuated.

(13) The virus of any one of the preceding embodiments, said virus being avirulent.

(14) The virus of any one of the preceding embodiments, said virus being capable of generating antibody responses in swine.

(15) The virus of embodiment (14), said antibody response being specific for strains of porcine reproductive and respiratory syndrome virus.

(16) The attenuated SRRP virus of any one of embodiments (5) to (10), obtained by attenuation of a virus having a nucleide acid sequence represented by SEQ ID NO: 2.

(17) A vaccine comprising the virus according to any one of the preceding embodiments.

ES 2 388 909 T3 (18) The vaccine of embodiment (17), further comprising a pharmacologically compatible carrier agent.

(19) An isolated nucleic acid sequence comprising at least 95% sequence identity with SEQ ID NO: 1.

(20) An isolated nucleic acid sequence comprising the sequence as shown in SEQ ID NO: 1.

(21) The vaccine as defined in embodiment (17) or (18) for use in immunizing swine against swine reproductive and respiratory syndrome, wherein said immunization comprises administering said vaccine to said swine.

The present invention overcomes the problems outlined above and provides attenuated atypical RSVR strains and corresponding improved modified live vaccines that confer effective immunity against newly discovered atypical RSVR strains. The term "effective immunity" refers to the ability of a vaccine to prevent RSVR infections in pigs, including atypical RSVR infections that result in important clinical signs of the disease. That is, immunized pigs may or may not be serologically positive for PRRSV, but do not exhibit any significant clinical symptoms. The term "atypical PRRSV" refers to these new SRRP strains that are substantially more virulent than typical PRRSV strains.

In preferred forms, the vaccine described herein includes live virus whose virulence has been attenuated. The resulting attenuated virus has been shown to be avirulent and to confer effective immunity. A particularly virulent strain of atypical SRRP (designated JA-142) that caused especially severe symptoms of SRRP and represents the dominant strain of atypical PRRS was selected for subsequent attenuation per passage. The resulting attenuated virus has been deposited with the American Type Culture Collection (ATCC), Rockville, MD on February 2, 1999 and was consistent with ATCC Accession No. VR-2638. This attenuated virus is a preferred Master Seed Virus (MSV) that has been passed and further developed as an effective PRRSV vaccine.

The name given to the unattenuated virus, JA-142, arises from the restriction enzyme pattern. The 1 represents the inability of the MLU I enzyme to cleave the virus at open reading frame 5 (ORF 5). The 4 represents cleavage by Hinc II at base pair positions 118 and 249 of ORF 5 and short contiguous sequences. The 2 represents the Sac II cleavage at the position of base pair 54 of ORF 5 and short contiguous sequences.

Passage of the virus to attenuation was accomplished using a new procedure that resulted in increased efficacy. Specifically, the virus remained in the logarithmic replication phase during all multiple passages of cell culture to substantially shorten the time to attenuation. This is achieved by ensuring that in each cell culture there is a substantial excess of initially uninfected cells over the number of viruses present. Thus, by transferring only small amounts of virus from one passage to another, logarithmic replication is ensured.

In practice, the procedure is typically initiated by inoculating several different cell cultures with progressively smaller viral aliquots (ie, less virus in each culture). For example, the starting cultures could contain viral aliquots of 200 µl, 20 µl, and 2 µl. After an initial short incubation period (eg ~ 24 hours), the same viral aliquots (in the example 200 µl, 20 µl, and 2 µl) from each cell culture are transferred to fresh individual cultures (previously not infected), while the starting cultures are monitored until a cytopathic effect (CPE) is observed or not. This procedure is continued in serial order for multiple passages, using the same viral aliquots in each case and preserving the cultures for observation of CPE. If all serial culture passages exhibit CPE after a selected number of passages are completed, the series with the highest viral aliquot (in the example, 200 µl and 20 µl) can be terminated, after which another series is used. of progressively smaller viral aliquots (eg, 2 µl, 0.2 µl, and 0.02 µl) and the procedure is repeated again, again maintaining the cell cultures after transfer for observation of the ECP.

At some point in this procedure of inoculation of successively smaller viral aliquots, no CPE will be observed in a given cell culture. When this occurs, the next higher level of viral aliquot showing ECP replaces the passage in which ECP was not observed, after which subsequent passages will be inoculated using the previously employed viral aliquots.

In view of the fact that a virus will tend to become more efficient in infecting cells and also to replicate to a higher infectivity titer for cell culture over time (which is especially true with RNA viruses such as RSVR), it will be observed that smaller and smaller viral aliquots are necessary to maintain infection during serial transfer. Using the smallest aliquot that maintains infection helps to ensure that viral replication remains in a log phase throughout the procedure.

ES 2 388 909 T3

The DNA sequence of the attenuated passage 201 virus was then determined using standard procedures. The sequence of this attenuated virus was designated as VSM JA-142 Pass N ° 201, and whose sequence is provided as SEQ ID N °: 1. The sequence of the virulent virus, JA-142, is provided as SEQ ID N °: 2 .

As used herein, the following definitions will apply: "Sequence identity", as known in the art, refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, specifically one reference sequence and a given sequence to be compared to the reference sequence. Sequence identity is determined by comparing the given sequence with the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the inter-strand pairing of said sequences. Following such alignment, sequence identity is determined on a position-by-position basis, eg, sequences are "identical" at a particular position if, at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give% sequence identity. Sequence identity can be easily calculated by known procedures, including, but not limited to, those described in Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred procedures for determining sequence identity are designed to give the greatest match between the sequences tested. Procedures for determining sequence identity are encoded in publicly available computer programs that determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J. et al., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF and col., J. Molec. Biol., 215: 403-410 (1990). The BLASTX program is publicly available from the NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, SF et al., J. Molec. Biol., 215: 403- 410 (1990)).

These programs optimally align sequences using default gap weights to produce the highest level of sequence identity between the given and reference sequences. By way of illustration, by a polynucleotide having a nucleotide sequence that has a "sequence identity" of at least, for example, 95% to a reference nucleotide sequence it is meant that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence can include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence that has an identity of at least 95% to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another. nucleotide, or a nucleotide number of up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These reference sequence mutations can appear at the 5 'or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, individually interspersed between the reference sequence nucleotides or at one or more contiguous groups within the reference sequence. Similarly, by a polypeptide having a given amino acid sequence that has, for example, at least 95% sequence identity to a reference amino acid sequence, it is meant that the given amino acid sequence of the polypeptide is identical. to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid modifications for every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence that has at least 95% sequence identity to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence can be deleted or substituted with another amino acid. , or an amino acid number of up to 5% of the total number of amino acid residues in the reference sequence can be inserted into the reference sequence. These modifications of the reference sequence may appear at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, individually interspersed between the residues of the reference sequence or in the one or more groups. contiguous within the reference sequence. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.

Similarly, "sequence homology", as used herein, also refers to a method of determining the degree of relationship of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above and gaps are introduced if necessary. However, contrary to "sequence identity", conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide that has a sequence homology of 95% with a sequence of

ES 2 388 909 T3 reference, 95% of the amino acid residues or nucleotides in the reference sequence must coincide, or comprise a conservative substitution, with another amino acid or nucleotide, or a number of amino acids or nucleotides of up to 5% of the Total amino acid or nucleotide residues, not including conservative substitutions, in the reference sequence can be inserted into the reference sequence.

A "conservative substitution" refers to the substitution of an amino acid or nucleotide residue with another amino acid or nucleotide residue that has similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

The term "isolated" means altered "by the hand of man" from its natural state, that is, if it appears in nature, has been modified or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated", but the same polynucleotide or polypeptide separated from coexisting materials from its natural state is "isolated", as the term is used herein. .

Preferably, sequences that share at least 95% sequence identity with SEQ ID NO: 1 are effective in conferring immunity to attenuated virus-vaccinated animals containing such homologous sequences.

Detailed description of the preferred embodiment

The following examples set forth preferred embodiments of the present invention. However, it should be understood that these examples are provided by way of illustration.

Example 1

Materials and Procedures

This example describes a passage procedure for attenuating viruses that maximizes attenuation efficiency by ensuring that the virus is preferentially in a logarithmic phase of replication. The virus was passaged (ie, an aliquot of nutrient medium including virus, unbound cells, and cell debris from a virus-infected cell culture was added to the nutrient medium from an uninfected culture) at daily intervals. Different amounts of virus were added to each interval using multiple cultures. For example, initially, 200 µl was transferred to an uninfected culture, 20 µl was added to a second non-infected culture and 2 µl to a third non-infected culture. The goal was to have a sufficient number of susceptible cells so that replication cycles could continue until the next transfer. The procedure was considered successful if the cells eventually showed CPE. However, because RSRV-induced CPE does not appear until some time after the logarithmic growth phase, passages were performed before it was known whether or not they would be ultimately successful ("blind passages"). Passages that resulted in virus-induced CPE were said to have resulted in a "take". If a pass did not result in a take, the pass was restarted at the highest dilution of the last pass that resulted in a take. As more and more passages were made, the virus became more adapted to replicate in the cell line and was less capable of producing disease symptoms in its original host. These changes are caused by random mutations that occur during replication.

Using this procedure, the following procedures were used to pass an exemplary virus in accordance with the present invention, VSM, JA-142. This strain was grown in MARC-145 cell cultures at daily intervals. Twenty-four well plates were used for the procedure to minimize the amount of cells and nutrient medium required and to simplify the multi-aliquot passage technique. Cells and nutrient medium were added to each well and the cells were allowed to form or nearly form (greater than about 70%) a confluent monolayer. The nutrient medium comprised approximately 90% Earle's balanced salt solution minimal essential medium (MEM), 10% fetal calf serum, and 0.05 mg / ml gentamicin sulfate. The volume of nutrient medium used was approximately 1 ml. Typically three wells of a column were used for each amount of virus that was transferred. An aliquot of nutrient medium from the previous passage was transferred to the first well of the column at 48 or 72 hours, after the cell cultures had been prepared, nutrient medium from the first well was transferred to the second well of the same column at 72 or 96 hours and to the third well of the same column at 96 or 120 hours. The plates were usually prepared twice a week, so that sometimes the fourth well of the column was used and sometimes it was not used. The passage conditions were kept at 37 ° C in a humid atmosphere containing CO2 at 5%.

Aliquots of different sizes (having different amounts of virus) were tested for each passage to determine if the amount of virus was sufficient to induce CPE. For example, a different series of aliquot transfers (passages) of 200 µl, 20 µl, and 2 µl, respectively, were used until the smallest aliquots consistently exhibited CPE, the goal being to transfer the smallest aliquot that produced CPE. When the smallest aliquot (for example, 2 pl) in the group of aliquots being tested gave as

ES 2 388 909 T3 systematically resulted in ECP, smaller amounts were tested (eg 0.2 µl and 0.02 µl). When a given dilution exhibited no CPE, that series of cultures was restarted with the next smallest amount that resulted in CPE at that passage (that is, if the 2 μl transfer was unsuccessful producing CPE in passage 25 but the transfer of 20 µl in pass 25 was successful, the 2 µl blot was repeated using 20 µl, 5 resuming the 2 µl blots for pass 26).

Using this procedure, the smallest amount of virus that needed to be transferred to obtain ECP was determined. The virus was successfully passaged at daily intervals using the following amounts of virus-infected nutrient medium (reflecting the highest dilution [ie, the smallest aliquot] that resulted in ECP, keeping in mind that other dilutions would work as well):

<td>Pass number</td><td>Amount Transferred</td>

<td> 42-83</td><td>20/200 pl (with alternation)</td>

ES 2 388 909 T3

Pass number

189-191

192

193-195

196

197 (continuation)

Amount Transferred

0.2 pl

0.02 pl

0.2 pl

0.02 p l

0.2 pl

Results and Discussion

Passage of the viruses using the above procedure resulted in an attenuated RSVR, JA-142. As is evident, the virus was more adapted to replicate in cell culture and therefore required a lesser amount of virus-infected nutrient medium to be transferred as passages continued. For transfers using a very small amount of virus-infected nutrient medium (eg 0.2 µl or 0.02 µl) a different dilution was required. This dilution was accomplished by adding a small amount of virus-infected nutrient medium to a larger quantity of nutrient medium. For example, to obtain a 0.2 µl blot, 2 µl of virus-infected nutrient medium was added to 20 µl of nutrient medium and 2 µl of this dilution was added to the next culture in the series. Using this strategy, the highest dilution resulting in an ECP was used and the time required to pass the virus was minimized. Passage at daily intervals ensured that the virus was always in a logarithmic replication phase. Daily blotting also ensured that there were an adequate number of cells for virus replication.

Because the mutations (which are likely cumulative) that are likely to result in attenuation only occur during replication, it is not an advantage to have substantially all cells infected or for replication to develop at a slower rate or stop earlier. of the next transfer. Based on previous PRRSV studies, the replication cycle was known to be approximately 8 hours, therefore transferring a minimal amount of virus from virus-infected nutrient medium to non-infected nutrient medium at daily intervals results in the virus always have many cells to replicate in.

As can be easily appreciated, the pass using this procedure results in time savings hitherto thought impossible (ie each pass required less time). This is especially important when a high number of passages are necessary for adequate viral attenuation. If each pass, using the old procedures, was performed at an interval of 3 days, a procedure requiring 200 passes would take 400 days less using the procedure of the present invention.

Example 2

Materials and Procedures

This example determined whether passage 200 of the PRRS virus, JA-142, would revert to virulent when passaged into the host animal six times. This study consisted of six groups. Five pigs from group 1 (main group) were inoculated intranasally with SRRP VSM, JA-142 passage 200, while three pigs from group 1A (control group) were inoculated intranasally with sterile diluent. The animals were given commercial feed and water ad libitum throughout the study. Pigs in both treatment groups were monitored daily for clinical signs (appearance, respiratory signs, feces, etc.). After six days, the animals were weighed, bled, and sacrificed. After scoring the lungs for lesions, lung washes were collected from each animal. Lung washes were frozen and thawed once, and a pool was prepared using 2.0 ml of serum and 2.0 ml of lung lavage from each animal within a group to prepare Retropaces 1 and 1A, respectively. This combination was used to challenge (intranasally) group 2 and group 2A animals, respectively. This procedure was repeated for groups 3 and 3A to 6 and 6A. Animals in each group were housed separately but under identical conditions.

After inoculation, blood samples were collected and body temperatures were monitored. Rectal temperatures for each animal were measured periodically from -1 DPE (days post-exposure) to 6 DPE and the average was calculated along with the temperatures of other animals in the same group. The health status of each animal was monitored daily for the duration of the study. The results were collected and scored on

ES 2 388 909 T3 form of daily observation. The scoring parameters are as follows:

1. Normal appearance = 0; depressed = 1; excited = 2; comatose / dead = 30.

2. Normal respiration = 0; sneezing = 1; cough = 1; fast / slow = 2; with difficulty = 3.

3. Normal stools = 0; dry = 1; loose = 2; fluids = 3.

Four. Normal eyes = 0; watery = 1; off = 2; sunken = 3.

5. Normal nostrils = 0; serous exudate = 1; red / swollen = 2; crusted ulcers = 3.

6. Normal mouth = 0; slime = 2; ulcers = 3.

7. Exercise

NA

8. Normal appetite = 0; decreased = 1; anorexic (none) = 3.

9. Others

Animals were also weighed prior to inoculation and at necropsy. Average weight gain was calculated for each group for comparison. Enzyme-linked immunosorbent assays (ELISA) and serum neutralization assays (NS) of SRRP were performed after exposures of the animals to test and control articles. Attempts were made to isolate PRRSV from serum samples in MA-104 cells. Before and after vaccination, total leukocyte counts were determined using a COULTER COUNTER MODEL Z1, Coulter Corp., Miami, FL. At necropsy, the lungs of each animal were scored. Lung scoring was performed by separating the lung into 7 sections and determining the percentage of lung involvement (the percentage of the lung area affected as shown by lesions or redness for each section and 10 multiplying by the approximate total lung area) from the percentage of total lung area covered by the section.

The parameters for pulmonary scoring are as follows:

% Implication of the Left Apical Lobe% of Implication of the Left Heart Lobe% of Implication of the Left Diaphragmatic Lobe% of Implication of the Right Apical Lobe% of Implication of the Right Heart Lobe% of Implication of the Right Diaphragmatic Lobe% of Implication of the Intermediate Lobe of the Intermediate Lung Right

X 0.10

X 0.25

X 0.10

X 0.25

X 0.10

Total (Sum of the values in the rightmost column)

ES 2 388 909 T3

Results and Discussion

Each group of pigs was monitored for six days after vaccination. Clinical scores were low in all groups. The results of the clinical scores are provided in Table 1.

TABLE 1

<td colspan="11">Daily Clinical Scores</td>

<td>Treatment</td><td>Group 1 Pig No.</td><td>Day 1</td><td>Day 0</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td><td>Day 6</td><td>Average</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0,4</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0,05</td>

<td>Saline solution</td><td> 550</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>Treatment</td><td>Group 2 Pig No.</td><td>Day 1</td><td>Day 0</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td><td>Day 6</td><td>Average</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0,4</td><td> 0,2</td><td> 0</td><td> 0</td><td> 0,075</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td>Group number 3 Pig No.</td><td>Day 1</td><td>Day 0</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td><td>Day 6</td><td>Average</td>

<td>Rewind 2</td><td> 548</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0,2</td><td> 0,2</td><td> 0</td><td> 0</td><td> 0,05</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>Treatment</td><td>Group 4 Pig No.</td><td>Day 1</td><td>Day 0</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td><td>Day 6</td><td>Average</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td></td><td>Average</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>Treatment</td><td>Team 5 Pig No.</td><td>Day 1</td><td>Day 0</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td><td>Day 6</td><td>Average</td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td>Team 5 Pig No.</td><td>Day 1</td><td>Day 0</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td><td>Day 6</td><td>Average</td>

<td></td><td>Average</td><td> 0,4</td><td> 0,4</td><td> 0,4</td><td> 0,4</td><td> 0,8</td><td> 0,4</td><td> 0,8</td><td> 0,8</td><td> 0,55</td>

<td></td><td>Average</td><td> 0</td><td> 0,08</td><td> 0,48</td><td> 0,48</td><td> 0,56</td><td> 0,48</td><td> 0,56</td><td> 0,56</td><td> 0,4</td>

<td>Treatment</td><td>Group 6 Pig No.</td><td>Day 1</td><td>Day 0</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td><td>Day 6</td><td>Average</td>

<td></td><td>Average</td><td> 0,8</td><td> 0,8</td><td> 0,8</td><td> 0,4</td><td> 0,8</td><td> 0,2</td><td> 0,2</td><td> 1,6</td><td> 0,7</td>

<td></td><td>Average</td><td> 0,666667</td><td> 0,56</td><td> 0,16</td><td> 0,08</td><td> 0,16</td><td> 0,04</td><td> 0,04</td><td> 0,32</td><td> 0,253333</td>

There were no significant differences between groups for rectal temperatures or daily weight gains. 5 All lung scores were negative.

Serologically, the ELISA S / P ratios and NS titers were negative throughout the test period of each group. Virus isolation was attempted in all serum samples and lung washes. On day 6, 60-100% of the serum samples from the groups administered JA-142, passage 200, and subsequent backpasses were positive. The groups administered saline were negative. In the first 10 three passages, virus was recovered in the lung lavages of only 20-40% of the pigs, but in the last three passages, the virus was recovered in 50-80% of the pigs.

Based on these data, JA-142 pass 200 did not revert to virulent when passed through pigs six times.

ES 2 388 909 T3

Example 3

Materials and Procedures

This example demonstrated that the safety attenuation level of VSM, JA-142, pass 200 did not change significantly during six backpasses in the host animal. The evaluation of the level of attenuation or safety was carried out using the pregnant sow model and controlling the effect on reproductive performance. This model is the most sensitive test system and does not depend on subjective factors for virulence testing. This example consisted of four groups (A, B, C and D) that had seven sows per group. Group A was inoculated intranasally with SRRP VSM, JA-142 passage 200. Group B was inoculated intranasally with JA-142 passage 200, retropase 6. Group C was inoculated intranasally with sterile diluent, to that will act as normal controls. Group D was inoculated intranasally with PRRSV JA-142 pass 4. Test articles (JA-142 challenge, pass 4) were administered at approximately 93 days gestation. Sow body temperatures were monitored for the first seven days after vaccination. Blood samples were collected from the sows once a week and at farrowing. Blood samples were collected and piglet weights were recorded at birth, 7 and 14 days of age. The health status of each animal was monitored daily for the duration of the study until and after parturition for 14 days. The farrowing performance was evaluated by observing the health status of the born piglets.

SRRP ELISA assays were performed after exposures of sows to the test article. SRRP ELISA assays were also performed on piglet sera weekly after farrowing. After exposure to the test article, attempts were made to isolate PRRSV from serum samples in MA104 cells. Rectal temperatures were measured periodically from 0 days post-vaccination (DPV) to 7 DPV, and the average temperature of each group was determined. Before and after inoculation, total leukocyte counts were determined as in Example 1. Clinical observations of sows were made as in Example 2, from -1 DPV to farrowing. Piglet clinical observations were made from farrowing to 14 days of age. Finally, at necropsy, the lungs of each piglet were scored to determine the percentage of lung involvement.

Results

The ELISA results indicate that the animals used in the present study had not received prior exposure to PRRSV. The animals that received virus inocula, groups A, B and D, presented seroconversion at 14 days post-treatment. Three sows from group B remained negative at 14 days post-treatment. At parturition, group B negative sows tested positive for PRRSV antibodies.

Pig ELISA results indicated that most piglet samples born to Group A and Group B sows were taken after they had suckled. Those pigs that were negative at zero days post-partum (0 DPP) tested positive at 7 DPP. All pigs born to sows in Group C were seronegative in the test throughout the study. Only a few group D pigs were tested, as most were stillborn or mummified. Half of those pigs that were tested were seropositive. This indicated that seronegative pigs were sampled before they suckled, or that they were unable to suckle. All piglets born to sows in group D died before 7 DPP. RSRV isolates from sows in groups A and B were sporadic. Although the ELISA test results indicated that these sows were successfully inoculated with the viral test articles, many remained negative for virus isolation from serum.

Most pigs born to sows in groups A and B tested positive for virus isolation during the study. The litter born from a sow from group A never tested positive and in the litter born from a sow from group B only two out of eight piglets tested positive for virus isolation. No virus was recovered from piglets born to sows of group C. Viruses were recovered from the majority (71%) of the piglets born to sows in group D.

Post-treatment rectal temperatures were standard. Groups that were treated with VSM, retropass 6, or sterile diluent had no measurements that exceeded 38.72 ° C (101.7 ° F). Group D, treated with JA-142, pass 4, had four (out of seven) sows that suffered temperatures exceeding 38.89 ° C (102 ° F), with one sow reaching 39.67 ° C (103.4 ° F) during one of the days. The weight gain behavior of piglets born to sows of groups A (treated with VSM) and B (treated with VSM, backpass 6) was higher than that of pigs born to control sows of group C. The average of Weight gain for the 14 day observation period was 3.58 kg (7.9 lb). For group A it was 3.49 kg (7.7 lb); for group B and group C it was 3.13 kg (6.9 lb). The difference in weight gain was not related to the size of the remaining litter at 14 days. The mean litter sizes at 14 days postpartum (WMD) were 9 for group A, 7 for group B and 10 for group C. No pig born to the sows of group D survived beyond 3 WMD. .

ES 2 388 909 T3

The leukocyte counts (WBC) for the sows in groups A, B and C remained relatively constant. The average percentages of the pre-exposure values were equivalent to or greater than 92% during the duration of the observation period. Three sows in Group D had WBC counts that were below the expected normal range (7-20 x 106/ ml).

Post-inoculation clinical scores were common for sows in groups A and B. Several sows in group C were observed to experience clinical signs over a period of several days. Most of the clinical symptoms observed were in the category of decreased appetite, respiratory symptoms, and depression. One sow from group C died on day 31 of the chronic bacterial pneumonia trial. Six of the seven sows in group D were observed to have clinical signs, mainly of varying degrees of severity, of loss of appetite, 10 ranging from diminished to anorexic. The results of the clinical score are provided in Table 2.

ES 2 388 909 T3

TABLE 2

<td rowspan="18">Sow Clinical Scores</td><td>CM</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td> 28</td><td>or</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td> 44</td><td>OR</td>

<td>Treatment</td><td>Group A</td><td>JA-142 VSM</td><td>Pass 200</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Group A</td><td>JA-142 VSM</td><td>Pass 200</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Group A</td>

ES 2 388 909 T3 (continued)

<td></td><td>JA-142 VSM</td><td>Pass 200</td><td></td><td></td><td></td><td></td><td></td><td>Treatment</td><td>B Group</td><td>Go back 6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>B Group</td><td>Go back 6</td><td></td>

ES 2 388 909 T3 (continued)

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>B Group</td><td>Go back 6</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Treatment</td><td>Group C</td><td>Thinner Sterile</td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td></td><td></td><td></td><td></td><td>Group C</td><td>Thinner Sterile</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Group C</td><td>Thinner Sterile</td><td></td><td></td><td></td><td></td><td></td><td>Treatment</td><td>Group D</td>

ES 2 388 909 T3 (continued)

<td></td><td>JA-142</td><td>Pass 4</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Group D</td><td>JA-142</td><td>Pass 4</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Group D</td><td>JA-142</td><td>Pass 4</td>

ES 2 388 909 T3 (continued)

ES 2 388 909 T3

The clinical observations of the piglets fell into two main categories, death and reduced appetite. There were no significant differences between groups A, B and C in the area of average litter deaths (MPC). Group A had an average of 1.3 MPC, Group B had an average of 2.4 MPC, Group C had an average of 2.0 MPC, and no pigs in Group D survived beyond three days postpartum. . The 5 clinical scores for the piglets are provided in Table 3.

ES 2 388 909 T3

TABLE 3

<td>Treatment</td><td>Group A</td><td>JA-142</td><td>Pass 200</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Group A</td><td>JA-142</td><td>Pass 200</td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>B Group</td><td>Go back 6</td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td>B Group</td><td>Go back 6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>N ° Pork</td><td> 249</td><td> 250</td><td> 426</td><td> 427</td><td> 428</td><td> 429</td><td>Prom.</td><td>I did not gesture.</td><td>N ° Pork</td><td></td><td> 24</td><td> 25</td><td> 46</td><td> 47</td><td> 48</td><td> 49</td><td> 50</td><td>IT</td>

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Treatment</td><td>Group C</td><td>Thinner Sterile</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>Treatment</td><td></td><td></td><td>Group C</td><td>Thinner Sterile</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>N ° Pork</td><td> 77</td><td> 78</td><td> 79</td><td> 80</td><td> 00</td><td></td><td> 142</td><td> 143</td><td> 144</td><td> 145</td><td>Prom.</td><td>N ° Pork</td><td></td><td> 891</td><td> 892</td><td>Prom.</td><td></td><td>Aborted</td>

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Treatment</td><td>Group D</td><td>JA-142</td><td>Pass 4</td><td></td><td></td><td></td>

ES 2 388 909 T3 (continued)

<td>N ° Pork</td><td></td><td> 883</td><td> 3- 00 00</td><td>Prom.</td><td></td><td>Aborted</td><td></td><td> 890</td><td>Prom.</td><td></td><td> 00 00 00</td><td> 889</td><td>Prom.</td><td></td><td>Aborted</td>

<td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ES 2 388 909 T3

The calving performance results provided the most dramatic differences and similarities between the various treatment groups. Since the treatments would not have an effect on litter size, the most appropriate way to compare farrowing results would be using percentage values. Group A had an average live / born percentage of 85% (SD +/- 9.6). Group B had an average live / born percentage of 89% (SD +/- 11.6). The control group (group C) had an average live / born percentage of 83.4% (SD +/- 7.9). The mean percentages for stillbirths for groups A, B and C were 8.8 (DT + / 9.66), 6.6 (DT +/- 9.7) and 14 (DT +/- 11.39) , respectively. The average percentages of mummified born to sows in groups A, B and C were 6.1 (DT +/- 6.01), 3.9 (DT +/- 4.45) and 2.6 (DT + / - 4.01), respectively. The mean percentages of live / births, stillbirths and mummified born to sows in group D were 8.7 (SD +/- 8.92), 10.7 (SD +/- 11.39) and 81.9 ( DT +/- 17.18), respectively.

The results of this example demonstrated the stability of the VSM, JA-142, passage 200, after passage in the host animal six times. There were no significant differences between the group of sows treated with VSM (group A) and the sows that were exposed to retropase 6 virus (group B) in the categories of farrowing performance, leukopenia, rectal temperatures and clinical observations of the sows. or piglets. Furthermore, the results in these same categories for groups A and B were comparable to those achieved by group C, which had been treated with sterile diluent. Finally, the performance of sows that had been exposed to the virulent parental virus of VSM, JA-142, passage 4, clearly illustrated the level of attenuation of the VSM and the absence of reversion to virulence by the retropase 6 virus JA-142.

Example 4

Materials and Procedures

This example evaluated the safety and the level of attenuation of administering a 10X concentration of VSM, JA-142, passage 201. The study was carried out in the pregnant sow model and the effect of this dosage on reproductive performance was controlled. The study consisted of three groups, A, C and D. Group A was inoculated intranasally with SRRP VSM, JA-142, passage 200. Group C was inoculated intranasally with sterile diluent to act as a group normal control. Group D was inoculated intranasally with JA-142 passage 201 10X. All inoculations were administered at approximately 93 days of gestation. Sow body temperatures were monitored for the first seven days after inoculation (vaccination). Blood samples were collected from the sows once a week and at farrowing. Before and after inoculation, total leukocyte counts were determined as in Example 2. The health status of each animal was monitored daily for the duration of the study until and after parturition for 14 days. Clinical observations of sows were made from -1 DPV until farrowing. The farrowing performance was evaluated by observing the health status of the born piglets. PRRSV ELISA assays were performed after exposures of sows to the test article. Attempts were made to isolate PRRSV from serum samples in MA-104 cells after exposure to the test article. Piglet clinical observations were made from farrowing to 14 days of age. Blood samples were collected from the piglets at birth, 7 and 14 days of age. PRRSV ELISA assays were performed on piglet sera weekly after farrowing. The piglets were also weighed at birth, on day 7 post-partum and at necropsy. At necropsy, the lungs of each piglet were scored to determine the percentage of lung involvement.

Results and Discussion

There were no significant differences between the groups administered a 10X dose of VSM, JA-142, passage 201, the groups administered a normal dose of VSM, JA-142, passage 200, and the groups who were administered sterile diluent. Therefore, based on the safety and attenuation of VSM, JA-142, pass 200 and in the absence of any significant difference in the results comparing these groups, a 10X dose of VSM, JA-142, was shown to pass 201 was safe, attenuated, and effective in inducing antibodies against PRRSV.

Example 5

Materials and Procedures

This example demonstrated that a minimum vaccine dose of PRRSV, JA-142, passage 205 representing VSM + 5 is effective in an experimental respiratory exposure model in fattening pigs. The pigs were divided into three groups. Group 1 was inoculated intramuscularly with SRRP VSM, JA-142 passage 205 at a titer of 2.0 log / dose. Group 2 was inoculated intramuscularly with sterile diluent. Group 3 acted as a normal control. Pigs

ES 2 388 909 T3 groups 1 and 2 were exposed to a PRRSV isolate with an RFLP pattern of 144 at day 28 post-vaccination. The body temperatures of the pigs were monitored for the first seven days after vaccination and daily after challenge. Each animal was weighed at vaccination, challenge, weekly throughout the study, and at necropsy. Blood samples were collected weekly after vaccination and every other day after challenge. The health status of each animal was monitored daily for the duration of the study. At necropsy, each animal was sacrificed and the lungs scored for percent lung involvement as in Example 2. PRRSV ELISA assays were performed after exposures of the pigs to the test and challenge articles. After exposure to the test articles, attempts were made to isolate PRRSV from serum samples in MA-104 cells. Virus isolation and ELISA results were analyzed using a χ analysis2 which tests whether the percentage of positive animals is the same in each group. White blood cell counts were performed as in Example 2.

Results and Discussion

Group 1 pigs (vaccinated pigs) did better in all aspects of this example than group 2 pigs (pigs given sterile diluent). Clinical scores, rectal temperatures, and percent lung involvement were all higher for pigs administered sterile diluent. White blood cell counts and weight gain were lower for pigs that received the sterile diluent. There was also a significant reduction in viremia, beginning on day 4 post-challenge in the vaccine group. On days 10 and 11 post-challenge, the number of viremia-positive animals further decreased in the vaccinated group, but remained the same in the group that received sterile diluent.

An ELISA was used to monitor anti-PRRSV serostatus before and after vaccination and challenge. All pigs were negative (S / P ratio <0.4) at the time of vaccination. All pigs, including vaccinated ones, were negative at 7 DPV (days post-vaccination). Seven days later, 21 of 22 vaccinated pigs tested positive for PRRSV antibody. Two pigs in group 1 continued to be negative during the pre-exposure period and presented seroconversion at 8 days post-challenge (8 DPE). All pigs in group 2 were negative on test day 0 and remained negative throughout the pre-exposure period. On test day 39 (8 DPE), 17 of the 22 exposed unvaccinated pigs (Group 2) were seropositive in the test. All pigs in group 3 (normal controls) remained seronegative throughout the study.

Virus isolations were made from sera before and after vaccination. Of the 22 vaccinated pigs, 17 were 2 DPV positive, 18 were 4 DPV positive, and 19 were 7 DPV positive. After vaccination, no vaccinia virus was recovered from one pig at all, and not up to 0 DPE from another. These results correspond to the seronegative status of these pigs during the post-vaccination observation period. At the time of challenge, 55% of vaccinated pigs were positive for viremia. After exposure, this percentage increased to 82% (at 2 DPE) and gradually decreased to 9% at 11 DPE. All pigs in group 2 were negative at 0 DPE and increased up to 82% positive at 2 DPE and 91% positive at 4 DPE. At 6 and 10 DPE, group 2 was approximately 82% virus positive and 73% of this group was 11 DPE positive. The normal controls, group 3, remained negative for the duration of the study.

Rectal temperature control showed an overall group increase experienced by group 2. Half of the pigs in this group experienced an increase of ~ 0.55 ° C (1 ° F) over the pre-exposure average for 2 or more. more days during the 11-day observation period. In comparison, only four of the 22 pigs in the vaccinated group experienced temperatures of ~ 0.55 ° C (1 ° F) above their pre-exposure average. The average length of the period during which these animals experienced elevated temperatures for two or more days was 2.2 days for Group 1 and 4 days for Group 2. None of the pigs in Group 3 experienced increases of ~ 0, 55 ° C (1 ° F) above your pre-exposure average for two days or longer.

Weight gain was monitored during the 11 day observation period. Group 3 pigs gained an average of 0.48 kg / day (1.06 pounds / day), Group 2 pigs gained an average of 0.43 kg / day (0.94 pounds / day), and pigs group 1 gained an average of 0.24 kg / day (0.53 pounds / day). Therefore, the exposed unvaccinated pigs gained only about 57% as much weight as did the exposed vaccinated pigs and only 50% as much weight as the control group.

Leukopenia (white blood cell counts) was monitored during the post-exposure observation period. Group 3 experienced a 5% reduction in the group average on test day 33 (2 DPE) compared to the pre-exposure average. For group 2, leukocyte counts decreased an average of 41% and did not

ES 2 388 909 T3 were returned to pre-exposure levels up to DPE. The vaccinated group experienced an average group decrease of 12% on test day 34 (3 DPE). Counts returned to the pre-exposure level the next day and remained the same at the pre-exposure level for the duration of the observation period.

Daily clinical observations were made from test day 28 (-4 DPE) to test day 42 (11 DPE). All pigs lacked any observable clinical signs during the pre-exposure period. Group 3 remained without any clinical signs for the duration of the post-exposure period. Five of the pigs in group 2 were observed to have post-challenge clinical signs. These signs were evident 6 EPDs and were not considered to be serious. Vaccinated pigs had only 1 clinical sign observed during the 11-day post-challenge observation period.

At the completion of the study, the lungs were evaluated for observable lung lesions. Group 3 had normal lungs and a group mean score of 0.02. Individual pig scores for group 2 ranged from a low of 33 to a high of 98 for a group average of 78.33. Scores for the vaccinated group ranged from 30 to a maximum of 90 with a group average of 53.20.

The data from this example demonstrated the efficacy of a modified live atypical SRRP viral vaccine. The vaccine was administered at a minimum dose of 2.0 log per dose containing the fifth passage beyond the VSM (JA-142, passage 205). The efficacy of the vaccine was demonstrated by significantly reducing the extent of lung lesions, the severity of post-exposure leukopenia, and post-exposure fever. Furthermore, a normal growth rate was maintained in vaccinated / challenged pigs compared to that achieved by normal control pigs, and significantly better than that achieved by challenged / unvaccinated pigs.

Example 6

Materials and Procedures

This example compared four groups, groups 1, 2 and 3, which had twenty pigs each, and group 4, which had 10 pigs. Group 1 was inoculated intramuscularly (IM) with SRRP VSM, JA-142, passage 205, at a titer of approximately 2.5 log / dose. Group 2 was inoculated intranasally with SRRP VSM, JA-142, passage 205, at a titer of approximately 5.0 log / dose. Group 3 was inoculated IM with sterile diluent. Group 4 acted as strict controls. Pigs were challenged with a PRRSV isolate from South Dakota State University (SDSU) with an RFLP pattern of 144 on day 28 post-vaccination. The body temperatures of the pigs were monitored daily after challenge. Each animal was weighed at vaccination, challenge, weekly for the duration of the study, and at necropsy. Blood samples were collected weekly after vaccination and every other day after challenge. The health status of each animal was monitored daily for the duration of the study. At the completion of the study, the animals were sacrificed and their lungs scored to determine the percentage of lung involvement.

PRRSV ELISA assays were performed after exposures of the pigs to the test and challenge articles. Attempts were also made to isolate PRRSV from serum samples in MA-104 cells after exposure to test articles. WBC counts and post-inoculation clinical observations were determined as in Example 2.

Results and Discussion

At zero days post-vaccination (DPV), all pigs in this example were serologically negative for PRRSV, as indicated by having a S / P ratio <0.4. At 14 DPV, 70% of the pigs in group 1 and 95% of the pigs in group 2 tested positive for the presence of anti-PRRSV antibody. Only one vaccinated pig in group 1 remained seronegative throughout the pre-exposure period. This pig became seropositive at seven days post-challenge (DPE). All pigs in groups 3 and 4 remained negative throughout the pre-exposure period. At nine DPE, all pigs in group 3, the group treated with sterile diluent, tested positive by ELISA for antibody against PRRSV. Normal controls, group 4, remained negative for the duration of the study.

Virus isolation results correlated well with serological results. Only one pig remained negative for virus isolation from serum and this corresponded to the seronegative state during the post-vaccination period. These results indicate a relationship between post-vaccination viremia and serological conversion with vaccine dosage. Group 2 was 100% seropositive at 14 DPV compared to 70% for group 1. The high-dose group (group 2) was 85% and 90% positive for viremia at 14 and 21 DPV,

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Post-challenge, 89% of Group 3 animals experienced temperatures that were ~ 0.55 ° C (1 ° F) or higher than pre-exposure values for two or more days. In group 1, 75% of the animals experienced temperatures of one degree or higher for two or more days. While only 45% of the animals in group 2 experienced elevated temperatures. In comparison, 30% of the animals in the normal control group (group 4) experienced elevated temperatures for two or more days during the 11-day observation period.

Treatment with the high dose of vaccine or the low dose of vaccine appeared to have no detrimental effect on growth performance during the post-vaccination period (-3 DPV to 28 DPV). The mean daily weight gain for groups 1 and 2 was 0.35 kg / day (0.77 lb / day) and 0.34 kg / day (0.76 lb / day), respectively. For a comparison, Groups 3 and 4 had average daily weight gains of 0.35 kg (0.77 lb) and 0.354 kg (0.78 lb), respectively. After challenge, the vaccinated groups had an average daily weight gain of 0.02 kg / day (0.05 lb / day) (group 1) and 0.07 kg / day (0.15 lb / day) ( group 2) more than the group treated with sterile diluent. Normal controls averaged 0.18 to 0.23 kg / day (0.4 to 0.5 lb / day) more daily weight gain than those vaccinated over the same time period.

Eighty-four percent (16 of 19) of group 3, the sterile diluent treatment group, experienced a 25% or greater decrease in their WBC count for one or more days after exposure. In normal controls, 3 out of 10 (30%) had experienced similar decreases. After exposure, in the vaccinated groups, the low dose (group 1) and the high dose (group 2), 11 of 20 (55%) and 3 of 20 (15%) had experienced a leukopenia of 25% for one or more days.

Clinical observations made prior to challenge indicated that the pigs were in good health. After exposure, the level of health status did not change significantly for those pigs that were exposed (groups 1, 2 and 3). Lethargy, respiratory signs, and loss of appetite were the clinical signs observed and these were described as mild in severity. The clinical signs described for a group 2 pig could be attributed to the bacterial pneumonia (see discussion below on lung lesions) that it was experiencing. The normal control group (group 4) had no observable clinical signs during the 11-day observation period.

At study completion, the pigs were euthanized and the lungs were observed for SRRP-like lesions to score the degree of lung involvement. The percentage of involvement was scored for each lobe, and then multiplied by the percentage of lung represented by total lung capacity. For example, 50% lung involvement for a diaphragmatic lobe was then multiplied by 25% to equal 12.5% of total lung capacity. The maximum score that could be obtained was 100. The group mean lung score for normal controls (group 4) was zero. The group mean score for the sterile diluent treatment group (group 3) was 70.08. The mean scores of vaccinated treatment groups were 48.83 for the low dose group (group 1) and 17.76 for the high dose group (group 2). One pig was observed to have a lung score of 62.5, the highest score within group 2. The lesions observed in the lungs of this pig were reported to be associated with bacterial pneumonia.

Based on the results of this study, both atypical PRRS VSM vaccine dosage levels reduced the severity of clinical signs associated with respiratory disease caused by PRRSV. A full field dose performed better than the minimal dose as indicated by the significant reduction in lung injury scores.

Example 7

Materials and Procedures

This example determined the sequence of the VSM, JA-142, from passage 201 attenuated, as well as the sequence of passage 3 of the virus from the field isolate, JA-142. The attenuated virus isolate was obtained from the master seed pool representing passage 201 in MA-104 simian cells of a PRRSV isolate from PRRS-affected pigs.

The virus was grown in 2621 cells, a monkey kidney cell line, also named MA-104 and USU-104 (Gravell et al., 181 Proc. Soc. Exp. Biol. Med. 112-1-19 (1986) , Collins et al., Isolation of Swine Infertility and

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Respiratory Syndrome Virus (Isolate ATCC VR-2332) in North America and Experimental Reproduction of the Disease in Gnotobiotic Pigs, 4 J. Vet. Diagnost. Invest. 117-126 (1992)) (the contents of which are hereby incorporated by reference). Cells were grown in 50 ml of Dulbecco's Modified Eagle's MEM medium (Life Technologies, Inc., Gaithersburg, MD), supplemented with 10% fetal calf serum and 50 pg / ml gentamicin (Sigma Chemical Co., St. Louis, MO) in an atmosphere of CO2 5% humidified at 37 ° C in 75 cm plastic tissue culture flasks2. Cells were maintained by passage at intervals of 5-7 days. The cells were detached from the surface with trypsin-versene and divided 1: 4. To infect the cells, the media was decanted and 1 ml of cell supernatant containing virus was added to a titer of approximately 105-106 infectious doses in tissue culture (TCID50) for 30 min. Thirty ml of fresh medium containing 4% fetal calf serum was added. The cells were incubated as described above for 5 days, at which time a cytopathic effect was evident in the culture. The virus-containing culture medium was centrifuged at 2000 rpm in a Beckman TJ6 centrifuge to pellet the cell debris.

Viral genomic RNA was purified by adding 1120 µl of prepared AVL buffer (QIAamp Viral RNA Isolation Kit) (QIAGEN, Inc. Valencia, CA) / carrier RNA to a 280 µl sample of virus-containing culture medium. The mixture was vortexed and incubated at room temperature for 10 min. 1120 µl of ethanol was added and the mixture was inverted several times. RNA was absorbed into the matrix of a QIAamp spin column by repeated centrifugation of 630 µl aliquots at 6,000 xg for 1 min. The column was washed with 500 µl of AW buffer and centrifuged to remove all traces of wash solution. RNA was eluted from the column with 60 µl of diethylpyrocarbonate-treated water at room temperature. The purified RNA was stored at -70 ° C or used immediately for cDNA synthesis.

For cDNA synthesis, viral RNA was heated at 67 ° C for 7 min, primed with random hexamers or RSVR-specific primers, and reverse transcription was performed with Superscript II RNase H (RT) reverse transcriptase (Life Technologies, Inc. .). Reactions contained MgCl2 5 mM, 1X standard buffer II (Perkin Elmer Corp. Wellesley, MA), 1 mM each of dATP, dCTP, dGTP, and dTTP, 1 unit / µl of RNase inhibitor, 2 units of RT, and 1 µl of RNA in a reaction of 40 µl. The reaction mixtures were incubated for 15 min at 42 ° C, for 5 min at 99 ° C and for 5 min at 5 ° C.

A polymerase chain reaction (PCR) was performed on the DNA fragments obtained for sequencing as follows: 10 μl portions of a cDNA reaction mix were combined with the following reagents, resulting in a reaction of 25 pl containing MgCl2 2 mM, 1X Conventional Buffer II (Perkin Elmer), 0.2 mM each of dATP, dCTP, dGTP and dTTP, 0.3 pM of 5 'and 3' RSVRP-specific primer, and 0.375 units of AmpliTaq Taq polymerase ( Perkin Elmer). Reactions were prepared by heating for 4 min at 93 ° C in a thermal cycler, then 35 cycles consisting of 50-59 ° C for 30 sec, 72 ° C for 30-60 sec, and 94 ° C for 30 sec. Specific times and temperatures varied depending on the annealing temperatures of the primers in each reaction, and the expected length of the amplified product. A final incubation was performed for 10 min at 72 ° C and the reactions were placed at 4 ° C. The PCR products were purified with a Microcon 100 kit (Amicon, Bedford, MA).

Rapid Amplification of cDNA Ends (RACE) PCR was performed to obtain the terminal sequence of the 5 'end of genomic RNA, based on the procedure of Frohman, MA., On Beyond Classic RACE (Rapid Amplification of cDNA Ends), 4 PCR Methods and Applications S40-S58 (1994) (the contents of which are hereby incorporated by reference). Viral RNA was isolated and converted to cDNA as described above, with random hexamers as primers. The reaction products were purified on a Microcon 100 column (Amicon). A poly (dA) tail was added to the 3 'end by incubating 10 µl of cDNA in a 20 µl volume containing 1X 4 buffer (New England Biolabs, Beverly, MA), CoCl2 2.5 mM, 0.5 mM dATP and 2 terminal transferase units (New England Biolabs), for 15 min at 37 ° C. The reaction was quenched by heating for 5 min at 65 ° C and then diluted to 200 µl with water.

PCR was performed using the Expand Large Template PCR Systemto (Boehringer Mannheim, Mannheim, Germany) in a 50 μl reaction volume containing 10 μl of diluted poly (dA) tailed cDNA, 1X buffer 3, 0.35 mM each of dATP, dCTP, dGTP and dTTP, MgCl2 0.625 mM, primer Qt 0.04 pM (Frohman, 1994), primer Qor 0.3 pM (Frohman, 1994), 5'-CGCCCTAATTGAATAGGTGAC-3 '0.3 pM and 0.75 µl of enzyme mixture. Reactions were heated to 93 ° C for 2 min in a thermal cycler and subjected to 25 cycles, each cycle consisting of 93 ° C for 10 s, 63 ° C for 30 s, and 68 ° C for 12 min. After 25 cycles, the reaction was incubated at 68 ° C for 7 min and kept at 4 ° C. An aliquot of the reaction was diluted 100 times and 5 µl of diluted product was added to a second PCR reaction containing, in 50 µl, 1 1X buffer, 0.35 mM each of dATP, dCTP, dGTP and dTTP, primer Qi 0.3 pM (Frohman, 1994), 5 '

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CCTTCGGCAGGCGGGGAGTAGTGTTTGAGGTGCTCAGC-3 '0.63 pM and 0.75 µl of enzyme mixture. Reactions were heated to 93 ° C for 2 min in a thermal cycler and subjected to 25 cycles, each cycle consisting of 93 ° C for 10 s, 63 ° C for 30 s, and 68 ° C for 4 min. After 25 cycles, the reaction was incubated at 68 ° C for 7 min and kept at 4 ° C. The reaction products were electrophoresed on a 1% agarose gel and the approximately 1500 bp band was purified using the QIAgen QXII Gel Purification Kit. The eluted DNA was cloned into the pGEM-T vector (Promega, Madison, WI) using standard procedures. Individual clones were isolated and cultured for plasmid DNA isolation using plasmid isolation kits from QIAgen.

Plasmid DNA and PCR products were combined with appropriate primers based on related RSRV sequences in Genbank or derived from known sequences, and subjected to automated sequencing reactions with cycle sequencing kits with Taq DyeDeoxy terminators (Applied Biosystems, Foster City, CA ) and a PR 2400 Thermal Cycler (Perkin Elmer) at the University of Minnesota Advanced Genetic Analysis Center. Reactions were electrophoresed on an Applied Biosystems 3700 DNA sequencer. Automatic nucleotide reading and sequence error correction were performed primarily with the Phred program (University of Washington Genome Center) and fragment assembly was primarily performed with the Phrap program (University of Washington Genome Center). Additional software including the Lasergene package (DNASTAR Inc., Madison, WI), the Wisconsin package version 9.1 (Genetics Computer Group, Madison, WI), and EuGene (Molecular Biology Information Resource, Houston, TX) was used to analyze the sequence. The final viral genomic sequence was assembled from approximately 100 PCR reactions and 428 DNA sequencing reactions.

Results

The results of Example 7 are provided as SEQ ID NO: 1 and 2 where SEQ ID NO: 1 represents the sequence of passage 201 of the Master Seed Virus, JA 142, and SEQ ID NO: 2 represents the sequence of the field-isolated virulent virus, JA 142, after three passages.

It is also disclosed

Point 1: In a multiple passage procedure for attenuating a virus that includes the steps of successively replicating said virus by inoculation and replication of the virus in respective individual cell cultures until attenuation is achieved, the improvement comprising removing the virus-containing samples from at least certain of said cell cultures while the virus is replicating at a logarithmic rate and prior to the induction of cytopathic effects in said certain cell cultures and inoculating the following respective cell culture passages with such samples.

Point 2: The procedure of point 1, said withdrawal stage occurring approximately 24 hours after inoculation of said cell cultures.

Item 3: The procedure of item 1, said cell culture comprising a monolayer of cells having a confluence greater than about 70%.

Point 4: The procedure of point 1, which further comprises the step of periodically analyzing said progeny virus to evaluate attenuation.

Point 5: The procedure of point 1, which also comprises the steps of preserving said certain cell cultures after said withdrawal step and observing whether the induction of cytopathic effects takes place in said preserved cell cultures.

Item 6: The procedure of item 1, said inoculation comprising the smallest number of viruses that ultimately results in CPE.

Item 7: An SRRP virus that has the accession number ATCC VR-2638.

Item 8: A virus attenuated by the procedure in item 1.

Point 9: The virus from point 8, said virus having been passed a minimum of 200 times in cell culture.

Item 10: The virus from item 9, said virus being substantially avirulent.

Point 11: The virus of point 10, said virus being capable of generating antibody responses in pigs.

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Point 12: The virus of point 11, said antibody response being specific for strains of porcine reproductive and respiratory syndrome virus.

Item 13: A vaccine comprising the virus from item 7.

Item 14: The vaccine of item 13, which further comprises a pharmacologically compatible carrier agent.

Item 15: The vaccine of item 13, said vaccine being capable of generating antibody responses in a host animal.

Item 16: The vaccine of item 15, including said porcine cattle host animal.

Item 17: The vaccine from item 15, said antibody response being specific for strains of porcine reproductive and respiratory syndrome virus.

Item 18: The vaccine from item 17, including such strains atypical porcine reproductive and respiratory syndrome strains.

Item 19: A virus that has a restriction enzyme cleavage pattern of 1-4-2 by the enzymes MLU 1, Hinc II, and Sac II, respectively, in open reading frame five.

Spot 20: The virus from spot 19 that is not cleaved by MLU 1 in open reading frame five.

Item 21: A vaccine comprising the virus from item 19.

Item 22: The vaccine of item 21, which further comprises a pharmacologically compatible carrier agent.

Item 23: The vaccine of item 21, said vaccine being capable of generating antibody responses in a host animal.

Item 24: The vaccine of item 23, including said porcine cattle host animal.

Item 25: The vaccine from item 23, said antibody response being specific for strains of porcine reproductive and respiratory syndrome virus.

Item 26: The vaccine from item 25, including these strains atypical porcine reproductive and respiratory syndrome.

Point 27: A procedure to immunize pigs against strains of the porcine reproductive and respiratory syndrome virus, said procedure comprising the step of administering the vaccine from point 21 to pigs.

Item 28: An isolated DNA sequence that has at least about 65% sequence identity to SEQ ID NO: 1.

Item 29: An isolated DNA sequence that has at least about 75% sequence homology to SEQ ID NO: 1.

Item 30: An isolated DNA sequence that has at least about 65% sequence identity to SEQ ID NO: 2.

Item 31: An isolated DNA sequence that has at least about 75% sequence homology to SEQ ID NO: 1.

Item 32: A PRRS virus that includes the sequence from item 28.

Item 33: A PRRS virus that includes the sequence from item 29.

Item 34: The virus from item 33, said virus being attenuated by the procedure from item 1.

Item 35: The virus from item 33, said virus having been passed a minimum of 200 times in cell culture.

Item 36: The virus from item 33, said virus being substantially attenuated.

Item 37: The virus from item 33, said virus being substantially avirulent.

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EP1183332A1	European Patent Office (EPO)	A1	2002-03-06
KR20020028875A	Republic of Korea	A	2002-04-17
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Porcine reproductive and respiratory syndrome vaccine, based on isolate JA-142

Abstract

Term

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DESCRIPTION

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TABLE 2

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Priority claims10

Members36

Numbers

Titles2

Classification