PRRSV antigenic sites identifying peptide sequences of PRRS...

Details PRRSV antigenic sites identifying peptide sequences of PRRS... PRRSV antigenic sites identifying peptide sequences of PRRS...

1999-11-05

2002-12-17

Salimi, Ali R. (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C424S204100, C424S211100, C536S023720, C530S327000, C530S326000, C530S328000

Reexamination Certificate

active

06495138

ABSTRACT:

TECHNICAL FIELD
The invention relates to the causative agent of Mystery Swine Disease, the PRRS virus, to peptide sequences identified in the PRRS virus, and to incorporating these sequences in vaccines and diagnostic tests.
BACKGROUND
PRRS virus (PRRSV) is the causative agent of a pig disease, currently called porcine reproductive and respiratory syndrome (PRRS). The virus is the causative agent of a pig disease, seen since approximately 1987 in the U.S. and since 1990 in Europe, known initially under various names such as Mystery Swine Disease, Swine Infertility and Respiratory Syndrome, and many more. The virus itself was also given many names, among which Lelystad virus (LV), SIRS virus, and many more, but is now mostly designated porcine reproductive and respiratory syndrome virus (PRRSV). It causes abortions and respiratory distress in pigs and was first isolated in Europe in 1991 (EP patent 587780, U.S. Pat. No. 5,620,691) and subsequently in the U.S. and many other countries throughout the world. PRRSV is a small enveloped virus containing a positive strand RNA genome. PRRSV preferentially grows in macrophages. In addition to macrophages, PRRSV can grow in cell line CL2621 and other cell lines cloned from the monkey kidney cell line MA-104 (Benfield et al.,
J. Vet. Diagn. Invest
. 4; 127-133, 1992). The genome of PRRSV, a polyadenylated RNA of approximately 15 kb was sequenced in 1993 (Meulenberg et al., Virology 192; 62-74, 1993). The nucleotide sequence, genome organization and replication strategy indicated that PRRSV is related to a group of small enveloped positive-strand RNA viruses, designated Arteriviruses. This group includes lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV). These viruses have a similar genome organization, replication strategy, morphology, and amino acid sequence of viral proteins. Arteriviruses contain a genome of 12.5 to 15 kb and synthesize a 3′ nested set of six subgenomic RNAs during replication. These subgenomic RNAs contain a leader sequence which is derived from the 5′ end of the viral genome. ORFs 1a and 1b comprise approximately two thirds of the viral genome and encode the RNA dependent RNA polymerase. Six smaller ORFs, ORFs 2 to 7, are located at the 3′ end of the viral genome. ORFs 2 to 6 likely encode envelope proteins whereas ORF7 encodes the nucleocapsid protein (Meulenberg et al,
Virology
206; 155-163, 1995).
PRRSV is the first Arterivirus for which it has been demonstrated that all six proteins encoded by ORFs 2 to 7 are associated with the virion. The 15-kDa N protein (encoded by ORF7) and the 18-kDa integral membrane protein M (ORF6) are not N-glycosylated, whereas the 29- to 30-kDa GP
2
protein (ORF2), the 45- to 50-kDa protein GP
3
protein (ORF3), the 31-to 35-kDa GP
4
protein (ORF4), and the 25-kDa protein GP
5
(ORF5) are. These proteins have also been detected in extracellular virus and lysates of cells infected with a North American isolate of PRRSV, ATCC-VR2332, and other isolates of PRRSV (other isolates of PRRSV are for example CNCM I-1140, ECACC V93070108, CNCM I-1387, CNCM I-1388, ATCC-VR2402, ATCC-VR2429. ATCC-VR2430, ATCC-VR2431, ATCC-VR2475, ATCC-VR2385, but many others are known).
We earlier described the isolation and characterization of a panel of PRRSV-specific MAbs that were specific for GP
3
, GP
4
, M and N (van Nieuwstadt et al., J. Virol. 70,4767-4772, 1996). Interestingly, MAbs directed against GP
4
were neutralizing, suggesting that at least part of the protein is exposed at the virion surface. Furthermore, most of the Mabs directed against N reacted with all PRRSV isolates tested.
PRRS in it self is a problem of major concern for the swine industry in most parts of the world. Introduction of PRRSV in pig herds will cause severe economic losses. Diagnostic testing against PRRS is widely practiced by many veterinarians and laboratories. Most diagnostic tests, such as IPMA, IFT, IFA, ELISA, each comprising suitable means of detection such as conjugated enzymes or fluorochromes, and other substrates, use interactions between antigen derived from PRRSV and antibodies directed against PRRSV to measure the presence of either PRRSV antigen or antibodies directed against PRRSV in a biological sample, such as blood, serum, tissue, tissue fluids, lavage fluids, urine, feces, that is sampled from the animal (such as a pig) to be tested. The antigen and/or antibodies used in these diagnostic tests, or diagnostic kits or assays, for PRRS diagnosis are only defined by their origin from, or by their reactivity with PRRSV. In principle this suffices for screening assays where a high specificity or sensitivity is not explicitly required. However, the ever continuing spread of PRRS has caused great concern among the pig industry, to the extent that it is deemed needed to eradicate PRRS from whole herds, or even from complete areas, regions, or countries where pigs are raised. A clear example of this need is the proposed eradication program relating to PRRS in Denmark. If one decides to completely eradicate PRRS then diagnostic tests are needed that exhibit higher specificity or sensitivity than the tests used today.
Vaccination against PRRS is also widely practiced. Several examples are known of modified live vaccines that are used, and also killed vaccines are known. However, a problem with live vaccines in general, and thus also with live PRRS vaccines, exists in that these vaccines have a tendency to spread to non-vaccinated pigs, thereby spreading instead of reducing detectable infection in pig herds, and thus being counter productive to complete eradication. If a line marker vaccine were used that could serologically be differentiated from the wild type virus, then this problem would be greatly reduced. Added disadvantages are that live vaccines sometimes cause anaphylactic reactions in the vaccinated pigs, because of undefined antigenic components. Although killed vaccines in general are reported to induce protection in the vaccinated pig, and have the additional advantage that they do not spread from pig to pig, a disadvantage of killed vaccines is that it may be hard to accrue sufficient antigenic mass in one dose of a vaccine to elicit a measurable and protective immune response. Especially killed vaccines that can induce measurable neutralizing antibody titers in pigs would be beneficial to have since measuring these neutralizing antibodies in vaccinated pig populations would help generate understanding about the level of protection obtained by vaccination in the pig herd. In addition, if one succeeds in assembling the necessary antigenic mass, this also means that more and other undefined antigenic mass is also present in the vaccine, which can also give rise to the anaphylactic reactions as described above. In this sense it would be beneficial to know which specific site on PRRSV is important peptide sequences needed for eliciting neutralizing antibodies. An advantage of the currently used vaccines originating from PRRSV isolates isolated in the U.S. is that such vaccines, albeit fully protective against and immunologically cross-reactive with European isolates of PRRSV, contain, as yet undefined, epitopes or antigenic sites by which they can be discerned from European isolates of PRRSV. Reciprocally, live vaccines originating from PRRSV isolates isolated in Europe, albeit fully protective against and immunologically cross-reactive with U.S. isolates of PRRSV, contain similar as yet undefined epitopes or antigenic sites by which they can be discerned from U.S. isolates of PRRSV.
If serological tests would be available which could discriminate (based on the small epitopic differences between PRRSV isolates) between pigs that are either vaccinated with a U.S. derived vaccine or infected with a European wild type of PRRSV (being vaccinated or not), or which could discriminate pigs that are either vaccinated with a European derived vaccine or infected with an U.S. wild type of PRRSV (being vaccinated or no), than ma

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