Methods and compositions for vaccinating piglets against PRRS-1 virus

Abstract

The present disclosure provides a vaccine comprising a modified live PRRS-1 virus attenuated in a cell expressing porcine CD163 for inducing protective immunity in a piglet of 60 hours old or more wherein the vaccine is intranasally administered to the piglet.

Description

Technical Field

[0001] The present invention generally relates to the field of vaccinating piglets against the porcine reproductive and respiratory syndrome (PRRS) virus. Background Art

[0002] PRRS is characterized by abortions, stillbirths, and other reproductive problems in sows and gilts, as well as respiratory diseases in piglets. The pathogen is the PRRS virus (PRRSV), which is a member of the family Arteriviridae and the order Nidovirales. Nidoviruses are enveloped viruses with genomes consisting of single-stranded positive-sense RNA. The genomic RNA of positive-strand RNA viruses plays a dual role in both the storage and expression of genetic information.

[0003] In the late 1980s, two different genotypes of the virus emerged almost simultaneously, one in North America and the other in Europe. Currently, the PRRS virus is endemic in almost all pig-producing countries and is considered one of the most economically important diseases affecting the global pork industry. European PRRS is commonly designated as "type 1" to distinguish it from the distantly related North American or "type 2" PRRS (PRRS-2). The various subtypes of European PRRS, designated as PRRS-1 viruses (all of which can be prevented against all aspects of the present invention), are further elaborated in: M.P. Murtaugh et al., Virus Research, Vol. 154, pp. 18-30, 2010; and M. Shi et al., Virus Research, Vol. 154, pp. 7-17, 2010.

[0004] Vaccines based on modified live PRRS-1 viruses have been commercially available. For example, PRRS MLV contains a modified live PRRS-1 virus grown in cells expressing porcine CD163. The virus is safe and effective for intramuscular injection in piglets as young as one day old. See, for example, U.S. Patent 11,090,376. It has been demonstrated that PRRS MLV (containing PRRS-1 subtype 1 virus) provides cross-protection against challenge with PRRS-1 subtype 2 virus and subtype 3 virus.

[0005] The present inventors have found that protective immunity can be induced in young piglets by intranasal administration of a modified live PRRS-1 virus grown in cells expressing porcine CD163. Summary of the Invention

[0006] On the one hand, the present invention provides a vaccine comprising a modified live PRRS-1 virus attenuated in cells expressing porcine CD163 for inducing protective immunity in piglets older than 60 hours of age, wherein the vaccine is administered intranasally to the piglets.

[0007] In certain embodiments, the genome of the modified live PRRS-1 virus comprises an RNA molecule that is SEQ ID NO:1 or at least 75% identical to SEQ ID NO:1, and further,

[0008] a) The amino acid sequence encoded by ORF1a of the genome of the modified live PRRS-1 virus in the vaccine contains: S, A or T at amino acid position 19, preferably S; Y, F or W at amino acid position 157, preferably Y; D or E at amino acid position 268, preferably D; H, R or K at amino acid position 294, preferably H; Y, F or W at amino acid position 416, preferably Y; S, A or T at amino acid position 742, preferably S; L, I, M or V at amino acid position 884, preferably L; P at amino acid position 908; K, R or H at amino acid position 916, preferably K; K, R or H at amino acid position 977, preferably K; S, A or T at amino acid position 1138, preferably S; F, Y or W at amino acid position 1160, preferably F; S, A or T at amino acid position 1500, preferably S; R, K or H at amino acid position 2094, preferably R;

[0009] P at amino acid position 2254; and L, I, M or V at amino acid position 2290, preferably L; and

[0010] b) The amino acid sequence encoded by ORF1b of the genome of the modified live PRRS-1 virus in the vaccine contains: S, A or T at amino acid position 567, preferably S; and H, R or K at amino acid position 912, preferably H; and

[0011] c) The amino acid sequence encoded by ORF2a of the genome of the modified live PRRS-1 virus in the vaccine contains: L, I, M or V at amino acid position 22, preferably L; F, Y, W at amino acid position 88, preferably F; M, L, I or V at amino acid position 94, preferably M; and F, Y or W at amino acid position 95, preferably F; and

[0012] d) The amino acid sequence encoded by ORF2b of the genome of the modified live PRRS-1 virus in the vaccine contains: L, I, M, or V at amino acid position 47, preferably L; and

[0013] e) The amino acid sequence encoded by ORF4 of the genome of the modified live PRRS-1 virus in the vaccine contains: T, S, or A at amino acid position 151, preferably T and

[0014] f) The amino acid sequence encoded by ORF5 of the genome of the modified live PRRS-1 virus in the vaccine contains: F, Y, or W at amino acid position 20, preferably F; and D or E at amino acid position 37, preferably D; and

[0015] g) The amino acid sequence encoded by ORF5a of the genome of the modified live PRRS-1 virus in the vaccine contains: V, L, I, or M at amino acid position 18, preferably V; and R, H, or K at amino acid position 35, preferably R.

[0016] In a subset of the examples, in the vaccines used as disclosed herein: the amino acid sequence encoded by ORF1a of the genome of the modified live PRRS-1 virus in the vaccine further contains: Y at amino acid position 24; and A at amino acid position 156; and the amino acid sequence encoded by ORF3 of the genome of the modified live PRRS-1 virus in the vaccine further contains S at amino acid position 52.

[0017] In certain examples, for any of the vaccines suitable for the vaccines disclosed above, the genome of the modified live PRRS-1 virus comprises an RNA molecule that is at least 90%, preferably at least 95% identical to SEQ ID NO:1. In the most preferred examples, the genome of the modified live PRRS-1 virus is identical to SEQ ID NO:1, or at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%) of the nucleotides differ from the corresponding nucleotides in SEQ ID NO:1 by silent mutations or conservative substitutions or any combination thereof.

[0018] In any of the embodiments of the vaccines containing the modified live PRRS-1 virus, the vaccines are administered intranasally to piglets that are older than 60 hours of age (including at least 64 hours of age, or at least 68 hours of age, or at least 72 hours of age, or at least 76 hours of age, or at least 80 hours of age, or at least 84 hours of age). At the same time, in certain embodiments, the piglets are about 21 days of age or younger, or about 14 days of age or younger, or about 10 days of age or younger, or about 7 days of age or younger, or about 5 days of age or younger.

[0019] In certain embodiments, the protective immunity induced by intranasally administering the vaccines disclosed herein to piglets older than 60 days of age includes at least one of reduced viremia, reduced viral shedding, lung lesion score, and lung lesion frequency.

[0020] In certain embodiments, the piglets are MDA positive. In other embodiments, the piglets are MDA negative. In the embodiments where the piglets are MDA negative, the protective response further includes increased weight compared to infected, unvaccinated piglets.

[0021] In certain embodiments, the vaccine is a single-dose vaccine. Detailed Description

[0022] To better explain the present invention, the following definitions are provided

[0023] The term "about" as applied to a reference number means that reference number plus or minus 10% of that value, except when the value is provided in a base-exponent expression (e.g., 10 3 ), in which case the term "about" means an exponent range within 10% of the value of the exponent. Taking 10 3 as an example, "about 10 3 " is between 10 2.7 and 10 3.3 .

[0024] The term "single-dose vaccine" or "vaccine effective as a single dose" etc. means the ability of the vaccine to induce protective immunity upon a single administration. The duration of such protective immunity is sufficient to keep the pigs from being vaccinated again for about six months or at any time before the slaughter of the pigs, whichever comes first.

[0025] The term "protective immunity" or "protective immune response" etc. means the ability of the vaccine to cause a reduction in the severity, frequency, or duration of at least one clinical sign of PRRS-1 virus infection. Such clinical signs include, but are not limited to, lung lesion severity, lung lesion frequency, oral shedding, nasal shedding, and viremia.

[0026] The term "effective amount" or "effective dose" refers to the amount of antigen that induces a protective immune response in a subject that has received the antigen and is challenged or infected with virulent PRRS-1 virus. In certain embodiments, the virulent PRRS-1 virus can be PRRS-1 virus subtype 1, or subtype 2 or subtype 3. In a more preferred embodiment, the virulent PRRS-1 virus is PRRS-1 virus subtype 1.

[0027] The term "vaccine" refers to a composition containing an effective amount of an antigen.

[0028] In one aspect, the present invention provides a vaccine comprising a modified live PRRS-1 virus attenuated in cells expressing porcine CD163 for inducing protective immunity in piglets at least three days old, wherein the vaccine is administered intranasally to the piglets.

[0029] Methods for preparing cells expressing porcine CD163 are known in the art. Suitable starting points include, but are not limited to, the simian kidney cell line MA-104 cells, baby hamster kidney BHK21 cells, and porcine kidney PK0809 cells. These cells can be transfected with a nucleic acid sequence encoding porcine CD163. Resulting clones, MARC-145 cells, BHK21-C12-26, and PK-9 cells are suitable non-limiting examples of cells expressing porcine CD163. For example, these cells have been disclosed in U.S. Patent No. 11,090,376. See also U.S. Patent No. 9,102,912, which refers to the designation of mammalian CD163 surface protein as the normal PRRS virus cell receptor).

[0030] As an illustration, to demonstrate that a particular transgenic modified strain has been attenuated, the experiment described below can be used. This illustration applies to both PRRS-1 and PRRS-2.

[0031] Each trial includes at least 10 sows per group, which are from PRRSV-free farms. The animals are tested and found to have no PRRS virus-specific serum antibodies and are PRRSV-negative. All animals included in the trial are from the same source and breed. The animal group allocation is random.

[0032] On day 90 of gestation, 10 5 TCID 50 1 ml of PRRSV is administered intranasally for challenge. Each test setup has at least three groups: one group for the wild-type virus; one test group for challenge with the potentially attenuated virus; and one strict control group.

[0033] When the strict control remains PRRSV-negative during the study and the number of healthy live piglets born in the wild-type challenge group is at least 25% less than that of the strict control, the study is considered valid.

[0034] Attenuation, in other words, a decrease in virulence, is defined as a statistically significant change in one or more variables that determine reproductive performance or other symptoms:

[0035] A significant decrease in at least one of the following variables for the test group (the potentially attenuated virus), compared to the unmodified parental strain-infected group, would be an indication of attenuation:

[0036] a) The frequency of stillbirths

[0037] b) Abortion on or before day 112 of gestation

[0038] c) The number of mummified piglets

[0039] d) The number of less active and weak piglets

[0040] e) Pre-weaning mortality

[0041] In addition, a significant increase in at least one of the following variables for the test group, compared to the unmodified parental strain-infected group, is preferred:

[0042] f) The number of piglets weaned per sow;

[0043] g) The number of healthy live piglets born per sow.

[0044] Alternatively, respiratory and other symptoms of PRRSV infection can be examined to determine attenuation.

[0045] In certain embodiments, the modified live PRRS-1 virus is the same as the virus used in PRRS MLV. This virus is a descendant of the PRRS-1 virus strain 96V198 that was attenuated in cells expressing porcine CD163. The genome of the virus used in PRRS MLV contains SEQ ID NO:1.

[0046] When identifying the preferred encoded amino acids, the corresponding / original amino acids of the wild isolate of strain 96V198 are also shown in parentheses, thus comparing the zero generation (wild type) with the 49th generation ( the genome of the PRRS-1 virus in PRRS MLV, SEQ ID NO:1). Thus, the amino acid sequence encoded by ORF1a of SEQ ID NO:1 contains:

[0047] S at amino acid position 19 (N);

[0048] Y at amino acid position 24 (F);

[0049] A at amino acid position 156 (T);

[0050] Y at amino acid position 157 (H);

[0051] D at amino acid position 268 (N);

[0052] H at amino acid position 294 (Y);

[0053] Y at amino acid position 416 (C);

[0054] S at amino acid position 742 (P);

[0055] L at amino acid position 884 (F);

[0056] P at amino acid position 908 (S);

[0057] K at amino acid position 916 (E);

[0058] K at amino acid position 977 (E);

[0059] S at amino acid position 1138 (P);

[0060] F at amino acid position 1160 (L);

[0061] S at amino acid position 1500 (P);

[0062] R at amino acid position 2094 (Q);

[0063] P at amino acid position 2254 (S); and

[0064] L at amino acid position 2290 (F).

[0065] The amino acid sequence encoded by ORF1b of SEQ ID NO:1 contains:

[0066] S at amino acid position 567 (N); and

[0067] H at amino acid position 912 (Q).

[0068] The amino acid sequence encoded by ORF2a of SEQ ID NO:1 contains:

[0069] L at amino acid position 22 (S);

[0070] F at amino acid position 88 (V);

[0071] M at amino acid position 94 (I); and

[0072] F at amino acid position 95 (L).

[0073] The amino acid sequence encoded by ORF2b of SEQ ID NO:1 contains:

[0074] L at amino acid position 47 (F).

[0075] The amino acid sequence encoded by ORF3 of SEQ ID NO:1 contains:

[0076] S at amino acid position 52 (T).

[0077] The amino acid sequence encoded by ORF4 of SEQ ID NO:1 contains:

[0078] T at amino acid position 151 (I).

[0079] The amino acid sequence encoded by ORF5 of SEQ ID NO:1 contains:

[0080] F at amino acid position 20 (L); and

[0081] D at amino acid position 37 (N).

[0082] The amino acid sequence encoded by ORF5a of SEQ ID NO:1 contains:

[0083] V at amino acid position 18 (A); and

[0084] R at amino acid position 35 (Q).

[0085] Any of the various sequence comparison algorithms and programs known in the art can be used to evaluate the nucleic acid sequence identity according to any of the embodiments described herein. For sequence comparison, typically one sequence serves as a reference sequence against which the test sequence is compared (e.g., a sequence disclosed herein, such as SEQ ID NO:1). Then, the sequence comparison algorithm calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0086] The percent identity between two nucleic acid sequences can be determined, for example, by comparing the sequence information using the computer program GAP, i.e., the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin Package Version 10.0 program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12:387-95). When calculating the percent identity, the sequences to be compared are generally aligned in a manner that maximizes the matches between the sequences. Preferred default parameters of the GAP program include: (1) the GCG implementation of a unary comparison matrix for nucleotides (containing a value of 1 for identity and 0 for non-identity), and the weighted amino acid comparison matrix of Gribskov and Burgess, ((1986) Nucleic Acids Res. 14:6745), as described in Atlas of Polypeptide Sequence and Structure, Schwartz and Dayhoff eds., National Biomedical Research Foundation, pp. 353-358 (1979), or other comparable comparison matrices; (2) for amino acid sequences, a penalty of 8 points for each gap and an additional penalty of 2 points for each symbol in each gap; or for nucleotide sequences, a penalty of 50 points for each gap and an additional penalty of 3 points for each symbol in each gap; (3) no penalty for terminal gaps; and (4) no maximum penalty for long gaps.

[0087] Sequence identity and / or similarity can also be determined by using the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, and computerized implementations of these algorithms (BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0088] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pair-wise alignment. A tree showing the clustering relationships used to create the alignment can also be drawn. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, 1987, Journal of Molecular Evolution (J. Mol. Evol.) 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, Computer Applications in the Biosciences (CABIOS) 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0089] Another example of a useful algorithm is the BLAST algorithm described in: Altschul et al., 1990, Journal of Molecular Biology (J. Mol. Biol.) 215:403-410; Altschul et al., 1997, Nucleic Acids Research (Nucleic Acids Res.) 25:3389-3402; and Karin et al., 1993, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. U.S.A.) 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program obtained from: Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses a number of search parameters, most of which are set to default values. The adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself based on the composition of the specific sequence and the composition of the specific database being searched for the sequence of interest; however, the values can be adjusted to increase sensitivity.

[0090] Another useful algorithm is Gapped BLAST, as reported in: Altschul et al., 1993, Nucleic Acids Research (Nucl. Acids Res.) 25:3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score; the threshold T parameter is set to 9; the two-hit method triggers gapless extension, charging a cost of 10 + k for the length of the gap for k; X u is set to 16, and X g is set to 40 for the database search phase and 67 for the output phase of the algorithm. Alignment of gaps is triggered by a score corresponding to approximately 22 bits.

[0091] Because the genetic code is degenerate, homologous nucleotide sequences can include any number of "silent" base changes, i.e., nucleotide substitutions that still code for the same amino acid.

[0092] Those skilled in the art will further recognize that changes in nucleic acid sequences that result in modifications of the amino acid sequence of the protein encoded by the nucleic acid sequence may have minimal (if any) effect on the resulting three-dimensional structure of the protein. For example, the codon for the amino acid alanine, a hydrophobic amino acid, may be replaced by a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue such as valine, leucine, or isoleucine. Similarly, changes that are expected to result in the replacement of one residue with a negatively charged residue, such as aspartic acid replacing glutamic acid; or one positively charged residue replacing another residue, such as lysine replacing arginine, may also produce proteins with substantially the same functional activity.

[0093] The following six groups each contain amino acids that are typical conservative substitutions for one another: [1] alanine (A), serine (S), threonine (T); [2] aspartic acid (D), glutamic acid (E); [3] asparagine (N), glutamine (Q); [4] arginine (R), lysine (K), histidine (H); [5] isoleucine (I), leucine (L), methionine (M), valine (V); and [6] phenylalanine (F), tyrosine (Y), tryptophan (W), (see, for example, U.S. Patent Publication 20100291549).

[0094] In certain embodiments, the genome of the modified live virus comprises SEQ ID NO:1 or is at least 75% identical (or at least 80% identical, or at least 85% identical, or at least 90% identical, or at least 95% identical, or at least 98% identical, or 100% identical) to SEQ ID NO:1.

[0095] In further embodiments, the genome of the modified live virus comprises SEQ ID NO:1 or is at least 75% identical (or at least 80% identical, or at least 85% identical, or at least 90% identical, or at least 95% identical, or at least 98% identical, or 100% identical) to SEQ ID NO:1, wherein further,

[0096] a) the amino acid sequence encoded by ORF1a of the genome of the modified live PRRS-1 virus in the vaccine comprises:

[0097] S, A, or T at amino acid position 19, preferably S;

[0098] Y, F, or W at amino acid position 157, preferably Y;

[0099] D or E at amino acid position 268, preferably D;

[0100] H, R or K at amino acid position 294, preferably H;

[0101] Y, F or W at amino acid position 416, preferably Y;

[0102] S, A or T at amino acid position 742, preferably S;

[0103] L, I, M or V at amino acid position 884, preferably L;

[0104] P at amino acid position 908;

[0105] K, R or H at amino acid position 916, preferably K;

[0106] K, R or H at amino acid position 977, preferably K;

[0107] S, A or T at amino acid position 1138, preferably S;

[0108] F, Y or W at amino acid position 1160, preferably F;

[0109] S, A or T at amino acid position 1500, preferably S;

[0110] R, K or H at amino acid position 2094, preferably R;

[0111] P at amino acid position 2254; and

[0112] L, I, M or V at amino acid position 2290, preferably L; and

[0113] b) the amino acid sequence encoded by ORF1b of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0114] S, A or T at amino acid position 567, preferably S; and

[0115] H, R or K at amino acid position 912, preferably H; and

[0116] c) the amino acid sequence encoded by ORF2a of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0117] L, I, M or V at amino acid position 22, preferably L;

[0118] F, Y, W at amino acid position 88, preferably F;

[0119] M, L, I or V at amino acid position 94, preferably M; and

[0120] F, Y or W at amino acid position 95, preferably F; and

[0121] d) The amino acid sequence encoded by ORF2b of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0122] L, I, M or V at amino acid position 47, preferably L; and

[0123] e) The amino acid sequence encoded by ORF4 of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0124] T, S or A at amino acid position 151, preferably T; and

[0125] f) The amino acid sequence encoded by ORF5 of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0126] F, Y or W at amino acid position 20, preferably F; and

[0127] D or E at amino acid position 37, preferably D; and

[0128] g) The amino acid sequence encoded by ORF5a of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0129] V, L, I or M at amino acid position 18, preferably V; and

[0130] R, H or K at amino acid position 35, preferably R.

[0131] These amino acids are non-conservative substitutions compared to the wild-type parental PRRS-1 virus.

[0132] In additional embodiments, in addition to the amino acids specified above, the ORF1a protein encoded by the genome of the modified live PRRS-1 virus in the vaccine further contains the following amino acids:

[0133] a) In ORF1a: Y at amino acid position 24; and A at amino acid position 156; and

[0134] b) In ORF3: S at amino acid position 52.

[0135] In a more specific subset of the embodiments, the genome of the modified live virus comprises SEQ ID NO:1 or is at least 75% identical (or at least 80% identical, or at least 85% identical, or at least 90% identical, or at least 95% identical, or at least 98% identical, 99% identical) to SEQ ID NO:1, wherein,

[0136] b) the amino acid sequence encoded by ORF1a of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0137] S at amino acid position 19;

[0138] Y at amino acid position 24;

[0139] A at amino acid position 156

[0140] Y at amino acid position 157;

[0141] D at amino acid position 268;

[0142] H at amino acid position 294;

[0143] Y at amino acid position 416;

[0144] S at amino acid position 742;

[0145] L at amino acid position 884;

[0146] P at amino acid position 908;

[0147] K at amino acid position 916;

[0148] K at amino acid position 977;

[0149] S at amino acid position 1138;

[0150] F at amino acid position 1160;

[0151] S at amino acid position 1500;

[0152] R at amino acid position 2094;

[0153] P at amino acid position 2254; and

[0154] L at amino acid position 2290; and

[0155] b) the amino acid sequence encoded by ORF1b of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0156] S at amino acid position 567; and

[0157] H at amino acid position 912; and

[0158] c) The amino acid sequence encoded by ORF2a of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0159] L at amino acid position 22;

[0160] F at amino acid position 88;

[0161] M at amino acid position 94; and

[0162] F at amino acid position 95; and

[0163] d) The amino acid sequence encoded by ORF2b of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0164] L at amino acid position 47; and

[0165] e) The amino acid sequence encoded by ORF3 of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0166] S at amino acid position 52; and

[0167] f) The amino acid sequence encoded by ORF4 of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0168] T at amino acid position 151; and

[0169] g) The amino acid sequence encoded by ORF5 of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0170] F at amino acid position 20; and

[0171] D at amino acid position 37; and

[0172] h) The amino acid sequence encoded by ORF5a of the genome of the modified live PRRS-1 virus in the vaccine contains:

[0173] V at amino acid position 18; and

[0174] R at amino acid position 35.

[0175] Preferably, nucleotide differences (other than nucleotides encoding the specific amino acids disclosed above) between the RNA genome of at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or 100%) of the modified live PRRS-1 virus described herein and SEQ ID NO: 1 result in

[0176] a) a silent mutation or

[0177] b) a conservative substitution or

[0178] c) a combination thereof.

[0179] Known techniques can be used to determine the effective dose of the modified live PRRS-1 virus of the present invention, taking into account factors that can be determined by those skilled in the art, such as the body weight of the animal to be vaccinated. The dose of the virus of the present invention in the vaccine of the present invention preferably ranges from about 10 1 to about 10 9 TCID 50 (50% tissue culture infective dose), more preferably about 10 1.5 to about 10 8 TCID 50 , more preferably about 10 2 to about 10 6 TCID 50 , more preferably about 10 2.2 to about 10 5.2 TCID 50 , or about 10 2.2 to about 10 4.2 TCID 50 per dose, about 10 2.2 to about 10 3.2 TCID 50 per dose.

[0180] The suitable dose volume range of the vaccine disclosed herein is from about 0.25 ml to about 10 ml, more preferably from about 0.5 ml to about 5 ml, or from about 1 ml to about 3 ml, or about 2 ml.

[0181] Thus, in a particularly preferred set of embodiments, the volume is about 2 ml and the dose is about 10 2.2 to 10 5.2 TCID 50 , for example about 10 2.2 TCID 50 .

[0182] The vaccines of the present invention can be formulated according to recognized practices to include carriers acceptable for pigs, such as standard buffers, stabilizers, diluents, preservatives, mucoadhesives, and / or solubilizers, and can also be formulated for sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, glucose, mannitol, sorbitol, and lactose, etc. Stabilizers include albumin, etc. Mucoadhesive polymers have various hydrophilic groups, such as hydroxyl, carboxyl, amide, and sulfate groups. These groups attach to mucus or cell membranes through various interactions, such as hydrogen bonding and hydrophobic or electrostatic interactions. Lectins and thiolate polymers are non-limiting examples of mucoadhesives. Other suitable vaccine carriers and additives, including those particularly useful in formulating live attenuated vaccines, are known or obvious to those skilled in the art. See, for example, Remington's Pharmaceutical Science, 18th Edition, 1990, Mack Publishing, which is incorporated herein by reference.

[0183] Previously, it has been demonstrated (see U.S. Patent 11,090,376) that a vaccine containing a modified live PRRS-1 virus is safe when administered to one-day-old piglets. When the vaccine is administered intramuscularly, the effectiveness of the vaccine has also been demonstrated in one-day-old piglets. However, the vaccine is ineffective when administered intranasally.

[0184] The inventors have found that the vaccine is effective when administered intranasally to piglets at about three days of age. Thus, the vaccines according to any of the embodiments disclosed above can be administered intranasally to piglets older than 60 hours of age, including but not limited to at least 64 hours of age, at least 68 hours of age, at least 72 hours of age, at least 76 hours of age, at least 80 hours of age, or at least 84 hours of age. At the same time, in certain embodiments, piglets older than any of the age threshold lower limits specified in this paragraph can be less than about 21 days of age or less than about 14 days of age, or less than about 10 days of age, or less than about 7 days of age, or less than about 5 days of age.

[0185] The vaccines disclosed herein when used according to any of the embodiments of the present invention can elicit protective immunity when administered intranasally to piglets generally older than 60 hours of age (as described in the above paragraph). There are various methods for measuring protective immunity. Suitable endpoints for measuring protective immunity include viremia, virus shedding, and lung lesions.

[0186] As described above, US11,090,376 discloses that intranasal administration of a modified live PRRS-1 virus attenuated in cells expressing porcine CD163 to one-day-old piglets is ineffective. The inventors of the '376 patent believe that the lack of efficacy is due to maternally-derived antibodies (or MDA). However, if the vaccine is administered to pigs at about 3 days of age or older, the MDA status of the piglets does not significantly interfere with the protective immunity induced by the vaccine.

[0187] In certain embodiments, induction of protective immunity is measured by at least one of the following: reduced viremia, reduced viral shedding (which can be nasal, oral, or oro-nasal), reduced extent of lung lesions, and reduced frequency of lung lesions. As disclosed in the examples, both PRRS-1 MDA-positive and PRRS-1 MDA-negative three-day-old piglets showed reduced viremia, reduced viral shedding (nasal viral shedding for PRRS-1 MDA-negative piglets and nasal and oral viral shedding for PRRS-1 MDA-positive piglets), reduced degree of lung lesions, and reduced frequency of lung lesions after challenge. In addition, PRRS-1 MDA-negative piglets showed higher body weight and higher weight gain compared to unvaccinated piglets challenged with PRRS-1.

[0188] Use of the vaccine as described herein results in the onset of immunity starting at about 21 days post-vaccination.

[0189] The present invention will now be described in the following non-limiting examples.

[0190] Examples

[0191] Example 1. Evaluation of the Efficacy of PRRS MLV Immunization in Piglets Inoculated Intranasally at Three Days of Age The purpose of this study was to determine if intranasal administration of PRRS MLV to piglets

[0192] would elicit a protective response. Intranasal administration of PRRS MLV

[0193] Animals were assigned to treatment groups and host sow groups according to a protocol provided by a Biometric Representative (BMR) designated by Zoetis Veterinary Medicines Research and Development (Zoetis VMRD). Prior to farrowing, four sows were randomly assigned to two rooms. Using a completely randomized design, treatment groups were randomly assigned to the individual rooms.

[0194] Cross-foster piglets with host sows according to the allocation plan provided by BMR so that each sow fosters piglets from all sows. Before the challenge, the sows are removed and the piglets are moved into a room and mixed in a single pen. The experimental unit is the room for the vaccination phase and the animals for the challenge phase. During the challenge phase, a generalized randomized block design is used with the farrowing sows as blocks.

[0195] Sows are fed once daily with a commercially available feed suitable for their age and physiological state (lactation). The feed is fed individually once a day. Piglets are nursed from birth until weaning (3 weeks of age). Thereafter, they have free access to feed suitable for their age. Both sows and piglets have free access to water.

[0196] Sows are transferred to the research facility approximately 10 days before farrowing and are housed in individual farrowing pens (two rooms) according to the allocation plan.

[0197] Piglets stay with their sows until weaning (before the challenge), after which the sows are removed and the piglets of T01 and T02 are mixed and housed in separate pens in the same room. On the day of vaccination (day 0), the piglets are cross-fostered, weighed, examined, and determined for their suitability to participate in the study. At the time of challenge, the piglets are 3 or 4 days old (less than 20% of the total number of piglets).

[0198] On day 0, the piglets are inoculated with saline (group T01) or vaccine (group T02) as described in Section 4. Each animal in group T02 is administered a volume of 2 mL of PRRS MLV (10 2.2 TCID 50 ) per 2 mL, and each animal in group T01 is administered a volume of 2 mL of the control material, intranasally (IN) with a needleless syringe, 1 mL in each nostril.

[0199] On day 21, the piglets are challenged with the virulent PRRS virus strain Olot / 91. On the day of challenge, the virus stock solution (10 5.8 TCID50 / mL) is thawed and diluted 1:9 with cell culture medium to achieve a target titer of 10 5.0 TCID50 / 2 mL. During the challenge, the virus is kept on ice. Pre-challenge and post-challenge titers are determined in porcine alveolar macrophages (PAM). The challenge material is administered intranasally (IN), 1 mL in each nostril

[0200] For each treatment, the frequency distributions of clinical signs before and after the challenge were calculated, and the time point data were collected. For each treatment, the frequency distribution of whether animals had ever had clinical signs for each phase (days ≤ DC (reference before challenge) and days > DC (after challenge)) was calculated. If animals had ever had clinical signs (after challenge), a generalized linear mixed model was used for analysis, which had a logit link with fixed effects of treatment and random effects of block. This model did not converge. Fisher's exact test was applied.

[0201] Viremia: Before statistical analysis, appropriate logarithmic transformations were used to transform the RT-qPCR data. The transformed data were analyzed using a general linear repeated measures mixed model with fixed effects: treatment, time point, and the interaction between treatment and time point, and random effects: block and animals within block by treatment (animal term). Given the significant treatment-by-time point interaction effect (P ≤ 0.05), pairwise treatment comparisons were made at each time point. The treatment least squares means and 95% confidence intervals were back-transformed for presentation. The area under the curve (AUC) of the transformed data was also calculated for each animal, log-transformed and analyzed using a general linear mixed model with fixed effects: treatment and random effects: block. Given the significant treatment effect (P ≤ 0.05), pairwise treatment comparisons were made. The treatment least squares means, standard errors, ranges, and 95% confidence intervals were presented. The percentage of viremia / shedding days was also calculated. It was also determined whether animals had ever had viremia or had ever shed at days ≤ DC (reference before challenge) and days > DC (after challenge).

[0202] Body weight was analyzed using a general linear repeated measures mixed model with fixed effects: treatment, time point, and the interaction between treatment and time point, and random effects: block and animals within block by treatment (animal term). The treatment least squares means, 95% confidence intervals, minimum, and maximum values calculated for the data at each time point were collected. Given the significant treatment effect or treatment-by-time point interaction, pairwise treatment comparisons were made between treatment groups. A parametric model-based estimate was used to estimate the average daily weight gain estimate. The average daily weight gain was compared by treatment.

[0203] The percentage of total lung lesions was calculated using the following formula: Percentage of total lung lesions = (0.10 × left cranial) + (0.10 × left middle) + (0.25 × left caudal) + (0.10 × right cranial) + (0.10 × right middle) + (0.25 × right caudal) + (0.10 × accessory lung). Prior to analysis, an arcsine square root transformation was applied to the percentage of total lung lesions. A generalized linear mixed model with fixed effects: treatment and random effects: block was used to analyze the transformed lung lesions. Given the significant treatment effect, pairwise comparisons were made between treatment groups. The back-transformed least squares means, their standard errors, 95% confidence intervals, and minimum and maximum values of the percentage of total lung lesions were calculated.

[0204] The frequency distribution of the lung lesion assessment scores was calculated for each treatment. A generalized linear mixed model for binomial data with fixed effects: treatment and random effects: block was used to analyze normal or non-normal scores. Since the main treatment effect was significant, pairwise treatment comparisons were made.

[0205] General linear repeated measures mixed model analysis was used to analyze rectal temperature, with fixed effects: treatment, time point, and treatment-by-time point interaction and random effects: block and animal within block by treatment (animal term). Treatment least squares means, 95% confidence intervals, minimum and maximum values were calculated for each time point data. Descriptive statistics, mean, standard deviation, and range were calculated for each treatment group and study day (before challenge). The frequency distribution of febrile animals (rectal temperature ≥ 40.5 °C) was calculated for each treatment group and time point data. It was determined whether the animals were febrile at day = DC and day > DC. If an animal was ever febrile, a frequency table for that animal was calculated for each time period, before challenge and day > DC.

[0206] Prior to statistical analysis, appropriate logarithmic transformations were used to transform the serological data. General linear repeated measures mixed model analysis was used to analyze the transformed serological data, with fixed effects: treatment, time point, and treatment-by-time point interaction and random effects: block and animal within block by treatment (animal term). Given the significant treatment or treatment-by-time point interaction effect (P ≤ 0.05), pairwise treatment comparisons were made at each time point. The treatment least squares means and 95% confidence intervals were back-transformed for presentation. In addition, it was determined whether seroconversion (S / P ratio ≥ 0.4) occurred at any time point during the study for each animal. The frequency distribution of whether an animal seroconverted was calculated for each treatment at each time point. Descriptive statistics, mean, standard deviation, and range were calculated for each treatment group and time point (before challenge).

[0207] Results

[0208] Viremia

[0209] The presence of PRRS virus in serum was monitored by RT-qPCR testing conducted according to local procedures. All pigs were negative for RT-qPCR PRRSV in serum before vaccination (day 0). At day 21 (before challenge), PRRSV RNA was detected in 100% of the pigs in group T02. At this time, all pigs in T01 remained negative for PRRSV. By day 23, two days after challenge, 100% of the piglets in the T01 control group had viremia, and viremia persisted during the monitoring period after challenge. During the monitoring period, 100% of the animals in T02 remained virus-positive. Before challenge, at day 21, due to the presence of residual vaccine virus, the viral load detected in the serum of group T02 was significantly (P<0.0001) higher than that of group T01. After challenge, at all monitoring time points after challenge (corresponding to 2, 4, 7, and 10 days after challenge), the viral load detected in the serum of group T02 was significantly lower (P≤0.0330) compared to group T01.

[0210] Table 2 summarizes the mean AUC of log viremia 10 days after challenge (days 21 to 31), as well as the change and range of the confidence interval (CI) for each treatment. The mean AUC value of PRRSV viremia as a whole period in group T02 was significantly (p<0.0001) lower compared to group T01, with mean values of 7.47±0.14 and 8.62±0.14, respectively.

[0211] Table 1. Back-transformed least-squares means of PRRSV RNA copies / ml in serum

[0212] Group D21 (Before attack) D23 D25 D28 D31 T01 47.5 34586961 42978817 17209023 6720302 T02 5432642 2139202 523266.1 344000.4 1924833 P value P<0.0001 P<0.0001 P<0.0001 P<0.0001 P＝0.0330

[0213] Table 2. AUC analysis of PRRSV RNA copies / ml in serum after challenge (days > 21)

[0214]

[0215] Lung lesions

[0216] The percentage of lung lesions for each treatment group is shown in Table 3. Comparison between treatment groups showed a significant difference (p = 0.0023) in the percentage of total lung lesions. The percentage of total lung lesions in the IVP vaccine-inoculated group (T02) was lower compared to the control (T01) group.

[0217] Table 3. Analysis of percentage of total lung lesions (%)

[0218]

[0219] Table 4 summarizes the visual lung scores by treatment. At necropsy, 85.7% (18 out of 21) of the piglets in the T01 control group had positive visual lung scores (score > 0), indicating that PRRSV challenge successfully induced lung lesions. The percentage of animals with positive scores (score > 0) in the IVP vaccinated (T02) group was 50.0% (11 out of 22), and was significantly lower (P = 0.0229) than the control (T01) group. Additionally, none of the animals vaccinated with IVP (T02) had a lung assessment score of 2, while 4.8% (one out of 21) of the animals from T01 had a lung assessment score of 2.

[0220] Table 4. Visual lung scores by treatment group

[0221]

[0222] Nasal and oral shedding

[0223] The presence of PRRS virus in nasal swab samples was monitored by RT-qPCR testing conducted according to local procedures. All pigs were negative for RT-qPCR PRRSV in nasal swab samples before vaccination (day 0). At day 21 (before challenge), PRRSV RNA was detected in nasal swab samples from 40.9% (nine out of 22) of the pigs in the T02 group. At this time, all T01 pigs remained negative for PRRSV. At day 23 (two days after challenge), 95.5% of the piglets from T01 had viral shedding via the nasal route, peaking four days after challenge (day 25), with 100% of the piglets from T01 shedding. Even though 100% of the piglets were vaccinated with IVP (T02) at one or more time points after challenge (days > 21, see Table 8), the percentage of animals with nasal shedding never reached 100% at one time point (see Table 5). Table 5 details the frequency of nasal shedding of animals in each treatment group at each time point, and Table 6 summarizes the frequency of those that ever shed.

[0224] Table 5. Percentage of animals with nasal shedding per day for each treatment

[0225] Group D0 D21 (Before attack) D23 D25 D28 D31 T01 0 0 95.5 100 100 85.7 T02 0 40.9 81.8 59.1 40.9 77.3

[0226] Table 6. Frequency distribution of those that ever shed in nasal swabs after challenge (days > 21)

[0227]

[0228] Before the challenge, i.e., on day 21, due to the presence of residual vaccine virus, the viral load detected in the nasal swab samples of pigs in group T02 was significantly (P = 0.0175) higher than that in pigs in group T01. After the challenge, the viral load detected in the nasal swabs of group T02 was significantly (P < 0.0001) lower than that of group T01 on days 23, 25, and 28 (corresponding to 2, 4, and 7 days post-challenge) (see Table 7), while there was no significant difference on day 31. Table 8 summarizes the mean AUC of the logarithm of the viral load in nasal samples for ten days post-challenge (days 21 to 31), as well as the change and range for each treatment. The mean AUC value for the entire time period was significantly (p < 0.0001) lower in group T02 compared to T01, with mean values of 5.31 ± 0.16 and 7.03 ± 0.10, respectively.

[0229] Table 7. Back-transformed least-squares means of PRRSV copies / ml in nasal swabs

[0230] Group D21 (Before attack) D23 D25 D28 D31 T01 49.7 497429.7 2795257 21684.8 3394.3 T02 305.6 10050.5 2719.6 552.3 1552.1 P value P<0.0175 P<0.0001 P<0.0001 P<0.0001 P＝0.3056

[0231] Table 8. AUC analysis of PRRSV RNA copies / ml in nasal swabs (days > 21 post-challenge)

[0232]

[0233] The presence of PRRS virus in oral swab samples was monitored by RT-qPCR performed according to local procedures. All pigs were negative for RT-qPCR PRRSV in oral swab samples before vaccination (day 0).

[0234] On day 21 (before challenge), PRRSV RNA was detected in the oral swab samples of 68.2% (15 out of 22 pigs) of pigs in group T02 due to the presence of residual vaccine virus. At this time, all T01 pigs remained negative for PRRSV. On day 23 (two days post-challenge), 90.9% of piglets from T01 shed the virus via the nasal route, peaking four days post-challenge (day 25), with 100% of piglets from T01 shedding.

[0235] The frequency of oral shedding during the monitoring period indicated that 100% of T02 piglets shed at one or more time points after the challenge (days > 21). However, the percentage of animals in group T02 shedding via the nasal route never reached 100% at a single time point.

[0236] Before the challenge, on day 21, due to the presence of residual vaccine virus, the viral load detected in the oral swab samples of pigs in T02 was significantly (P<0.0001) higher than that in pigs in T01. After the challenge, the viral load detected in the oral swab samples of the T02 group was significantly (P≤0.05) lower than that of the control (T01) group on days 25 and 28 (corresponding to four and seven days after the challenge), while the differences on days 23 and 31 were not significant. Table 9 summarizes the back-transformed least-squares means of the PRRSV copy number / ml in oral swabs. Table 10 summarizes the average AUC of the logarithm of the viral load in oral samples ten days after the challenge (days 21 to 31), as well as the change and range for each treatment. The difference in the average AUC value for the entire time period was not statistically significant (P = 0.0690).

[0237] Table 9. Back-transformed least-squares means of PRRSV copy number / ml in oral swabs

[0238] Group D21 (Before attack) D23 D25 D28 D31 T01 50.0 12913.4 147949.3 16716.8 8776.0 T02 6855.0 30940.0 18966.5 1924.3 2320.4 P value P<0.0001 P<0.2013 P<0.003 P<0.0018 P＝0.0542

[0239] Table 10. AUC analysis of PRRSV RNA copy number / ml in oral swabs after challenge (days > 21)

[0240]

[0241] Rectal temperature

[0242] During all monitored days after the challenge, the difference in the least-squares means of rectal temperature between T01 and T02 was not significant (not shown).

[0243] Body weight

[0244] Before the challenge (day 21) and at the end of the study, ten days after the challenge (day 31), the average body weight in the T02 group was significantly (P≤0.05) higher than that in the control (T01) group. Table 11 summarizes the least-squares means of body weight (kg) by treatment and time point. From the day of the challenge (day 21) to the end of the study (ten days after the challenge, day 31), the average daily gain (ADG) for T01 piglets was 0.11 kg / day, and for T02 piglets was 0.15 kg / day. The difference in ADG between T01 and T02 (0.04 kg / day) was statistically significant (P = 0.0245). Table 12 summarizes the results of ADG.

[0245] Table 11. Body weight analysis - Least-squares means (Kg) by treatment group and time point

[0246]

[0247] Table 12: Body weight analysis - Average daily gain (Kg / day) by treatment group and time period

[0248] Group From day 21 to day 31 T01 0.11 T02 0.15 P value P＝0.0245

[0249] Serology

[0250] Before vaccination on day 0 after birth, all piglet serum tests were negative (S / P < 0.4). Before challenge (day 21), 100% of the piglets from the control (T01) group remained seronegative for PRRSV (S / P < 0.4), while 100% of the piglets from T02 tested positive for PRRSV-specific antibodies. At the end of the study, ten days after challenge (day 31), all piglets from T02 had seroconverted, and 100% of the piglets from T02 remained seropositive for PRRSV (data not shown).

[0251] Before challenge (day 21), the mean level of PRRSV-specific antibodies in the control (T01) group was < 0.4, indicating seronegativity; while the mean level of PRRSV-specific antibodies for T02 was 1.719, indicating seropositivity (≥ 0.4) for this group. The difference in mean S / P ratios between treatment groups was statistically significant (P < 0.0001). At the end of the study, ten days after challenge (day 31), the mean level of PRRSV-specific antibodies in both treatment groups was > 0.4, indicating that both T01 and T02 were seropositive. Nevertheless, the mean level of PRRSV-specific antibodies in T02 was statistically higher (P < 0.0001) than that in T01. See Table 13.

[0252] Table 13. Summary of serological least squares means (LSM) by group and study day

[0253] Group D21 (Before attack) D31 T01 -0.001 1.412 T02 1.719 2.031 P value P<0.0001 P<0.0001

[0254] Example 2. Evaluation of the potential impact of maternally derived antibodies on the efficacy of PRRSMLV administered intranasally to 3-day-old seropositive pigs against challenge with a PRRSV-1 European strain (Olot / 91 strain)

[0255] The objective of this study was to evaluate the efficacy of a PRRS MLV vaccine administered intranasally (IN) to three-day-old seropositive piglets against challenge with a PRRSV-1 European strain (Olot / 91 strain) in the presence of maternally derived antibodies (MDA).

[0256] Animal housing was similar to that described in Example 1, except that sixty piglets were included in the study. Piglets aged 2 to 4 days were used in the study (less than 20% of the 4-day-old animals), and the piglets were seropositive for PRRS. The presence of specific antibodies against PRRSV before vaccination (MDA+ condition) was determined by ELISA assay as well as by serum neutralization assay.

[0257] Similar to the setting in Example 1, on day 0, saline (1 ml per nostril) was administered to the piglets in group T01, and PRRS MLV (1 ml per nostril, 10 2.2 TCID 50 ) was administered to the piglets in group T02. On day 69 (when maternally derived antibodies could not be detected), the piglets were challenged with PRRS-1 strain Olot / 91 (1 ml per nostril, 10 50 TCID50 / 2 mL).

[0258] Using the same procedures as described in Example 1, viremia and shedding, lung injury, lung injury score, body weight, rectal temperature, and serology were determined and analyzed.

[0259] Results

[0260] Viremia

[0261] The presence of PRRSV in serum was monitored by RT-qPCR assay performed according to local procedures. All pigs were negative for RT-qPCR PRRSV in serum before vaccination (day 0) (if ≤50 PRRSV RNA copies / mL).

[0262] On day 69 (before challenge), PRRSV RNA was detected in 76.7% (23 out of 30 pigs) of the pigs in group T02. At this time, all T01 pigs remained negative for PRRSV. By day 71, 2 days after challenge, 82.1% (23 out of 28) of the piglets from T01 developed viremia, reaching a peak 5 days after challenge (day 74), at which time 96.4% of the T01 piglets were positive. See Table 14. The incidence of viremia at any time point during the entire monitoring period confirmed that 100% of the pigs in group T01 developed viremia after challenge. However, the incidence of viremic animals in group T02 never exceeded 76.7%, and one animal remained negative for PRRSV during the monitoring period (data not shown).

[0263] Table 14. Percentage of viremic animals for each treatment per day

[0264] Group D<0 D69 (Before attack) D71 D74 D76 D78 T01 0 0 82.1 96.4 78.6 82.1 T02 0 76.7 71.9 53.1 59.4 68.8

[0265] Before challenge, on day 69, due to the presence of residual vaccine virus, the viral load in group T02 was significantly (P < 0.0001) higher than that in group T01. After challenge, the viral load detected in the serum of group T02 was significantly (P ≤ 0.05) lower than that in the T01 control group on days 74 and 76 (corresponding to 5 and 7 days after challenge), while the differences were not significant on days 71 and 78. (See Table 15).

[0266] Table 16 summarizes the mean area under the curve (AUC) of log viremia from 9 days post-challenge (days 69 to 78), as well as the change and range for each treatment. Compared to the T01 control group, the mean AUC value of PRRSV viremia in the T02 group was significantly (P < 0.0001) lower over the entire time period, with means of 5.69 ± 0.20 and 6.87 ± 0.16, respectively.

[0267] Table 15: Back-transformed least squares means of PRRSV RNA copies / mL in serum

[0268]

[0269]

[0270] Table 16. AUC analysis of PRRSV RNA copies / mL in serum post-challenge (days > 69)

[0271]

[0272] Lung lesions

[0273] The percentage of lung lesions for each treatment group is shown in Table 17. Comparison between treatment groups revealed a significant difference (P = 0.0175) in the percentage of total lung lesions. The percentage of total lung lesions in the T02 group was lower compared to the T01 group.

[0274] Table 17. Analysis of percentage of total lung lesions (%)

[0275]

[0276] Table 18 summarizes the visual lung scores by treatment. At necropsy, 64.3% (18 out of 28) of the piglets in the T01 control group had a positive visual lung score (score > 0), indicating successful induction of lung lesions by PRRSV challenge. The percentage of animals with a positive score (score > 0) in T02 was 34.4% (11 out of 32), significantly lower (P = 0.0265) than the T01 group (64.3% (18 out of 28) with a positive score). More importantly, none of the animals inoculated with T02 had a score of 2 (moderate lesions), while 10.7% (3 out of 28) of the animals from T01 had a score of 2.

[0277] Table 18. Visual lung scores by treatment group

[0278]

[0279] Virus shedding

[0280] All pigs were negative for RT-qPCR PRRSV in nasal swab samples before vaccination (day 0) (if ≤50 PRRSV RNA copies / mL). At day 69 (before challenge), PRRSV RNA was detected in nasal swab samples from 18.8% (6 out of 32 pigs) of the pigs in the T02 group. At this time, all T01 pigs remained negative for PRRSV. By day 71 post-challenge, 2 days after challenge, 7.1% (2 out of 28) of the piglets from T01 shed virus via the nasal route, peaking at 5 days post-challenge (day 74), at which time 96.4% (27 out of 28) of the piglets (T01 group) inoculated with saline were positive for shedding. The frequency of nasal shedding at any time during the monitoring period confirmed that 100% of the piglets in the T01 group shed virus at one or more time points post-challenge (>69 days). Nevertheless, the frequency of nasal virus shedding in the T02 group never exceeded 56.3% (see Table 19), and 9.4% of the animals remained negative for shedding during the monitoring period.

[0281] Table 19. Percentage of animals with nasal shedding per treatment per day

[0282] Group D0 D69 (Before attack) D71 D74 D76 D78 T01 0 0 7.1 96.4 96.4 46.4 T02 0 18.8 43.8 37.5 56.3 50

[0283] Before challenge, some animals in T02 were positive due to the presence of vaccine virus, but the difference in viral load detected in nasal swab samples was not significant. After challenge, the viral load detected in nasal swabs from pigs in the T02 group was still significantly (P = 0.0018) higher than that of piglets inoculated with saline (T01 group) at day 71 (corresponding to 2 days post-challenge). However, the viral load detected in nasal swab samples from the T02 group was significantly (P < 0.0001) lower than that of the T01 group at days 74 and 76 (corresponding to 5 and 7 days post-challenge) (see Table 20), while the difference was not significant at day 78.

[0284] Table 21 summarizes the mean AUC of the logarithm of the viral load in nasal samples 9 days post-challenge (days 69 to 78), as well as the change and range for each treatment. The mean AUC value for the entire time period was significantly (P < 0.0001) lower in the T02 group compared to T01, with mean values of 4.89 ± 0.23 and 6.53 ± 0.10, respectively.

[0285] Table 20: Back-transformed least-squares means of PRRSV copies / mL in nasal swabs

[0286] Group D69 (Before attack) D71 D74 D76 D78 T01 54.1 71.0 333749 380652.4 601.5 T02 93.9 467.8 344.8 1754.1 1535.5 P value p＝0.0578 P＝0.0018 P<0.0001 P<0.0001 P＝0.2852

[0287] Table 21. AUC analysis of PRRSV RNA copies / mL in nasal swabs post-challenge (days >69)

[0288]

[0289]

[0290] The presence of PRRSV in oral swab samples was monitored by RT-qPCR testing conducted according to local procedures. All pigs had negative RT-qPCR PRRSV in oral swab samples before vaccination (day 0) (if ≤50 PRRSV RNA copies / mL). At day 69 (before challenge), PRRSV RNA was detected in oral swab samples of 21.9% (seven out of 32 pigs) of the pigs in T02. At this time, all T01 pigs remained negative for PRRSV. By day 71 post-challenge, 2 days after challenge, 10.7% of the piglets from T01 shed virus via the oral route, peaking at 5 days after challenge (day 74), when 89.3% of the piglets vaccinated with saline (T01 group) were positive for shedding. The frequency of oral shedding at any time during the monitoring period confirmed that 100% of the piglets vaccinated with saline (T01 group) shed virus at one or more time points after challenge (>69 days). Nevertheless, the frequency of animals with oral shedding in the T02 group never exceeded 68.8% (see Table 22), and 9.4% of the animals remained negative for shedding during the monitoring period. Table 22 details the frequency of orally shedding animals in each treatment group at each time point.

[0291] Table 22. Percentage of nasally shedding animals per treatment per day

[0292] Group D0 D69 (Before attack) D71 D74 D76 D78 T01 0 0 10.7 89.3 82.1 85.7 T02 0 21.9 36.7 50.0 37.5 68.8

[0293] Before challenge, on day 69, the viral load detected in oral swab samples of pigs in the T02 group was significantly (P = 0.0085) higher than that of pigs vaccinated with saline (T01 group) due to the presence of residual vaccine virus. After challenge, the level of viral load detected in oral swabs of pigs in the T02 group remained significantly (P ≤ 0.05) higher than that of piglets in T01 at day 71 (corresponding to 2 days after challenge). However, the viral load detected in oral swab samples of the T02 group was significantly (P ≤ 0.05) lower than that of the T01 control group at days 74 and 76 (corresponding to 5 and 7 days after challenge) (see Table 23), with no significant difference at day 78. Table 24 summarizes the mean AUC of the logarithm of the viral load in oral samples for nine days after challenge (days 69 to 78), as well as the change and range for each treatment. The mean AUC value for the entire period was significantly (P = 0.0212) lower in T02 piglets compared to T01 piglets, with means of 4.33 ± 0.16 and 4.81 ± 0.12, respectively.

[0294] Table 23: Back-transformed least-squares means of PRRSV RNA copies / mL in nasal swabs

[0295] Group D69 (Before attack) D71 D74 D76 D78 T01 52.4 78.8 5372.9 1803.8 3928.0 T02 105.6 211.5 351.4 308.7 2778.6 P value p＝0.0085 p＝0.0308 P<0.0001 P＝0.0077 P＝0.6287

[0296] Table 24. AUC analysis of PRRSV RNA copies / mL in oral swabs after challenge (days > 69)

[0297]

[0298] Rectal temperature

[0299] Before challenge, on day 69, the rectal temperature in group T02 was significantly higher (P < 0.0001) than that in group T01, with the least-squares means being 40.0 °C and 39.5 °C, respectively. However, since no animals had a fever, the means were within the expected range of the variable, and considering that the animals had just been mixed, it was considered irrelevant. On day 74, the least-squares mean rectal temperature of piglets in group T01 was significantly (P = 0.0007) higher than that of piglets in group T02. However, no animals in either treatment group had a fever on that day. On other monitoring days after challenge, there were no significant differences in rectal temperature between groups.

[0300] Body weight

[0301] Comparison of the least-squares means between the two groups showed no significant differences (P > 0.05) in body weight before challenge (day 69) and at the end of the study 9 days after challenge (day 78). However, from the day of challenge (day 69) to the end of the study 9 days after challenge (day 78), the average daily gain for piglets in group T01 was 0.69 Kg / day, and the average daily gain for piglets in group T02 was 0.56 Kg / day. The difference in ADG (0.14 Kg / day) between T01 and T02 was statistically significant (P = 0.0226). Table 25 summarizes the results of ADG.

[0302] Table 25: Body weight analysis - Average daily gain (Kg / day) by treatment group and time period

[0303] Group From day 69 to day 78 T01 0.69 T02 0.56 P value P＝0.0226

[0304] Serology

[0305] After birth, at day <0 (<3 days of age), PRRSV-specific antibodies were detected by ELISA in 90.9% (30 out of 33) of the piglets from group T01 and 96.9% (31 out of 32) of the piglets from group T02 (see Table 26). According to the protocol, all piglets should be seropositive for PRRSV (MDA+) before vaccination. Four piglets in which PRRSV-specific antibodies were not detected by ELISA were confirmed to be serologically positive for PRRSV (serum neutralization titer >2) by serum neutralization test.

[0306] Before challenge (day 69), 92.9% (26 out of 28) of the piglets from the T01 control group were seronegative for PRRSV by ELISA (S / P < 0.4). Among the piglets from group T02, only 3.3% (1 out of 30) of the animals tested negative for PRRSV-specific antibodies. At necropsy (day 78), 64.3% (18 out of 28) of the piglets from the T01 control group had seroconverted, while 100% of the piglets from group T02 were seropositive for PRRSV.

[0307] Table 27 summarizes the serological least squares means for each treatment at each time point. Before challenge (day 69), the mean level of PRRSV-specific antibodies in group T01 was <0.4, indicating seronegativity; while the mean level of PRRSV-specific antibodies for group T02 was 1.880, indicating seropositivity (≥0.4) for this group. The difference in mean S / P ratios between treatment groups was statistically significant (P < 0.0001). At the end of the study (day 78 = 9 days post-challenge), the mean level of PRRSV-specific antibodies in both treatment groups was >0.4, indicating seropositivity in both T01 and T02. Nevertheless, the mean level of PRRSV-specific antibodies in T02 was statistically higher than that in T01.

[0308] Table 26. Distribution of percentage of seroconversion (S / P ratio ≥0.4) for each treatment at each time point

[0309]

[0310] Table 27. Summary of serological least squares means (LSM) by group and study day

[0311] Group D69 (Before attack) D78 T01 0.156 0.767 T02 1.880 1.840 P value P<0.0001 P<0.0001

[0312] All publications (both patent publications and non-patent publications) cited in this specification represent the level of skill of those skilled in the art to which the present invention pertains. All of these publications are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

[0313] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments merely illustrate the principles and applications of the present invention. Accordingly, it is to be understood that numerous modifications may be made to the exemplary embodiments and other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A vaccine comprising a modified live PRRS-1 virus attenuated in cells expressing porcine CD163 for inducing protective immunity in piglets over 60 hours of age, wherein the vaccine is administered intranasally to the piglets.

2. The vaccine according to claim 1, used as claimed in claim 1, wherein the genome of the modified live PRRS-1 virus comprises an RNA molecule that is SEQ ID NO:1 or at least 75% identical to SEQ ID NO:1, and wherein further, a) the amino acid sequence encoded by ORF1a of the genome of the modified live PRRS-1 virus in the vaccine contains: S, A or T at amino acid position 19, preferably S; Y, F or W at amino acid position 157, preferably Y; D or E at amino acid position 268, preferably D; H, R or K at amino acid position 294, preferably H; Y, F or W at amino acid position 416, preferably Y; S, A or T at amino acid position 742, preferably S; L, I, M or V at amino acid position 884, preferably L; P at amino acid position 908; K, R or H at amino acid position 916, preferably K; K, R or H at amino acid position 977, preferably K; S, A or T at amino acid position 1138, preferably S; F, Y or W at amino acid position 1160, preferably F; S, A or T at amino acid position 1500, preferably S; R, K or H at amino acid position 2094, preferably R; P at amino acid position 2254; and L, I, M or V at amino acid position 2290, preferably L; and b) the amino acid sequence encoded by ORF1b of the genome of the modified live PRRS-1 virus in the vaccine contains: S, A or T at amino acid position 567, preferably S; and H, R or K at amino acid position 912, preferably H; and c) the amino acid sequence encoded by ORF2a of the genome of the modified live PRRS-1 virus in the vaccine contains: L, I, M or V at amino acid position 22, preferably L; F, Y, W at amino acid position 88, preferably F; M, L, I or V at amino acid position 94, preferably M; and F, Y or W at amino acid position 95, preferably F; and d) the amino acid sequence encoded by ORF2b of the genome of the modified live PRRS-1 virus in the vaccine contains: L, I, M or V at amino acid position 47, preferably L; and e) the amino acid sequence encoded by ORF4 of the genome of the modified live PRRS-1 virus in the vaccine contains: T, S or A at amino acid position 151, preferably T; and f) the amino acid sequence encoded by ORF5 of the genome of the modified live PRRS-1 virus in the vaccine contains: F, Y or W at amino acid position 20, preferably F; and D or E at amino acid position 37, preferably D; and g) the amino acid sequence encoded by ORF5a of the genome of the modified live PRRS-1 virus in the vaccine contains: V, L, I or M at amino acid position 18, preferably V; and R, H or K at amino acid position 35, preferably R.

3. The vaccine according to claim 2, used as claimed in claim 2, wherein a) the amino acid sequence encoded by ORF1a of the genome of the modified live PRRS-1 virus in the vaccine further contains: Y at amino acid position 24; and A at amino acid position 156; and b) the amino acid sequence encoded by ORF3 of the genome of the modified live PRRS-1 virus in the vaccine further contains S at amino acid position 52.

4. The vaccine according to claim 2 or claim 3, used as claimed in claim 2 or claim 3, wherein the genome of the modified live PRRS-1 virus contains an RNA molecule that is at least 90% identical to SEQ ID NO:

1.

5. The vaccine according to claim 1, used as claimed in claim 1, wherein the genome of the modified live PRRS-1 virus contains an RNA molecule that is at least 90% identical to SEQ ID NO:

1.

6. The vaccine according to claim 1, used as claimed in claim 1, wherein the genome of the modified live PRRS-1 virus contains an RNA molecule that is at least 95% identical to SEQ ID NO:

1.

7. The vaccine according to any one of claims 2 to 6, used as claimed in any one of claims 2 to 6, wherein at least 50% of the different nucleotides between the genome of the modified live PRRS-1 virus and SEQ ID NO:1 result in a) silent mutations or b) conservative substitutions or c) a combination thereof.

8. The vaccine according to any one of claims 1 to 7, used as claimed in any one of claims 1 to 7, wherein the piglets are at least 64 hours old.

9. The vaccine according to any one of claims 1 to 8, used as claimed in any one of claims 1 to 8, wherein the piglets are at least 68 hours old.

10. The vaccine according to any one of claims 1 to 9, used as claimed in any one of claims 1 to 9, wherein the piglets are at least 72 hours old.

11. The vaccine according to any one of claims 1 to 10, used as claimed in any one of claims 1 to 10, wherein the piglets are at least 76 hours old.

12. The vaccine according to any one of claims 1 to 11, used as claimed in any one of claims 1 to 11, wherein the piglets are at least 80 hours old.

13. The vaccine according to any one of claims 1 to 12, when used as described in any one of claims 1 to 12, wherein the piglets are at least about 84 hours old.

14. The vaccine according to any one of claims 1 to 13, when used as described in any one of claims 1 to 13, wherein the piglets are 28 days old or younger.

15. The vaccine according to any one of claims 1 to 14, when used as described in any one of claims 1 to 14, wherein the piglets are 21 days old or younger.

16. The vaccine according to any one of claims 1 to 15, when used as described in any one of claims 1 to 15, wherein the piglets are 14 days old or younger.

17. The vaccine according to any one of claims 1 to 16, when used as described in any one of claims 1 to 16, wherein the piglets are 10 days old or younger.

18. The vaccine according to any one of claims 1 to 17, when used as described in any one of claims 1 to 17, wherein the piglets are 7 days old or younger.

19. The vaccine according to any one of claims 1 to 18, when used as described in any one of claims 1 to 18, wherein the piglets are 5 days old or younger.

20. The vaccine according to any one of claims 1 to 19, when used as described in any one of claims 1 to 19, wherein the protective immunity includes at least one of a reduction in viremia, a reduction in viral shedding, a lung lesion score, and a frequency of lung lesions.

21. The vaccine according to any one of claims 1 to 20, when used as described in any one of claims 1 to 20, wherein the piglets are PRRS-1MDA positive or PRRS-1MDA negative.

22. The vaccine according to claim 21, when used as described in claim 21, wherein the piglets are PRRS-1MDA positive.

23. The vaccine according to claim 21, when used as described in claim 21, wherein the piglets are PRRS-1MDA negative.

24. The vaccine according to claim 23, when used as described in claim 23, wherein the protective response further includes an increase in body weight compared to unvaccinated infected piglets.

25. The vaccine according to any one of claims 1 to 24, when used as described in any one of claims 1 to 24, wherein the vaccine is a single-dose vaccine.

26. The vaccine according to any one of claims 1 to 24, for use as claimed in any one of claims 1 to 24, wherein the effective dose of the modified live PRRS-1 virus in the vaccine is at least 10 2.0 TCID 50 .

27. The vaccine according to claim 25, for use as claimed in claim 25, wherein the effective dose of the modified live PRRS-1 virus in the vaccine is from about 10 2.2 TCID 50 to about 10 6.0 TCID 50 .

28. The vaccine according to claim 25, when used as described in claim 25, wherein the effective dose of the modified live PRRS-1 virus in the vaccine is from about 10 2.2 TCID 50 to about 10 5.2 TCID 50 .

29. The vaccine according to claim 25, used as claimed in claim 25, wherein the effective dose of the modified live PRRS-1 virus in the vaccine is about 10 2.2 TCID 50 or about 10 2.5 TCID 50 or about 10 2.8 TCID 50 or about 10 3.1 TCID 50 or about 10 3.4 TCID 50 or about 10 3.7 TCID 50 or about 10 4.0 TCID 50 or about 10 4.4 TCID 50 or about 10 4.8 TCID 50 or about 10 5.2 TCID 50 or about 10 5.6 TCID 50 .

30. The vaccine according to any one of claims 1 to 29, when used as described in any one of claims 1 to 29, wherein the volume of the vaccine administered to the piglets is from about 0.5 ml to about 5 ml.

31. The vaccine according to any one of claims 1 to 30, when used as described in any one of claims 1 to 30, wherein the volume of the vaccine administered to the piglets is about 2 ml.

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