ZP000506 INFLUENZA VACCINES FIELD OF THE INVENTION [0001] The invention is the field of influenza vaccines. BACKGROUND [0002] Canine influenza (CI), or dog flu, is a highly contagious viral infection affecting dogs and also cats. Influenza viruses belong to the family Orthomyxoviridae. Canine influenza is a Type A influenza virus and is further identified based on the composition of two specific proteins in the lipid outer layer of the capsid: hemagglutinin (HA) and neuraminidase (NA). At present, two subtypes of canine influenza virus have been identified in the United States: H3N8 and H3N2. [0003] Influenza viruses are able to quickly change and give rise to new subtypes that can infect different species. Both subtypes of canine influenza virus identified in the U.S. can be traced to influenza viruses known to infect species other than dogs. At some point, these viruses acquired the ability to infect dogs and be transmitted from dog to dog. Transmission of H3N2 canine influenza viruses to cats from infected dogs has been reported also. [0004] Canine H3N8 influenza was first identified in Florida in 2004 in racing greyhounds. It is thought this strain developed from an equine H3N8 influenza strain that jumped from horses to dogs. Since being detected in 2004, canine H3N8 influenza has been identified in dogs in most U.S. states and the District of Columbia. [0005] Canine H3N2 influenza was first identified in the United States in March 2015 following an outbreak of respiratory illness in dogs in the Chicago area. Prior to this, reports of canine H3N2 influenza virus were restricted to South Korea, China and Thailand. It was initially identified in dogs in Asia in 2006-2007 and likely arose through the direct transfer of an avian influenza virus – possibly from among viruses circulating in live bird markets – to dogs. [0006] Canine influenza virus (CIV) infects and replicates inside cells in the respiratory tract from the nasal lining to the terminal airways. The inflammatory response to the infection results in rhinitis, tracheitis, bronchitis and bronchiolitis. The pathologic process results in death of the epithelial cells lining the respiratory tract, exposing the underlying basement membrane. This
ZP000506 predisposes the respiratory tract to secondary bacterial infections that contribute to nasal discharge and coughing. [0007] Virtually all dogs exposed to canine influenza virus become infected, with approximately 80% developing clinical signs of disease. The approximately 20% of infected dogs that do not exhibit clinical signs of disease can still shed the virus and spread the infection. Mortality is not a common outcome (less than 10%). [0008] Like other mammalian influenza viruses, canine influenza virus causes an acute respiratory infection in dogs. There is no “season” for canine influenza, and infections can occur any time of the year. Canine influenza virus infection often resembles canine infectious tracheobronchitis ("kennel cough"), which is caused by one or more bacterial or viral infections, including Bordetella bronchiseptica and parainfluenza virus. [0009] Vaccines are available for both H3N8 and H3N2 canine influenza. A bivalent vaccine offering protection against both viruses is also available. Currently, there are no canine influenza vaccines approved for use in cats. Vaccination can reduce the risk of a dog contracting canine influenza. Vaccination may not all together prevent an infection, but it may reduce the severity and duration of clinical illness. Accordingly, there is need for improved vaccines against canine influenza virus. [0010] In addition, Highly Pathogenic Avian Influenza (HPAI) has been detected in multiple mammals. Most recently, Federal officials announced that a highly pathogenic H5N1 strain had been detected in dairy cows. As of April 30, 2024, the USDA has since confirmed infections in 34 dairy herds in nine states. SUMMARY OF THE INVENTION [0011] In one aspect, the instant application provides a vaccine comprising: a) an antigen, the antigen comprising an inactivated canine influenza virus; and b) an adjuvant, the adjuvant comprising a triterpenoid saponin, a sterol and a CpG-containing immunostimulatory oligonucleotide. In certain embodiments, the adjuvants in the vaccines disclosed herein do not contain one, two, or three of a quaternary ammonium compound, a polyacrylic acid polymer and/or a glycolipid adjuvant. Most preferably, the adjuvants in the vaccines disclosed herein
ZP000506 consist essentially of or consist of the triterpenoid saponin, the sterol, and the CpG-containing immunostimulatory oligonucleotide. [0012] In certain embodiments, the triterpenoid saponin comprises an immunologically active saponin fraction from the bark of Quillaja Saponaria which, preferably, is present in the amount of 10-30 micrograms per dose. In further embodiments, the sterol is cholesterol, which, preferably, is present in the amount of amount of 10-30 micrograms per dose. [0013] In a further subset of embodiments the CpG containing immunostimulatory oligonucleotide comprises a 5' TLR activation domain and at least two palindromic regions, one palindromic region being a 5' palindromic region of at least 6 nucleotides in length and connected to a 3' palindromic region of at least 8 nucleotides in length either directly or through a spacer. Preferably, the CpG containing immunostimulatory oligonucleotide comprises a 5’ nucleotide which is a 5'-Iodo-2'-deoxyuridine or 5-Ethyl-2'-deoxyuridine and/or one or more phosphorothioate bonds. In particularly preferred embodiments, the CpG containing immunostimulatory oligonucleotide comprises SEQ ID NO: 8. [0014] The vaccines disclosed herein comprise the inactivated canine influenza virus comprises (or consist of) a H3N2 canine influenza virus or a combination of H3N2 and H3N8 canine influenza virus. [0015] In additional or alternative embodiments, the inactivated canine influenza virus comprises an inactivated influenza virus that cross-protects against an HPAI virus, such as, for example an H5 influenza virus, more preferably H5N1 influenza virus. In certain embodiments, the inactivated influenza virus that cross-protects against H5N1 HPAI virus is an inactivated H5N2 influenza virus. [0016] In further embodiments, the vaccine that contains an inactivated canine influenza virus and/or an inactivated influenza virus which cross-protect against the HPAI virus may further comprise one or more antigens selected from the group consisting of a CAV-2 antigen, a CPI antigen, and a B bronchiseptica antigen. [0017] In another aspect, the invention provides a method of protecting a subject against an influenza virus, the method comprising administering to said subject the vaccine described herein. In certain embodiments, the subject is in need of being protected against H3N2 influenza
ZP000506 virus and/or H3N8 influenza virus and the antigen in the vaccine disclosed herein comprises an in activated H3N2 virus and/or H3N8 influenza virus, respectively. In other embodiments, the subject is in need of being protected from HPAI virus such as H5N1 influenza virus, and the antigen in the vaccine comprises an inactivated H5N2 influenza virus. [0018] In certain embodiments, the vaccine disclosed herein is administered by a subcutaneous or an intramuscular injection. [0019] In particularly preferred embodiments of this second aspect of the invention, the subject in need of being protected is a mammal, more preferably dog or a cat. DETAILED DESCRIPTION Definitions [0020] "About" or "approximately," when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. With regard to time period, the term “about” refers to the indicated value and to a range within 10 % of the indicated value (e.g., “about 8 months” includes 8 months as well as 8 months plus or minus 10%), except the upper limit of “about 11 months” is 12 months and the upper limit of “about 12 months” is 12.5 months. [0021] "Adjuvant" means any substance that increases the humoral or cellular immune response to an antigen. Adjuvants are generally used to accomplish two objectives: the controlled release of antigens from the injection site, and the stimulation of the immune system. [0022] "Antigen" or "immunogen" refers to any substance that is recognized by the animal's immune system and generates an immune response. The term includes killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term "antigen" also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, individually or in any combination thereof. The term antigen also includes antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).
ZP000506 [0023] "Buffer" means a chemical system that prevents change in the concentration of another chemical substance, e.g., proton donor and acceptor systems serve as buffers preventing marked changes in hydrogen ion concentration (pH). A further example of a buffer is a solution containing a mixture of a weak acid and its salt (conjugate base) or a weak base and its salt (conjugate acid). [0024] "Consisting essentially" as applied to the adjuvant formulations refers to formulation which does not contain unrecited additional adjuvanting or immunomodulating agents in the amounts at which said agent exert measurable adjuvanting or immunomodulating effects. Preferably, if present, such unrecited additional adjuvanting or immunomodulating agents are in the amount that is below detection threshold. [0025] "Dose" refers to a vaccine or immunogenic composition given to a subject in a single administration. [0026] "Immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. Immune responses can usually be determined using standard immunoassays, cell-based assays, and neutralization assays, which are known in the art. [0027] "Immunologically effective amount" or "effective amount to produce an immune response" of an antigen is an amount effective to induce an immunogenic response in the recipient. The immunogenic response may be sufficient for diagnostic purposes or other testing or may be adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a disease agent. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of an animal to an immunogenic composition may be evaluated, e.g., indirectly through measurement of antibody titers, cytokine assays, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strains, whereas the protective immunity conferred by a vaccine can be evaluated by measuring, e.g., appearance, severity and/or duration of clinical signs such as mortality, morbidity, fever, overall physical condition, and overall health and performance of the subject. The immune response may comprise, without limitation, induction of cellular and/or humoral immunity.
ZP000506 [0028] "Immunogenic" means evoking an immune or antigenic response. Thus, an immunogenic composition would be any composition that induces an immune response. [0029] "Pharmaceutically acceptable" refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use. [0030] The term “protective immune response” refers to the immune response, elicited by an immunogenic composition or a vaccine in a subject, wherein upon the challenge with the pathogen against which the animal was immunized, the subject does not get infected (complete protection) or exhibits symptoms of lesser magnitude and/or duration compared to the non- immunized animal (partial protection). In a particularly preferred embodiment of partial protection, the immunized and challenged subject does not shed the coronavirus, or the magnitude and/or the duration of shedding is decreased. Thus, protective immune response prevents the infection and/or lessens the symptoms and/or the duration of the infection. [0031] The term “treating” refers to reducing or alleviating magnitude and/or duration of at least one symptom of an existing coronavirus infection. [0032] The term “vaccine” refers to an immunogenic composition that elicits protective immune response in the subject. when administered to a subject, induces or stimulates a protective immune response. A vaccine can render an organism immune to a particular disease, in the present case coronavirus infection, and more particularly SARS-CoV-2 infection. The vaccine of the present invention thus induces an immune response in a subject which is protective against subsequent SARS-CoV-2 challenge. A vaccine comprising the antigen and the adjuvant of the invention may be capable of inducing a cross-protective immune response against a plurality of coronavirus genotypes. ANTIGENS [0033] The antigens in the vaccines disclosed herein are influenza antigens. [0034] The vaccines disclosed herein may contain H3N2 canine influenza antigen, preferably an inactivated H3N2 canine influenza virus. In certain embodiments, the H3N2 CIV strain is NJ62,
ZP000506 however other H3N2 CIV strains are also suitable. Optionally, the vaccines disclosed herein may also contain a H3N8 antigen, preferably in the form of inactivated H3N8 canine influenza virus. [0035] In other embodiments, the vaccines disclosed herein may contain Highly Pathogenic Avian Influenza (HPAI) antigens, e.g., H5 and H7 subtypes of Influenza A virus, most preferably H5N2 influenza virus which has been shown to cross-protect against H5N1 HPAI. In other embodiments the antigen protects against H5N6, H7N2 or H1N1 virus and can be an inactivated H5N6, or an inactivated H7N2 or an inactivated H1N1 virus, respectively. [0036] Preferably, the influenza antigens are provided in the form of inactivated or attenuated viruses but other forms of the antigens (e.g., DNA vaccines and/or subunit vaccines) can also be used. Generally, H3N2 influenza antigens may be provided in the amount of at least 500 HA units, e.g., about 1000 to about 12000 HA units per dose, or about 1500 HA units, or about 2000 HA units, or about 2500 HA units, or about 3000 HA units, or about 3500 HA units, or about 4000 HA units, or about 4500 HA units, or about 5000 HA units, or about 5500 HA units, or about 6000 HA units, or about 6500 HA units, or about 7000 HA units, or about 7500 HA units, or about 8000 HA units, or about 8500 HA units, or about 9000 HA units, or about 9500 HA units, or about 10000 HA units, or about 10500 HA units, or about 11000 HA units, or about 11500 HA units per dose. HPAI influenza antigens may be provided in the amount of at least about 500 HA units per dose, e.g., about 500 to about 20000 HA units, or about 500 to about 15000 HA units, or about 500 to about 10000 HA units, or about 500 to about 7500 HA units, or about 500 to about 5000 HA units, or about 500 to about 2500 HA units, or about 600 to about 2000 HA units, or about 700 to about 1500 HA units, or about 800 to about 1200 HA units or about 1000 units. [0037] The choice of additional antigens depends on the subject animals. For example, in canine vaccines, in addition to the influenza antigens the vaccine may further comprise one or more of non-influenza antigens such as Canine Parainfluenza Virus (CPI) antigen, or Canine Adenovirus Type 2 (CAV-2) antigen, or Bordetella bronchiseptica antigen. These non-influenza antigens may be provided, independently from each other, in an attenuated form, or an inactivated form, or as a part of a subunit vaccine or DNA vaccine. [0038] Thus, in some embodiments, the vaccines disclosed herein comprise an HPAI inactivated virus. In other embodiments, the vaccines disclosed herein comprises an HPAI inactivated virus
ZP000506 and one or more of CPI virus antigen, CAV-2 virus antigen and/or Bordetella bronchiseptica antigen. [0039] In other embodiments, the vaccines disclosed herein comprise H3N2 canine influenza virus, optionally, in combination with H3N8 influenza virus. In yet other embodiments, the vaccines disclosed herein comprise HPAI virus and H3N2 canine influenza virus, and optionally also H3N8 canine influenza virus. In yet other embodiments, applicable to the vaccines with any combination of the influenza antigens disclosed above, the vaccines disclosed herein further comprise one or more of CPI virus antigen, CAV-2 virus antigen and/or Bordetella bronchiseptica antigen. [0040] If the modified live CAV-2 is present, one dose may contain the following amounts: from 102.5 TCID50 to about 108 TCID50, more preferably, from 103 TCID50 to about 108 TCID50, more preferably, from 103 TCID50 to about 106 TCID50, from 103 TCID50 to about 105.5 TCID50, from 103 TCID50 to about 105 TCID50, from 103 TCID50 to about 104.5 TCID50, from about 103.5 TCID50 to about 105.5 TCID50, from 103.5 TCID50 to about 105 TCID50, from 103.5 TCID50 to about 104.5 TCID50, from 103.5 TCID50 to about 104 TCID50, from about 103.7 TCID50 to about 105.5 TCID50, from 103.7 TCID50 to about 105 TCID50, from 103.7 TCID50 to about 104.5 TCID50, from 103.7 TCID50 to about 104 TCID50, or from 103.7 TCID50 to about 104 TCID50. [0041] If the modified live CPI is present, one dose of the vaccine may contain the following amount of the CPI: 104.5 TCID50 to about 108 TCID50, more preferably 105.5 TCID50 to about 108 TCID50, or 106.5 TCID50 to about 108 TCID50, or 107.5 TCID50 to about 108 TCID50, or 104.5 TCID50 to about 107 TCID50105.5 TCID50 to about 106 TCID50, more preferably 106.5 TCID50 to about 108 TCID50, or 106.5 TCID50 to about 107 TCID50. [0042] If B bronchiseptica antigen, such as whole-cell bacterial extract, is present in the vaccine, one dose, e.g., about 0.5 to 2 milliliters, or about 1 ml, may contain at least 0.1 micrograms per milliliter, more preferably from 0.1 to about 1.5 micrograms per milliliter, e.g., 0.25, 0.5, 0.75, 1, 1.25 or about 1.5 micrograms per milliliter. Thus, one dose would contain from 0.1 micrograms to about 1.5 micrograms, e.g., 0.25, 0.5, 0.75, 1, 1.25 or about 1.5 micrograms.
ZP000506 [0043] The methods of attenuating or inactivating bacteria and viruses are well known. Moreover, products containing such viruses and bacteria are commercially available and include VANGUARD® line of product, e.g. VANGUARD® DAPPi or VANGUARD® B Oral. [0044] In vaccines intended for cats, additional antigens may further contain other influenza viruses, e.g., H3N2 influenza virus. In other embodiment, the vaccine is monovalent, i.e., only contains H5N2 influenza virus. As mentioned above, H5N2 virus protects against H5N1 HPAI virus. If H3N2 influenza virus is present in the vaccine, it may be provided in the amounts described above. In certain embodiments, it may be present in the amount of about 1500 HA units per dose. [0045] In vaccines intended for cattle, additional antigens may contain, in addition to HPAI, antigens that elicit protective immune response against one or more of Infectious Bovine Rhinotracheitis virus, Parainfluenza type 3 virus, Respiratory Syncytial virus, BVDV-1, BVDV-2, or Bovine Influenza virus type D. Preferably, these antigens are inactivated virus. If Infectious Bovine Rhinotracheitis virus, Parainfluenza type 3 virus, and/or Respiratory Syncytial virus are present in the inactivated form, the amount of each of said inactivated viruses may be independently selected from 105.5 to 108.5 TCID50, more preferably from about 106.0 to about 108.0 TCID50, or from about 106.5 to about 107.5 TCID50. If BVDV-1 or BVDV-2 is present the amount of each virus may be from about 2500 to about 5000 Relative Unites per dose. If Influenza D is present, each dose may contain from about 500 to about 20000 HA units. [0046] In vaccines intended for swine, additional antigens may further contain in addition to HPAI, antigens that elicit protective immune response against other influenza viruses known to infect swine, including without limitations H1N1, H1N2 and H3N2 swine influenza. In certain embodiments, these antigens may be in the form of inactivated viruses, i.e., include an inactivated H1N1 influenza virus, an inactivated H1N2 influenza virus, and an inactivated H3N2 influenza virus. If present, these viruses may be provided in the amount of about 5000 to about 20000 HA units. Additionally or alternatively, the vaccine may further comprise antigens that elicit immune response against one or more antigens selected from the group consisting of PRRS- 1 virus, PRRS-2 virus, Mycoplasma hyopneumoniae, Lawsonia intracellularis, and Porcine Circovirus type 2. PRRS-1 virus and PRRS-2 virus may be provided in the form of inactivated virus.
ZP000506 PCV-2 antigen may be provided as an inactivated virus comprising ORF-1 of PCV-1 and ORF2 of PCV2. Mycoplasma hyopneumoniae and Lawsonia intracellularis antigens may be provided in the form of bacterins or whole cell bacterial extracts. [0047] In vaccines intended for horses, additional antigens may further contain in addition to HPAI, other influenza subtypes, including, without limitations H3N8 equine influenza virus. Alternatively or additionally, the vaccine may contain antigen(s) eliciting protective response against one or more of West Nile virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Venezuelan equine encephalomyelitis virus; and tetanus. In certain embodiments, the antigens eliciting protective response against West Nile virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Venezuelan equine encephalomyelitis virus are provided in the form of the respective inactivated viruses. ADJUVANTS [0048] The vaccines described herein also contain an adjuvant comprising a triterpenoid saponin, a sterol, and a CpG containing immunostimulatory oligonucleotide. More preferably, the adjuvant consists of the triterpenoid saponin, the sterol, and the CpG containing immunostimulatory oligonucleotide. Saponins [0049] Suitable saponins include triterpenoid saponins. These triterpenoids a group of surface- active glycosides of plant origin and share common chemical core composed of a hydrophilic region (usually several sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure. Because of these similarities, the saponins sharing this chemical core are likely to have similar adjuvanting properties. Triterpenoids suitable for use in the adjuvant compositions can come from many sources, either plant derived or synthetic equivalents, including but not limited to, Quillaja saponaria, tomatine, ginseng extracts, mushrooms, and an alkaloid glycoside structurally similar to steroidal saponins. [0050] If a saponin is used, the adjuvant compositions generally contain an immunologically active saponin fraction from Quillaja Saponaria, preferably, from the bark of Quillaja Saponaria. The saponin may be, for example, Quil A or another purified or partially purified saponin preparation, which can be obtained commercially. Thus, saponin extracts can be used as mixtures
ZP000506 or purified individual components such as QS-7, QS-10, QS-17, QS-18, and QS-21. In one embodiment the Quil A is at least 85% pure. In other embodiments, the Quil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure. [0051] In vaccines suitable for smaller animals such as cats and dogs, the triterpenoid saponin (e.g., Quil A or a purified fraction hereof) may be present in the vaccine in the amount of 10-50 µg per dose, or 10-40, or 10-25 or 15-25, or about 20 µg per dose. In vaccines suitable for larger animals, such as horses, pigs, sheep, cattle or goats, the triterpenoid saponin may be present in the amount of 20-500 µg per dose, or 20-400, or 40-300 or 50-150, or 40-100, or 100-300, or about 100 µg per dose. Sterols [0052] Sterols share a common chemical core, which is a steroid ring structure[s], having a hydroxyl (OH) group, usually attached to carbon-3. The hydrocarbon chain of the fatty-acid substituent varies in length, usually from 16 to 20 carbon atoms, and can be saturated or unsaturated. Sterols commonly contain one or more double bonds in the ring structure and also a variety of substituents attached to the rings. Sterols and their fatty-acid esters are essentially water-insoluble. In view of these chemical similarities, it is thus likely that the sterols sharing this chemical core would have similar properties when used in the vaccine compositions of the instant invention. Sterols are well known in the art and can be purchased commercially. For example, cholesterol is disclosed in the Merck Index, 12th Ed., p. 369. Suitable sterols include, without limitations, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. [0053] In the vaccines particularly suitable for smaller animals, such as cats and dogs, the sterol (e.g., cholesterol) may be present in the vaccine in the amount of 10-50 µg per dose, or 10-40, or 10-25 or 15-25, or about 20 µg per dose. In vaccines suitable for larger animals, such as horses, pigs, sheep, cattle or goats, the triterpenoid saponin may be present in the amount of 20-500 µg per dose, or 20-400, or 40-300 or 50-150, or 40-100, or 100-300, or about 100 µg per dose. CpG-containing immunostimulatory oligonucleotides [0054] The adjuvant component of the vaccine also comprises an immunostimulatory oligonucleotide. Immunomodulatory oligonucleotides according to the invention comprise CpG (and are also referred to as “CpG containing immunostimulatory oligonucleotides”, “CpG
ZP000506 oligonucleotides” or simply “CpGs”). The effect of CpG containing oligonucleotides on the immune system has been known for over 20 years. [0055] Generally, the CpGs suitable for the invention are between 15 and 100 bases long, e.g., between 15 and 50 bases long, or between 18 and 40 bases long or between 20 and 30 bases long, or 20-24 bases long. [0056] Several classes of CpG have been described, including A-class CpGs, B-class CpGs, C-class CpGs, and P-class CpGs. In certain embodiments, the CpG containing immunostimulatory oligonucleotide is a P-class CpG. P-class CpGs are characterized by the presence of one or more TLR-9 activating motif(s) and two palindromes or two complementarity areas. Preferably, the one or more TLR-9 activating motifs are at the 5’ of the oligonucleotide and may be completely or partially be incorporated into the 5’ palindrome or the 5’ complementarity area. TLR-9 activating motifs are known and include, without limitations, TCG, TTCG, TTTCG, TypR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5’ palindrome or the 5’ complementary area is at least 6 bases long. The 3’ palindrome or the 3’ complementary area is at least 8 bases long and is generally rich in C and G. These structural features of the P-class CpGs confer the ability to spontaneously self-assemble into concatamers either in vitro and/or in vivo. [0057] In order to increase lipophilicity of the CpG oligonucleotides, at least one lipophilic substituted nucleotide analog may be included, preferably at the 5’ end of the oligonucleotide. The P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E- modification refers to ethyl-modified nucleotide(s). Thus, E-modified P-class immunostimulatory oligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5’ nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazol, O-Methylation, modification with proynyl-dU, inosine modification, 2- bromovinyl attachment (preferably to uridine). [0058] The oligonucleotides modified by an addition of a lipophilic moiety are generally described in US 20100166780.
ZP000506 [0059] In certain embodiments, CpGs according to the invention comprise the modified backbone including, without limitations, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl-modifications), and phosphodiester modifications. [0060] Suitable non-limiting examples of modified P-class immunostimulatory oligonucleotides are provided below (“*” refers to a phosphorothioate bond, “-” refers to a phosphodiester bond, “JU” refers to 5’-Iodo-2’-deoxyuridine and “EU” refers to 5-Ethyl-2’-deoxyuridine). SEQ ID NO: 1 5’ T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3’ SEQ ID NO: 2 5’ T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3’ SEQ ID NO: 3 5’ T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3’ SEQ ID NO: 4 5’ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3’ SEQ ID NO: 5 5’ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3’ SEQ ID NO: 6 5’ JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3’ SEQ ID NO: 7 5’ EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3’ SEQ ID NO: 8 5’ JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3’ SEQ ID NO: 9 5’ JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3’ SEQ ID NO: 10 5’ T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3’ [0061] In certain embodiments, the CpG oligonucleotide according to the invention comprises any one of SEQ ID NOs 1-10 or an oligonucleotide comprising at least 15 consecutive bases of any one of SEQ ID NOs 1-10. In the most preferred embodiment, the vaccine comprises an oligonucleotide comprising at least 15 consecutive bases of SEQ ID NO: 8 (e.g., at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23). [0062] In certain embodiments, the CpG-containing immunostimulatory oligonucleotide comprises SEQ ID NO: 8 and/or 20-23-oligonucleotide-long fragments of SEQ ID NO: 8. In certain embodiments, the CpG-containing immunostimulatory oligonucleotide may be present as a heterogenous population containing SEQ ID NO: 8 and 20-23-oligonucleotide-long fragments of SEQ ID NO: 8. The homogeneity of said CpG oligonucleotide preparation may be at least 60%, i.e., the amount SEQ ID NO: 8 or other reference sequence comprising at least 60% of all CpG oligonucleotide amount by weight and shorter fragments (preferably 20-23-oligonucleotide-long fragments) of the reference sequence comprise the remaining percentage. Thus, the homogeneity of the CpG oligonucleotide preparation may be at least 65%, at least 70%, at least
ZP000506 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. The homogeneity may be determined by HPLC, and 60% refers to the ratio of the main peak to areas of all peaks including impurities. [0063] In certain embodiments, particularly suitable for smaller animals, such as cats and dogs, the CpG-containing immunostimulatory oligonucleotide (e.g., SEQ ID NO: 8 and shorter fragments thereof as described above) may be present in the vaccine in the amount of 10-50 µg per dose, or 10-40, or 10-25 or 15-25, or about 20 µg per dose. In vaccines suitable for larger animals, such as horses, pigs, sheep, cattle or goats, the triterpenoid saponin may be present in the amount of 20-500 µg per dose, or 20-400, or 40-300 or 50-150, or 40-100, or 100-300, or about 100 µg per dose. [0064] In certain embodiments, the adjuvants disclosed herein do not contain one, two or all three of the following compounds: a quaternary ammonium compound, a polyacrylic acid polymer, and/or a glycolipid-type adjuvant. [0065] Quaternary ammonium adjuvants are ammonium-based adjuvants containing a nitrogen atom linked to four hydrocarbon groups. In practice, hydrocarbon groups are generally limited to alkyl or aryl groups. In a set of embodiments, the quaternary ammonium compounds are composed of four alkyl chains, two of which are C10-C20 alkyls and the remaining two are C1-C4 alkyls. In certain embodiments, the quaternary ammonium is dimethyldioctadecylammonium (DDA) bromide, chloride or another pharmaceutically acceptable counter ion. In another embodiment, the quaternary ammonium adjuvant is avridine. [0066] Polyacrylic acid polymers are polymers of acrylic acid cross-linked with methacrylic acid, polyalkenyl ethers or divinyl glycol and include, without limitations, CARBOPOL®. [0001] Glycolipid-type adjuvants include, without limitations, base and salt forms of compounds encompassed by Formula I and generally described in US Patent Publication 20070196384 (Ramasamy et al).
ZP000506 Formula I wherein, R1 is
to 20 carbon atoms; X is -CH2-, -O- or -NH-; R2 is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R3, R4, and R5 are independently hydrogen, -SO4 2-, -PO4 2-, -COC1-10 alkyl; R6 is L-alanyl, L-alpha- aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L- hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenyalany, L-prolyl, L- seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers. [0067] Examples of a glycolipid type adjuvants are, without limitation, N-(2-Deoxy-2-L- leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamide (BAY®1005, or R1005) or a salt (e.g., an acetate) thereof. EXCIPIENTS [0068] Other components of the vaccines described herein can include pharmaceutically acceptable excipients, such as carriers, solvents, and diluents, isotonic agents, buffering agents, stabilizers, preservatives, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like. The vaccines can also contain antibiotics or preservatives, including, for example, gentamicin, m15erthiolate, or chlorocresol. The various classes of antibiotics or preservatives from which to select are well known to the skilled artisan. METHODS OF VACCINE ADMINISTRATION [0069] Administration of the vaccines disclosed herein leads to the induction of protective immune response against canine influenza viruses of types H3N2 and H3N8 (if present) and thus preventing a subject in need thereof from being infected with the canine influenza virus, or, if
ZP000506 said subject still gets infected, for reduction of the number and/or severity of the symptoms of the canine influenza infection. In certain embodiments, the subject is a canine subject (e.g., dogs, coyotes, wolves, jackals) of a feline subject (including domestic cats and felines in zoos such as leopards, cheetahs, lions, tigers, ocelots, lynxes, etc). More preferably, the subject is a dog or a cat, most preferably, the subject is a dog. In other embodiments, particularly suitable for HPAI vaccine, the subject is a marine mammal, preferably a pinniped, such as a walrus, a seal or a sea lion. [0070] In further embodiments, particularly appropriate for the HPAI vaccine described herein, the subject is a ruminant, such as an ovine, caprine, or bovine. In yet further embodiments, the subject is an equine or a porcine animal. [0071] The vaccines disclosed herein may be administered according to the following regimen: a prime dose followed by the boost (or booster) dose about 14 to about 42 days after the prime dose. In different embodiments, the booster dose is administered about 14 to about 28 days, or about 21 days after the prime dose. In certain embodiments, this regimen provides at least a six- month duration of immunity after the booster dose, and preferably, at least a 12-month duration of immunity (e.g., 6 month-long, 7 month-long, 8 month-long, 9 month-long, 10 month-long, 11 month-long duration of immunity). Thus, in certain embodiments, semi-annual or annual revaccinations are envisioned. [0072] The vaccines according to the invention may be formulated for and be administered to the subject by any known routes, including the oral, intranasal, mucosal, topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). Administration can also be achieved using needle-free delivery devices. Administration can be achieved using a combination of routes, e.g., the prime administration using a parental route and the boost administration using a mucosal route. In the currently preferred embodiments, the vaccines disclosed herein are administered intramuscularly or subcutaneously. [0073] The invention will now be described in the following non-limiting examples. EXAMPLES Example 1. H3N2 CIV
ZP000506 [0074] The objective of this study was to aid in the adjuvant selection for the development of the CIV H3N2 subtype killed vaccine. Dogs were vaccinated with experimental CIV H3N2 killed vaccines formulated with selected adjuvants and challenged with H3N2 Strain SY29 and evaluated for efficacy and safety of the experimental vaccines. 40 healthy, 8-9 weeks old beagle dogs that were negative for CIV antigen and antibody were randomly divided into 4 groups, with 10 dogs per group. Group T01 was the control group and received a dose of 1ml/dog of PBS subcutaneously on D0 and D21, Groups T02~T04 were the experimental immunized groups that received a H3N2 killed vaccine formulated with different adjuvants, QCT (T02), RT (T03) and REHYDRAGEL® (T04). These animals were also vaccinated on D0 and D21 with a 1ml/dog dose subcutaneously. Injection site observations, temperatures and clinical observations were monitored twice on vaccination days (pre- and post-vaccination) and then once daily for D1-7 and D22-28 [0075] On D42 (21 days post 2nd vaccination), all dogs were challenged with stain SY29 at a dose of 3×107.55 EID50/3ml/dog via aerosolization and observed continuously for 7 days post challenge. Clinical observations and temperatures were monitored twice on D42-48 and once on D41 and D49. Nasal swabs were collected on D–1 (prior to vaccination), D42 (prior to challenge) and daily from D43-49. Blood samples were collected from all animals on Day -1 (prior to 1st vaccination) and on Days 7, 14, 21 (prior to 2nd vaccination), 28, 35, 42 (prior to challenge) and 49. On D49, all dogs were anesthetized and euthanized via exsanguination. At necropsy the dogs were observed for clinical pathology and lung scores were collected, along with samples for histopathological examination. [0076] Study design and vaccine formulations are summarized in Tables 1 and 2, respectively. Table 1. Study design summary Treatment Animal Treatment drugs Challenge material groups numbers Name Titer Dose& Route1 Name Dose& Route T01 10 PBS NA 1ml/dose, Strain 3×107.55 T02 10 CIV H3N2 10240HA/ml subcutaneous SY29 EID50/3ml/dog, Killed-QCT injection on
ZP000506 T03 10 CIV H3N2 10240HA/ml D0 (right aerosolization Killed- -RT shoulder) and on D42 T04 10 CIV H3N2 10240HA/ml D21 (eft Killed- shoulder) REHYDRAGEL ® Table 2. Vaccine formulation Treatment Formulation groups T01 NA T02 BEI Inactivated CIV H3N2 NJ62 strain formulated with QCT adjuvant (0.02 mg Quil-A, 0.02 mg Cholesterol, 0.02 mg CpG SEQ ID NO: 8 per dose), phosphate buffer Q.S. in 1 ml dose. T03 BEI Inactivated CIV H3N2 NJ62 strain formulated with RT adjuvant (0.25 mg glycolipid BAY®1005 acetate, 0.05 mg CpG SEQ ID NO: 8 per dose), phosphate buffer Q.S. in 1 ml dose. T04 BEI Inactivated CIV H3N2 NJ62 strain formulated with 5% REHYDRAGEL®，0.063% PBS buffer Q.S. in 1 ml dose. [0077] CIV vaccinated (T02/T03/T04) dogs were seronegative to CIV H3N2 prior to vaccination on Day 0 and control (T01) vaccinated dogs remained seronegative (≤ 8) to H3N2 following vaccination until the time of challenge. All animals were CIV antigen negative by nasal swab prior to Day 0 and prior to Challenge. Eighty percent (80%) or more of the control (T01) vaccinated dogs showed signs of clinical disease following challenge, thus validating the challenge model. [0078] Criteria for satisfactory demonstration of efficacy was protection 7 days post challenge, after continuous observation. Protection was defined as the absence of the following two criteria in the vaccinated animals: (1) Clinical signs (two or more clinical signs present on two or more consecutive days post- challenge) including fever (≥39.5℃), nasal discharge, coughing, sneezing, mucopurulent ocular discharge, retching, increased respiratory noises, and depression; and (2) Virus Shedding (virus shedding by virus isolation from nasal swabs collected being positive on two or more consecutive days from day 3-7 post-challenge).
ZP000506 [0079] Adjuvant Selection was evaluated by observing injection site reactions and systemic adverse events post-vaccination for the multiple formulations and the level of protection post challenge. [0080] Vaccine and challenge potency are summarized in tables 3 and 4, respectively. Table 3. Potency of vaccine formulations Vaccine Target Actual Potency T02 H3N2 killed vaccine w/ QCT ≥2560 HA/dose 10240 HA/dose T03 H3N2 killed vaccine w/ RT ≥2560 HA/dose 10240 HA/dose T04 H3N2 killed vaccine w/ RT ≥2560 HA/dose 10240 HA/dose Table 4 Potency of challenge material (log10EID50/3ml)/dog Challenge Actual Potency Actual Potency Target material Pre-Challenge Post- Challenge CIV H3N2 strain 3×107.5 3×107.5 3×107.6 SY29 [0081] Safety: none of the animals in groups T01-T04 exhibited injection site swelling or signs of pain after first or second vaccinations. Clinical Scores [0082] Clinical observations, mean clinical scores, and statistical analyses are summarized in Tables 5-7. Table 5. Summary of clinical observations after vaccination by treatment groups Treatmen Vaccinati Nasal Ocular Respira Retchi Sneezi Cough Depression Fever t groups on times discharge Discharge tion ng ng st T01 1 0/10 0/10 0/10 0/10 1/10* 0/10 0/10 0/10 (Control) 2nd 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 T02 1st 0/10 0/10 0/10 0/10 1/10* 0/10 0/10 0/10 (QCT) 2nd 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1st 0/10 0/10 0/10 0/10 1/10* 0/10 0/10 0/10 T03 (RT) 2nd 0/10 0/10 0/10 0/10 1/10* 0/10 0/10 0/10
ZP000506 T04 1st 0/10 0/10 0/10 0/10 2/10* 0/10 0/10 0/10 (REHYDR AGEL®) 2nd 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 Note: * mild ocular discharge. Table 6. Summary of mean total clinical scores post-challenge by treatment groups Scores of clinical signs Treatment groups Mean St. dev. Median Minimum Maximum T01 (Control) 46.2 19.6 43 16 75 T02 (QCT) 10.2 5.9 12 1 17 T03 (RT) 12.7 10.5 10 1 33 T04 (REHYDRAGEL®) 15.0 11.2 13 0 38 [0083] A total clinical score was calculated for each animal by summing the scores for temperature, cough, depression, nasal discharge, ocular discharge, respiration, retching and sneezing across the observations post-challenge. Clinical signs scores were scored as 1 (Mild), 2 (Moderate), and 3 (Severe). Temperature was scored as 1 for each instance of fever (≥ 39.5℃). P-values below 0.05 indicate statistically significant differences. Table 7 Significance analysis of scores of clinical signs post-challenge by treatment groups Treatment groups P value T01-T02 <0.0001* T01-T03 <0.0001* T01-T04 0.0001* T02-T03 0.5149 T02-T04 0.2387 T03-T04 0.6386 * P-values below 0.05 indicate statistically significant differences. Viral Shedding. [0084] The total shedding duration was defined as the number of days (last day virus detected – first day virus detected + 1) that an animal had CIV virus detected in the nasal swabs from day 1- 7 post-challenge will be calculated for each animal. If the animal did not have virus detected, the number of days will be 0.
ZP000506 [0085] Virus Shedding was defined as continuous shedding for two or more consecutive days from day 3-7 post-challenge. [0086] Shedding duration and statistical analyses are summarized in tables 8 and 9. P-values below 0.05 indicate statistically significant differences. Table 8. Summary of shedding duration post-challenge by treatment groups Treatment groups Mean St. dev. Median Minimum Maximum T01 (Control) 4.5 1.1 5 3 6 T02 (QCT) 0.7 0.8 1 0 2 T03 (RT) 1.4 1.3 1 0 4 T04 (REHYDRAGEL®) 1.0 1.3 1 0 4 Table 9. Significance analysis of duration of shedding post-challenge by treatment groups Treatment groups P value T01-T02 <0.0001* T01-T03 <0.0001* T01-T04 <0.0001* T02-T03 0.1909 T02-T04 0.5701 T03-T04 0.4499 * P-values below 0.05 indicate statistically significant differences. [0087] HI antibody titers and statistical analyses are provided in Tables 10 and 11, respectively. P-values below 0.05 indicate statistically significant differences. Table 1 Summary of HI antibody titers in each group (GMT) by treatment groups Treatment groups D7 D14 D21 D28 D35 D42 D49 T01 (Control) 4.6 4.3 4.0 4.0 4.0 4.0 10.9 T02 (QCT) 17.1 21.9 29.9 568.1 494.6 388.0 1097.5 T03 (RT) 21.1 22.6 19.7 315.2 294.1 163.1 1060.1 T04 (REHYDRAGEL®) 8.9 17.1 30.9 265.0 222.9 200.9 675.6 Table 11. Significance analysis of HI titers by treatment groups Treatment P value groups D7 D14 D21 D28 D35 D42 D49
ZP000506 T01-T02 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 T01-T03 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 T01-T04 0.0091 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 T02-T03 0.5240 0.9154 0.2032 0.0720 0.1121 0.0084 0.9154 T02-T04 0.0483 0.4651 0.9168 0.0224 0.0170 0.0483 0.1448 T03-T04 0.0070 0.3845 0.1582 0.5866 0.3845 0.5141 0.1582 P-values below 0.05 indicate statistically significant differences. [0088] Lung sore was calculated with the following equation: Percent consolidation = 0.53× [(0.35× right cranial lobe) + (0.15× right middle lobe) + (0.40× right caudal lobe) + (0.10× accessory lobe)] + 0.47× [(0.30× left cranial lobe-cranial part) + (0.25× left cranial lobe- caudal part) + (0.45× left caudal lobe)]. CIV-related histopathological lesion in lung was defined by the presence of microscopic lesions consistent with CIV infection in one or more lobes of the lung, particularly evidence of broncho interstitial inflammation with epithelial degeneration/necrosis and/or hyperplasia. [0089] Tables 12-15 summarize the findings related to lung lesions post-challenge. P-values below 0.05 indicate statistically significant differences. Table 2 Frequency distribution of lung lesion post-challenge by treatment groups Scores of lung gross lesions Treatment groups Mean St. dev. Median Minimum Maximum T01 (Control) 42.84 22.04 46.91 2.4 70.7/7 T02 (QCT) 11.15 16.41 0.38 0.0 41.2 T03 (RT) 11.84 14.83 8.76 0.0 46.0 T04 (REHYDRAGEL®) 7.89 12.57 0.20 0.0 28.9 Table 13. Frequency distribution of lung scores＞5 post-challenge by treatment groups Treatment groups Animal number Lung scores＞5 T01 (Control) 10 9/10 T02 (QCT) 10 4/10 T03 (RT) 10 5/10 T04 (REHYDRAGEL®) 10 3/10
ZP000506 Table 14. Significance analysis of lung scores by treatment groups Treatment groups P value T01-T02 <0.0001* T01-T03 <0.0001* T01-T04 <0.0001* T02-T03 0.7377 T02-T04 0.5168 T03-T04 0.3284 * P-values below 0.05 indicate statistically significant differences. Table 15. Frequency distribution of histopathological lesions in lung post-challenge by treatment groups Treatment groups Animal number Histopathological lesions T01 (Control) 10 10/10 T02 (QCT) 10 4/10 T03 (RT) 10 7/10 T04 (REHYDRAGEL®) 10 5/10 [0090] CIV-related histopathological lesion in trachea was defined by the presence of microscopic lesions consistent with CIV infection, particularly evidence of respiratory epithelial degeneration/necrosis+/- hyperplasia with attendant mucosal inflammation. Histopathological lesions in trachea/bronchi observed post-challenge are summarized in tables 16-18. Table 16. Frequency distribution of gross lesions observed in tracheal/bronchial lymph nodes post-challenge by treatment groups Gross Frequency of enlargement Frequency of discoloration Treatment groups lesions/Total Mild Moderate Marked Mild Moderate Marked T01 (Control) 10/10 2/10 3/10 4/10 6/10 2/10 1/10 T02 (QCT) 3/10 1/10 2/10 0/10 2/10 0/10 0/10 T03 (RT) 6/10 2/10 0/10 2/10 3/10 2/10 1/10 T04 3/10 1/10 0/10 1/10 3/10 0/10 0/10 (REHYDRAGEL®)
ZP000506 Table 17. Frequency distribution of gross lesions observed in tonsils post-challenge by treatment groups Gross Frequency of enlargement Frequency of discoloration Treatment groups lesions/Total Mild Moderate Marked Mild Moderate Marked T01 (Control) 1/10 0/10 0/10 1/10 0/10 1/10 0/10 T02 (QCT) 2/10 0/10 1/10 1/10 1/10 1/10 0/10 T03 (RT) 4/10 1/10 1/10 1/10 3/10 1/10 0/10 T04 2/10 2/10 0/10 0/10 1/10 0/10 0/10 (REHYDRAGEL®) Table 18. Frequency distribution of histopathological lesions in trachea post-challenge by treatment groups Treatment groups Animal number Histopathological lesions T01 (Control) 10 10/10 T02 (QCT) 10 3/10 T03 (RT) 10 4/10 T04 (REHYDRAGEL®) 10 6/10 [0091] After challenge, the protection rates of three immunized groups T02~T04 were 90% (9/10), 80% (8/10) and 90% (9/10), respectively. In addition, compared with the control group, the total score of clinical symptoms and the score of lung lesion were significantly decreased and the virus shedding duration was significantly shortened in the three immunized groups. Besides, compared with T03 (RT) and T04 (REHYDRAGEL®) groups, the clinical scores and virus shedding duration of T02 group (QCT) were lower; the severity of clinical pathology and/or histopathology lesions in the lungs, trachea, tracheal/bronchial lymph nodes and tonsils were milder; and the HI antibody titers in sera were relatively higher in T02 group than T03 and T04 groups. Taken together, these results demonstrate advantages of adjuvant QCT over RT and REHYDRAGEL®. [0092] In conclusion, the protective efficacy of T02 group (QCT adjuvant) was superior compared to the other two immunized groups. Example 2. HPAI virus vaccine - dogs
ZP000506 [0093] The objective of the study was to determine the safety and serology of H5N2 vaccines when administered to dogs. The study utilized the antigen H5N2 which is a poultry vaccine inactivated virus which was genetically modified from the Highly Pathogenic Avian Influenza virus which cross-protects against Highly Pathogenic Avian Influenza virus H5N1. [0094] Purpose-bred beagles of approximately 63-67 weeks of age were used in this study. All animals were in good general health and have not received any CIV vaccinations. One animal in T01 had an HAI titer of 10 on Day -1, and one animal in T02 had a titer of 40 on Day -1. Since dogs had no exposure to H5N2 prior to the study, it is assumed that this is due to assay variability on a non-validated assay. Twenty dogs were housed in one room, two dogs per double pen with one dog from each treatment group represented in each pen. [0095] Feed was consistent with the standard practices of the testing facility. Diet was dry food suitable for the age and nutritional requirements of the animals, moistened if necessary and provided ad libitum at least once daily through the course of the study. Canned food or non- medicated nutritional supplements were given as needed. Lot numbers of dry and canned food and supplements were documented. Water was available ad libitum at all times. Dogs may have been given water bowls in addition to the automatic watering system during the first few weeks upon arrival to the vaccination phase facility. [0096] Treatment group 01 (T01) was administered 1.0 mL 1000 H5N2 HA Dose with 5% REHYDRAGEL®. Treatment group 02 (T02) was administered 1.0 mL 1000 H5N2 HA Dose with adjuvant QCT containing 20 µg Quil A, 20 µg cholesterol and 20 µg SEQ ID NO: 8 per dose. Vaccines were administered subcutaneously on the right scapular region on Day 0 and on the left scapular region on Day 21 for all animals. [0097] Injection Site Observations and Tympanic Temperatures were recorded prior to and 2-6 hours post-vaccination on vaccination days, in addition to once daily for seven days post- vaccination. There was no injection site swelling or pain for all animals during the study. No animals from treatment group T01 displayed a fever post-vaccination. Three dogs from treatment group T02 did display fevers post-vaccination. [0098] Blood samples for serology were collected via venipuncture on Days -1, 20, and 35.
ZP000506 [0099] Antibody titers: Treatment group 01 had a back-transformed least square means of 5 on Day -1, 11 on Day 20, and 65 on Day 35. Treatment group 02 had a back-transformed least square means of 6 on Day -1, 14 on Day 20, and 113 on Day 35. There was no significant difference between the treatment groups on Days -1 and 20, but there was significant difference (P=0.0729) on Day 35, with T02 (QCT adjuvant) having higher LSM antibody titers (Table 19). Refer to Table 20 for treatment comparisons of antibody titers. Table 39. Back-Transformed Treatment Least Squares Means, Standard Errors, and Ranges of Antibody Titers Day BT Least BT BT Lower BT Upper Treatment Of Number Of Squares Standard Range 90% 90% Number Study Observations Mean Error Confidence Confidence Bound Bound -1 10 5 0.8 5 to 10 4 7 T01 20 10 11 3.3 5 to 40 6 18 35 10 65 14.0 20 to 160 45 93 -1 10 6 0.9 5 to 40 5 8 T02 20 10 14 4.3 5 to 40 9 23 35 10 113 24.4 40 to 320 79 162 T01: 1000 H5N2 HA Dose with 5% Rehydragel® T02: 1000 H5N2 HA Dose with QCT Table 40. Treatment Comparisons of Antibody Titers Day of Contrast P-value Significance Study of P-value -1 T01 vs T02 0.5239 No 20 T01 vs T02 0.5154 No 35 T01 vs T02 0.0729 Yes [00100] There was no significant difference in antibody titers between the treatment groups on Days -1 and 20, but there was significant difference (P=0.0729) on Day 35, with the QCT adjuvant group having higher titers. [00101] No animals were observed with injection site swelling or pain during the study. [00102] Animals in treatment group T01 did not display any fevers during the study. 20% of treatment group T02 had fevers after the first vaccination. 30% of treatment group T02 had fevers after the second vaccination.
ZP000506 [00103] Both vaccine formulations were safe and effective for use in dogs while the QCT adjuvant produced higher antibody titers against H5N2. Example 3. HPAI virus vaccine - cats [00104] The objective of the study was to determine the safety and serology of H5N2 vaccines when administered to cats. The study utilized the antigen H5N2 which is a poultry vaccine inactivated virus which was genetically modified from the Highly Pathogenic Avian Influenza virus H5N1 formulated with adjuvants that have been proven to be safe and effective in cats. [00105] Specific pathogen-free and unvaccinated for CDV cats of approximately 57-58 weeks of age were used in this study. Sixteen cats were group-housed in 4 pens containing 3 or 5 cats per pen in one room. Cats were fed with a diet according to their age requirements and provided fresh water ad libitum. Canned commercial cat food may have been eventually offered as a treat. Cats were acclimated for at least 7 days prior to use in the study. [00106] Treatment group 01 (T01) was administered 1.0 mL 1000 H5N2 HA Dose with 5% REHYDRAGEL®. Treatment group 02 (T02) was administered 1.0 mL 1000 H5N2 HA Dose with QCT containing 20 µg Quil A, 20 µg cholesterol and 20 µg SEQ ID NO: 8 per dose. Vaccines were administered subcutaneously on the right rear leg on Day 0 and on the left rear leg on Day 21 for all animals. [00107] Immediate Local and System Reactions were recorded for approximately 30 minutes post- vaccination on vaccination days. No animals displayed clinical signs of either immediate local reactions or immediate systemic reactions. [00108] Injection Site Observations and Tympanic Temperatures were recorded prior to and 2-6 hours post-vaccination on vaccination days, in addition to once daily for seven days post- vaccination. No animals displayed injection site reactions post-vaccination. No animals from T01 and six animals from T02 had fevers after the first vaccination. One animal from T01 and five animals from T02 had fevers after the second vaccination. [00109] Blood samples for serology were collected via venipuncture on Days -22 and 35. [00110] Antibody titers: There was no significant difference in antibody titers (P=1.0000) between treatment groups on Day -22 as all animals were negative to H5N2. There was significant
ZP000506 difference (P=0.0002) between treatment groups by the end of the study, with animals vaccinated with the QCT adjuvanted vaccine producing much higher titers. See Table 21. Table 25. Back-Transformed Treatment Least Squares Means, Standard Errors, and Ranges BT Leas BT Lower BT Upper Treatment Day Of Number Of t BT 90% r Study Observations Squares Stan 90% Numbe dard Range Mean Error Confidence Confidence Bound Bound T01 -22 8 2.5 0.4 2.5 to 2.5 2.0 3.2 35 8 640.0 91.8 320.0 to 1280.0 501.5 816.7 T02 -22 8 2.5 0.4 2.5 to 2.5 2.0 3.2 35 8 1522.2 218.4 1280.0 to 5120.0 1192.8 1942.5 T01: 1000 H5N2 HA Dose with 5% REHYDRAGEL® T02: 1000 H5N2 HA Dose with QCT [00111] All cats were negative to H5N2 prior to vaccination. There was a significant difference between antibody titers of the two groups (P < 0.05) with the animals vaccinated with the QCT adjuvanted vaccine producing much higher titers over the REHYDRAGEL®- adjuvanted vaccine. [00112] No animal was observed with injection site pain or swelling during the study. [00113] No animals in T01 were recorded with fever after the first vaccination, however, several animals had fever after the second vaccination. In the T02 vaccine group, animals had fevers following both vaccinations. Fevers were mild and considered to be normal post-vaccination. [00114] Both vaccine formulations were safe and effective for use in cats while the QCT adjuvant produced higher antibody titers against H5N2. Example 3. HPAI vaccine in cattle [00115] Eight 2 to 3 month old calves were enrolled for this immunogenicity study. [00116] Calves were pre-screened for HI antibody titers in a Hemagglutination Inhibition Assay. All calves were sero-negative and had a HI antibody titer which was <1:40 (negative). [00117] On Days 0 and 21, all 8 calves were vaccinated via the SC route with 2mL of the experimental QCT+H5N2 inactivated vaccine. The vaccine contained 40 micrograms of QUIL ATM, 40 micrograms of cholesterol, and 40 micrograms of CpG of SEQ ID NO: 8 per dose, in 0.063% PBS (phosphate-buffered saline). The antigen was present in the amount of 2000 HA units per dose.
ZP000506 [00118] Response to vaccination was monitored by testing serum from all calves on Day 14, 21, 28, 35 and 42 in a Hemagglutination inhibition assay. By Day 14, 25% of the calves had met the recognized criteria for sero-protection (titers ≥1:40 dilution) for Influenza vaccine. On each of days 28, 35, and 42, 100% of the calves had met this threshold and the mean titers were above the seroconversion level. One calf has been bled for serum and thus removed from the calculations of the mean titer. All calves showed titers that were ≥1:320 dilution at either of the time points on or after day 28. [00119] All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. [00120] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.