FELINE LEUKEMIA VIRUS VACCINE

Abstract

The present invention provides a vaccine for feline leukemia virus and methods of making and using the vaccine alone, or in combinations with other protective agents.

Claims

1.-19. (canceled)

20. A method comprising: (a) administering a primer vaccine to a feline subject, the primer vaccine comprising a first Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle (RP) that encodes a first feline leukemia virus (FeLV) glycoprotein 85 (gp85) or an antigenic fragment thereof, and a first pharmaceutically acceptable carrier, and (b) administering a booster vaccine to the feline subject, the booster vaccine comprising a second VEE alphavirus RNA RP that encodes a second FeLV gp85 or an antigenic fragment thereof, and a second pharmaceutically acceptable carrier.

21. The method of claim 20, wherein the booster vaccine is administered at least 21 days after the primer vaccine is administered.

22. The method of claim 21, wherein the booster vaccine is administered between about 21 days and about 1 year after the primer vaccine is administered.

23. The method of claim 20, wherein the booster vaccine and the primer vaccine are administered via the same route of administration.

24. The method of claim 23, wherein the primer vaccine and the booster vaccine are both administered via subcutaneous injection or oral administration.

25. The method of claim 20, wherein the primer vaccine, or the booster vaccine, or both the primer vaccine and the booster vaccine, comprise less than about 2.010.sup.6 alphavirus RNA RPs, or between about 1.010.sup.5 to about 2.010.sup.6 alphavirus RNA RPs.

26. The method of claim 20, wherein the first FeLV gp85 encoded in the first alphavirus RNA RP present in the primer vaccine is from a different strain of FeLV compared to that of the second FLV gp85 encoded in the second alphavirus RNA RP present in the booster vaccine.

27. The method of claim 20, wherein the first VEE alphavirus RNA RP in the primer vaccine, or the second VEE alphavirus RNA RP in the booster vaccine, or both the first VEE alphavirus RNA RP and the second VEE alphavirus RNA RP, further comprise at least one of: (a) a nucleic acid construct that encodes at least one non-FeLV antigen for eliciting protective immunity to a non-FeLV pathogen, or (b) a nucleic acid construct that encodes a feline calicivirus (FCV) antigen which originates from a virulent systemic feline calicivirus or an antigenic fragment thereof, or (c) a nucleic acid construct that encodes a feline calicivirus (FCV) antigen which originates from a classical (F9-like) feline calicivirus or an antigenic fragment thereof.

28. The method of claim 20, wherein the primer vaccine, or the booster vaccine, or both the primer vaccine and the booster vaccine, are non-adjuvanted, or do not comprise an adjuvant.

29. The method of claim 20, wherein the primer vaccine, or the booster vaccine, or both the primer vaccine and the booster vaccine, further comprise at least one non-FeL V antigen for eliciting protective immunity to a non-FeL V feline pathogen.

30. The method of claim 29, wherein the non-FeLV feline pathogen is selected from the group consisting of feline herpesvirus (FHV), feline calicivirus (FCV), feline pneumovirus (FPN), feline parvovirus (FPV), feline infectious peritonitis virus (FIPV), feline immunodeficiency virus, borna disease virus (BDV), feline influenza virus, feline panleukopenia virus (FPLV), feline coronavirus (FCOV), feline rhinotracheitis virus (FVR), Chlamydophila felis, and any combination thereof.

31. The method of claim 30, wherein the non-FeLV antigen is a killed or attenuated non-FeLV antigen selected from the group consisting of a killed or attenuated feline herpesvirus (FHV), a killed or attenuated feline calicivirus (FCV), a killed or attenuated feline pneumovirus (FPN), a killed or attenuated feline parvovirus (FPV), a killed or attenuated feline infectious peritonitis virus (FIPV), a killed or attenuated feline immunodeficiency virus, a killed or attenuated borna disease virus (BDV), a killed or attenuated feline influenza virus, a killed or attenuated feline panleukopenia virus (FPLV), a killed or attenuated feline coronavirus (FCoV), a killed or attenuated feline rhinotracheitis virus (FVR), a killed or attenuated Chlamydophila felis, and any combination thereof.

32. The method of claim 31, wherein the attenuated non-FeLV antigen is a modified live feline pathogen selected from the group consisting of a modified live Chlamydophila felis, a modified live feline rhinotracheitis Virus (FVR), a modified live feline calicivirus (FCV), a modified live feline panleukopenia virus (FPL), a modified live feline herpesvirus (FHV), a modified live feline pneumovirus (FPN), a modified live feline parvovirus (FPV), a modified live feline infectious peritonitis virus (FIPV), a modified live feline immunodeficiency virus, a modified live borna disease virus (BDV), a modified live feline coronavirus (FCOV), a modified live feline influenza virus, and any combination thereof.

33. The method of claim 20, wherein the primer vaccine, or the booster vaccine, or both the primer vaccine and the booster vaccine, further comprise an alphavirus RNA RP comprising a nucleotide sequence encoding at least one protein antigen or an antigenic fragment thereof that originates from a non-FeL V antigen.

34. The method of claim 33, wherein the protein antigen or an antigenic fragment thereof originates from a non-FeLV feline pathogen selected from the group consisting of feline herpesvirus (FHV), feline calicivirus (FCV), feline pneumovirus (FPN), feline parvovirus (FPV), feline infectious peritonitis virus (FIPV), feline immunodeficiency virus, borna disease virus (BDV), feline influenza virus, feline panleukopenia virus (FPLV), feline coronavirus (FCoV), feline rhinotracheitis virus (FVR), Chlamydophila felis, and any combination thereof.

35. The method of claim 20, wherein the first FeLV gp85, the second FeLV gp85, or both the first FeLV gp85 and the second FeLV gp85, comprise an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

36. The method of claim 20, wherein the antigenic fragment of the first FeLV gp85 or the antigenic fragment of the second FeLV gp85 is gp70, or gp45, or an antigenic fragment of gp70 or gp45.

37. The method of claim 20, wherein the primer vaccine is administered to the feline subject at about 8 weeks of age.

38. An alpha RNA replicon particle (RP) comprising a heterologous nucleic acid sequence, the heterologous nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO:10, wherein the RP is a Venezuelan Equine Encephalitis (VEE) RP.

39. The RP of claim 38, wherein the VEE RP is TC-83 VEE RP.

40. The RP of claim 39, wherein the heterologous nucleic acid sequence further comprises a nucleic acid that encodes one or more non-FeLV antigens.

Description

EXAMPLES

Example 1

Incorporation of the Coding Sequences for FELV GP85 into the Alphavirus RNA Replicon Particles

Introduction

[0065] RNA viruses have been used as vector-vehicles for introducing vaccine antigens, which have been genetically engineered into their genomes. However, their use to date has been limited primarily to incorporating viral antigens into the RNA virus and then introducing the virus into a recipient host. The result is the induction of protective antibodies against the incorporated viral antigens. Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such alphavirus replicon platforms have been developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993) the contents of which are hereby incorporated herein in their entireties], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991), the contents of which are hereby incorporated herein in their entireties]. Moreover, alphavirus RNA replicon particles are the basis for several USDA-licensed vaccines for swine and poultry. These include: Porcine Epidemic Diarrhea Vaccine, RNA Particle (Product Code 19U5.P1), Swine Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 1905.D0), and Prescription Product, RNA Particle (Product Code 9PP0.00).

Alpha Virus RNA Replicon Particle Construction

[0066] An amino acid sequence for FeLV gp85 were used to generate codon-optimized (feline codon usage) nucleotide sequences in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, CA). Accordingly, a synthetic gene was designed based on the amino acid sequence of gp85. The construct (gp85_wt) was wild-type amino acid sequence [SEQ ID NO: 2], codon-optimized for feline, with flanking sequence appropriate for cloning into the alphavirus replicon plasmid.

[0067] The VEE replicon vectors designed to express FELV gp85 were constructed as previously described [see, U.S. Pat. No. 9,441,247 B2; the contents of which are hereby incorporated herein by reference in their entireties], with the following modifications. The TC-83-derived replicon vector pVEK [disclosed and described in U.S. Pat. No. 9,441,247 B2] was digested with restriction enzymes AscI and PacI. A DNA plasmid containing the codon-optimized open reading frame nucleotide sequence of the FeLV gp85 genes, with 5 flanking sequence (5-GGCGCGCCGCACC-3) [SEQ ID NO: 9] and 3 flanking sequence (5-TTAATTAA-3), was similarly digested with restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clone was re-named pVHV-FeLV gp85. The pVHV vector nomenclature was chosen to refer to pVEK-derived replicon vectors containing transgene cassettes cloned via the AscI and PacI sites in the multiple cloning site of pVEK.

[0068] Production of TC-83 RNA replicon particles (RP) was conducted according to methods previously described [U.S. Pat. No. 9,441,247 B2 and U.S. Pat. No. 8,460,913 B2; the contents of which are hereby incorporated herein by reference]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, WI). Importantly, the helper RNAs used in the production lack the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91 (Pt 7): 1723-1727 (2010)]. Purified RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro SFM cell culture media (Thermo Fisher, Waltham MA). Following overnight incubation, alphavirus RNA replicon particles were purified from the cells and media by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, MN), washing with phosphate buffered saline containing 5% sucrose (w/v), and finally eluting the retained RP with 400 mM NaCl buffer. Eluted RP were formulated to a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers.

Example 2

Comparative Efficacy and Safety of FELV Vaccines in Cats

[0069] A vaccine comprising an alphavirus RNA replicon particle (RP) comprising the capsid protein and glycoproteins of the avirulent TC-83 strain of Venezuelan Equine Encephalitis Virus (VEE) and encoding the FeLV viral glycoprotein (gp85), was formulated in 5% sucrose. The liquid vaccine was frozen for storage before use. This vaccine was compared with a commercially available vaccine comprising a recombinant canary pox encoding FeLV, as shown in Table 1 below. Five groups of eight feline subjects were vaccinated either with a single dose at 8-9 weeks, or in a prime/boost regimen of 8-9 weeks of age and then 21 days later. The doses for each experimental vaccinate group is provided in Table 1 below.

TABLE-US-00003 TABLE 1 VACCINATION PROTOCOL Vaccinate No. of Vaccination Group Animals Vaccine RP/dose Days 1 8 RP-FeLV 4.35 10.sup.8 0, 21 2 8 RP-FeLV 3.55 10.sup.7 0, 21 3 8 RP-FeLV 1.5 10.sup.8 21 (one shot) 4 8 PureVax .sup.# Does not apply 0, 21 5 8 Placebo none 0, 21 .sup.# A vaccine containing a recombinant canary pox encoding FeLV sold by Merial

[0070] All cats were subcutaneously vaccinated with 1.0 mL of their respective vaccine regimen. Cats were 8-9 weeks of age at the time of the initial vaccination (including cats in Group 3). The cats of Group 4 were vaccinated at the times provided with the quantity of vaccine as directed on the label of the commercial vaccine. Following the vaccination the cats were observed for adverse reactions to the vaccines by observing the general health daily, as well as palpating the site of injection for the two days following each vaccination and twice per week for two weeks following each vaccination. No adverse reactions were observed for any of the vaccines.

[0071] All cats were challenged with a virulent culture of FeLV four weeks after the booster vaccination (four weeks after the one-shot vaccination for the Group 3 cats). The cats were challenged on four separate days over one week (study days 49, 52, 54 and 56) by administering 1.0 mL of challenge virus by the oronasal route (0.3 mL in each nostril and 0.4 mL orally). Three weeks after challenge serum samples were collected each week through ten weeks post-challenge. Serum samples were tested by ELISA for the presence of FeLV p27 antigen. An animal is considered infected with FeLV if it is persistently antigenemic. Antigenemia is defined as a positive p27 ELISA result for three consecutive weeks or on five or more occasions during the eight week testing period. An FeLV vaccine must protect 75% of the cats vaccinated with the test product for USDA licensure. In addition, in order for the challenge to be regarded as valid, 80% of the control cats must be persistently antigenemic [see, Shipley et al., JAVMA, Vol. 199, No. 10, (Nov. 15, 1991)]. The results of the challenge are summarized in the Table 2 below.

TABLE-US-00004 TABLE 2 VACCINATION AND CHALLENGE Treatment % Cats % Cats Group Vaccine RP dose Antigenemic Protected 1 RP-FeLV 4.35 10.sup.8 0% 100% 2 RP-FeLV 3.55 10.sup.7 0% 100% 3 RP-FeLV 1.5 10.sup.8 13% 87% (one shot)* 4 PureVax .sup.# Does not apply 43% 57% 5 Placebo Does not apply 88% 12% .sup.# A vaccine containing a recombinant canary pox encoding FeLV sold by Merial *All other groups received a two-dose regimen, see, Table 1 above.

[0072] As Table 2 demonstrates, the RP-FeLV vaccines protected 100% of the cats when administered in a two-dose regimen (i.e., primary and booster vaccination) at both doses tested. Moreover, the RP-FeLV vaccine protected 87% of the cats when administered as a single dose. In direct contrast, the commercially available vaccine only protected 57% of the cats, even with a two-dose regimen. In addition, the challenge is regarded as valid because greater than 80% of the control cats were persistently antigenemic [see, Table 2]. Finally, all of the RP-FeLV vaccine formulations were found safe in cats.

Example 3

Determination of the Dose Dependence of an RP-FELV Vaccine by Vaccination and Challenge

[0073] The RP-FeLV vaccine of Example 2 was formulated in a vaccine formulation that included enzymatically hydrolyzed casein (NZ-amine), gelatin, and sucrose. The vaccine was then lyophilized. Four groups of ten cats each were vaccinated as summarized in Table 3 below:

TABLE-US-00005 TABLE 3 VACCINATION PROTOCOL Treatment No. of Vaccination Group Animals Vaccine RP/dose Days 1 10 RP-FeLV 1.1 10.sup.5 0, 21 2 10 RP-FeLV 2.1 10.sup.6 0, 21 3 10 RP-FeLV 6.5 10.sup.7 0, 21 4 10 Non-vaccinated None NA Controls

[0074] All cats were vaccinated with 1.0 mL of respective test product, subcutaneously. The cats were 8-9 weeks of age at the time of initial vaccination. Following the vaccination the cats were observed for adverse reactions to the vaccines by observing their general daily health, as well as palpating the site of injection for the two days following each vaccination and twice per week for the two weeks following each vaccination. No adverse reactions to any of the vaccines were observed.

[0075] All of the cats were challenged with a virulent culture of FeLV three weeks after the booster vaccination. Cats were challenged on four separate days over one week (study days 42, 45, 47 and 49) by administering 1.0 mL of challenge virus by the oronasal route (0.3 mL in each nostril and 0.4 mL orally). Three weeks after challenge serum samples were collected each week through twelve weeks post-challenge. Serum samples were tested by ELISA for the presence of FeLV p27 antigen. An animal is considered infected with FeLV if it is found to be persistently antigenemic. Antigenemia is defined as a positive p27 ELISA result for three consecutive weeks, or on five or more occasions during the eight-week testing period. For USDA licensure an FeLV vaccine must protect 75% of the cats vaccinated with the test product. For the challenge to be considered valid, 80% of the control cats must be persistently antigenemic [Shipley et al., JAVMA, Vol. 199, No. 10, Nov. 15, 1991]. The results of the challenge are summarized in the Table 4 below:

TABLE-US-00006 TABLE 4 DOSE DEPENDENCE OF RP-FELV Treatment % Cats % Cats Group Vaccine RP/dose Antigenemic Protected 1 RP-FeLV 1.1 10.sup.5 10% 90% 2 RP-FeLV 2.1 10.sup.6 0% 100% 3 RP-FeLV 6.5 10.sup.7 0% 100% 4 Non-vaccinated None 90% 10% Controls

[0076] In this study of short term immunity, the minimum protective dose of the RP-FeLV vaccine for 100% protection of the cats was between about 1.010.sup.5 to about 2.010.sup.6 RPs, when administered in a two dose (primary and booster vaccination) regimen. The challenge was valid because at least 80% of the control cats were persistently antigenemic. All RP-FeLV vaccine formulations tested were safe in cats.

[0077] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0078] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.