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Definitions

• the present invention is related to the techniques used in the prevention and treatment of diseases, preferably of the avian type and more particularly, it is related to recombinant vaccines comprising an inactivated viral vector, which has inserted an exogenous nucleotide sequence that encodes a protein with antigenic activity of a disease; and, a pharmaceutically acceptable carrier, adjuvant or excipient.
• vaccines against viral pathogens are formulated by isolating the corresponding virus for later use for the production of a vaccine, administering it to animals or humans by various formulations.
• vaccine formulations that use complete and active viruses that have shown low pathogenicity in the field, or whose pathogenicity has been attenuated in the laboratory, but which, however, when supplied, cause a sufficient antigenic reaction to provide protection against viral strains of the same species with greater pathogenicity.
• Newcastle disease is of viral origin and highly contagious, and can even be lethal. This disease affects domestic and wild birds causing high morbidity and mortality.
• ENC is caused by a virus of the family Paramyxoviridae, of the genus Avulavirus. According to their degree of pathogenicity and virulence, the strains are classified as: lentogenic, mesogenic and velogenic, that is, low, moderate and high pathogenicity respectively (Office International des Epizooties (2008). Newcastle Disease. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animáis, Office International des Epizooties, France, p. 576-589). The sources of transmission of the ENC virus are multiple.
• velogenic type ENC viruses WENC
• the incubation period for velogenic type ENC viruses (WENC) causing high mortality is approximately 21 days, and with respiratory and / or nervous signology such as gasping and sneezing and incoordination, wings bristling, dragging of the legs, crooked head, neck, tics, circle displacements, depression, lack of appetite and complete paralysis.
• respiratory and / or nervous signology such as gasping and sneezing and incoordination, wings bristling, dragging of the legs, crooked head, neck, tics, circle displacements, depression, lack of appetite and complete paralysis.
• One of the strategies used for the control and prevention of ENC is precisely the use of active virus vaccines, normally made from lentogenic strains.
• Live vaccines against ENC induce protection at the level of the respiratory mucosa and have been used by industry for more than 50 years.
• These active virus vaccines are mainly based on the use of the lentogenic viruses of the Hitchner B1 and LaSota strains, the latter being the most popular vaccine (Op. Cit, Office International des Epizooties (2008), Newcastle).
• the active viruses may be inactivated due to the components of an emulsion
• the stability of emulsion active vaccines is limited, so they are normally used in other types of formulations, or are supplied by mixtures. in situ, which hinders its application in large-scale poultry farming.
• influenza virus The main problem with active viruses is that it is not always possible to use them as vaccines because of their high capacity for genetic variation, recombination with other active viruses or predisposition to changes in their pathogenicity, such as influenza virus.
• Influenza is a respiratory disease that affects both mammals and birds. The appearance of an influenza virus strain in a given population can have serious consequences for individuals, both for domestic birds as well as for humans or other mammals. The virus, when it infects domestic chickens and mammals, mutates rapidly to adapt to this new population and during that evolutionary process of adaptation can result in very important biological changes in the same virus that give rise to fatal results for the host and the animal or human population.
• AI avian influenza
• VIA AI viruses
• VABP low pathogenicity AI virus
• the VIA can be classified according to two of the virus's external proteins: the first one is the hemoagglutinin that is of great importance, since it is responsible for the response of neutralizing antibodies in infected or vaccinated birds and for which 16 different subtypes or serotypes have been reported; The second protein is the neuraminidase of which 9 different subtypes have been reported. Particularly, the most important viruses for birds are those whose hemoagglutinin contains serotypes H5 and H7 that when mutated at high pathogenicity are capable of producing mortality close to 100%.
• AI disease in birds has two forms of clinical presentation: the first one is low pathogenic avian influenza (IABP) that can cause mild disease, sometimes expressed by poor appearance of the plumage, reduction in egg production; but mainly the IA is important in birds due to the high mutagenic capacity of the virus that in these birds invariably gives rise to the second presentation that is high pathogenic avian influenza (IAAP) capable of causing mortalities close to 100%.
• IABP low pathogenic avian influenza
• IAAP high pathogenic avian influenza
• the clinical signs of AI are variable and are influenced by the subtype of virus involved, the pathogenicity thereof, the immune status and the affected avian species.
• the incubation period for VIAAP is 21 days and the clinical signs range from conjunctivitis, elevation of the temperature characterized by feathering, depression, prostration and death.
• the most frequently described lesions are: pulmonary congestion, hemorrhages and edema.
• the VIA Once the VIA has been introduced into a poultry farm, it is excreted into the environment by feces and respiratory fluids. The transmission and dissemination of the virus to other birds occurs primarily through direct contact with the secretions of infected birds, especially feces, food, water, contaminated equipment and clothing. The susceptibility to infection and the manifestation of clinical signology of the disease is very variable.
• inactivated virus vaccines usually in emulsion.
• vaccines made with inactivated AI virus stimulate a strong immune response at the systemic level and have had positive results for the control of both forms of AI.
• Vaccination is used not only to prevent the clinical signs of the disease, but also, to reduce, as far as possible, the viral excretion of infected birds into the environment. The reduction of viral excretion decreases the opportunity for the spread of the virus from vaccinated birds that become infected to susceptible uninfected birds (Swayne, D. and Kapczynski, D. (2008). Vaccines, Vaccination and Immunology for avian influenza viruses in poultry .In Avian Influenza. Ed. By David Swayne. Blackwell Publishing, USA, p. 407-451.)
• the emulsion inactivated virus vaccines have greater stability, which allows a better handling of the vaccine and a longer shelf life of the vaccine. It is for this reason that ENC vaccines have also been formulated with virus inactivated in emulsion.
• inactivated virus vaccines require a concentration of virus much higher than an active virus, usually at least 10 times more, to achieve the same antigenic activity, since precisely the virus has been manipulated to eliminate its ability to replicate, so that the totality of the antigen that will be required to cause the immune response should be present from the moment the vaccine is given, since the organism will not replicate normally to the virus and consequently, its quantity will not be increased .
• the use of recombinant vaccines is one of the most significant advances in the field of biotechnology.
• the ability to isolate and splice (or recombine) specific fragments of DNA from an organism, the size of a gene and transfer them to another by means of a DNA vector or plasmid to produce an antigen capable of inducing the formation of antibodies protectors has led to the introduction of new vaccines.
• the recombinant technology provides very important advantages in the case of diseases, such as the AI described above, in which there is no possibility of using complete active viruses due to its high mutagenic capacity and where the use of the virus Complete inactivation always constitutes a risk if the inactivation process was inadequate.
• the recombinant vaccines in their active form, by having inserted the nucleotides necessary for the expression of the antigens against the disease of interest, can be safely administered to induce local immunity at the level of the respiratory mucosa in an active viral vector of a disease of low pathogenicity, which would be impossible to perform by using the non-recombinant live virus because of the risks that this would entail.
• recombinant vaccines Another advantage of recombinant vaccines is that the viral vector that is used does not normally correspond to the disease with respect to the one they protect, which facilitates its use in the veterinary area of diagnostic and prevention techniques of the type that allow vaccinated animals to be differentiated of infected animals, better known as DIVA (Capua, I. et. al. "Development of a DIVA (differentiating infected from vaccinated animáis) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza".
• DIVA Capua, I. et. al. "Development of a DIVA (differentiating infected from vaccinated animáis) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza.
• the vaccines that are currently used for the control of AI completely inactivated and emulsified viruses in oil
• other similar diseases prevent the mortality caused by VIAAP but do not prevent infection and replication of VIA in birds, by Therefore, the decrease in excretion and spread of the virus is partially achieved.
• recombinant active virus vaccines are traditionally formulated with virus concentrations approximately 10 times higher than that used for the non-recombinant active virus vaccine corresponding to the viral vector used, in order to achieve adequate exposure of the antigenic sites of the microorganism of interest.
• recombinant vaccines have not been used nactivated, as this would imply achieving viral vector concentrations 100 times higher than those required for the normal virus (10 times higher than that of the active recombinant virus), which is industrially very complicated. Consequently, these active recombinant virus vaccines have also not been widely used in emulsion, because the stability is limited and the emulsion does not represent advantages in this aspect due to the active nature of the active viral vector.
• a vaccine comprising an inactivated recombinant viral vector, which has inserted an exogenous nucleotide sequence that codes for an antigenic site of a disease of interest; and, a pharmaceutically acceptable emulsion vehicle, adjuvant or excipient, provides adequate protection against said disease of interest by using a viral vector titer similar to that required for a recombinant active virus vaccine based on the same viral vector.
• the exogenous nucleotide sequence is selected from sequences for antigenic sites against influenza, infectious laryngotracheitis, infectious bronchitis, infection of the Fabrizio pouch
• an antigen selected from avian influenza, laryngotracheitis, infectious bronchitis, infection of the pouch is used.
• Fabrizio (Gumboro), hepatitis, PRRS and circovirus.
• the exogenous nucleotide sequence consists of the gene that codes for the hemoagglutinin (HA) of the avian influenza virus, selected from the 16 subtypes of hemoagglutinin or immunogenic variant of the influenza virus, which more preferably it codes for at least one of the subtypes H1, H2, H3, H5, H6, H7 or H9 of said protein.
• HA hemoagglutinin
• the H5 protein gene is obtained from the Mexican H5N2 subtype avian influenza virus or the H5N1 subtype of Asian origin, with excellent protection of both modalities being observed towards the mortality generated by VIAAP subtype H5N2.
• said viral vector in the preferred embodiment in which said viral vector where the exogenous nucleotide sequence is inserted, is the Newcastle disease virus (rNDV), said viral vector It is preferably selected from vaccine strains, such as the LaSota, Ulster, QV4, B1, CA 2002, Roakin, Komarov, Clone 30, VGGA strains or strains of genetic groups I to V of Newcastle disease.
• the recombinant virus is from the LaSota strain (rNDV / LS).
• the adenovirus is selected from avian and porcine adenoviruses, and more preferably between type 9 avian adenovirus (rFAdV / 9) and type 5 porcine adenovirus (rSAdV / 5).
• the result achieved by the vaccine of the present invention is unexpected, since in the case of recombinant vaccines in the viral vector, it is traditionally thought that replication of the viral vector in the host cells is necessary for that the recombinant protein is expressed in a sufficient amount in order to stimulate an adequate immunogenic response, however, in the present invention, the result obtained indicates that the antigenic protein of the disease of interest is adequately expressed and in sufficient quantity on the surface of the vector virus, and its inactive presence alone makes possible an adequate antigenic and protective response against said disease of interest.
• the recombinant vaccine of the present invention achieves a local immune response at the level of the respiratory mucosa of the birds, as well as an immune response at the systemic level, which can be differentiated by specific laboratory tests, from immune responses induced by the contact of birds with complete viruses, whether they are vaccines or field, which represents an important advance for epidemiological effects.
• the vaccine is formulated to be administered subcutaneously, however any systemic route such as intramuscular or intradermal could be used successfully.
• the vehicle preferably used for the vaccine is a liquid vehicle, more preferably, a water-in-oil emulsion is used, but it is also possible to successfully use other types of adjuvants or modulators of the immune response.
• Figure 1 is a graph of the results of mortality (M) and morbidity index (IM) of Example 6A generated by the challenge with a velogenic ENC virus (WENC).
• Figure 2 is a graph of the mortality (M) and morbidity index (IM) results of Example 6A generated by the challenge with a high pathogenic AI virus (VIAAP) subtype H5N2.
• Figure 3 is a graph of the mortality (M) and morbidity index (IM) results of Example 6B, generated by the challenge with a WENC.
• Figure 4 is a graph of the mortality (M) and morbidity index (IM) results of Example 6B, generated by the challenge with a VIAAP subtype H5N2.
• Figure 5 is a graph of the mortality (M) and morbidity index (IM) results of Example 6C, generated by the challenge with a WENC.
• Figure 6 is a graph of the mortality (M) and morbidity index (IM) results of Example 6C, generated by the challenge with a VIAAP subtype H5N2.
• Figure 7 is a graph of the mortality (M) and morbidity index (IM) results of Example 6D, generated by the challenge with a WENC.
• Figure 8 is a graph of the mortality results (M) and morbidity index (MI) of the
• Example 6D generated by the challenge with a VIAAP subtype H5N2.
• a vaccine comprising an inactivated viral vector, which has inserted a nucleotide sequence that codes for a disease of interest; and, a pharmaceutically acceptable carrier, adjuvant or excipient, provides adequate protection against the disease of interest by using a viral vector titer similar to that required for an active virus vaccine based on the same viral vector.
• the viral vector is inactivated, being understood as inactivated that the recombinant virus that functions as a viral vector and contains the nucleotide sequence that codes for the antigenic site of the disease of interest has lost the property of replicating.
• Inactivation is achieved by physical or chemical procedures well known in the state of the art, preferably by chemical inactivation with formaldehyde or beta-propiolactone (Office International des Epizooties (2008). Newcastle Disease. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animáis Office International des Epizooties, France, p. 576-589). In the opposite sense, it is understood that an active or live virus maintains its ability to replicate.
• the viral vector preferably selected from adenovirus or paramyxovirus, is inactivated and has an exogenous nucleotide sequence inserted that encodes at least one antigenic site of a disease of interest, preferably at least one disease selected from influenza, infectious laryngotracheitis. , infectious bronchitis, infection of the Fabrizio (Gumboro) bag, hepatitis, viral rhinotracheitis, infectious coryza, Mycoplasma hyopneumoniae, pasterellosis, Porcine Reproductive and Respiratory Syndrome (PRRS), circovirus, bordeteliosis, parainfluenza or any other antigen whose size allows its insertion in the corresponding viral vector.
• a disease of interest preferably at least one disease selected from influenza, infectious laryngotracheitis.
• infectious bronchitis infection of the Fabrizio (Gumboro) bag
• hepatitis viral rhinotracheitis
• infectious coryza infectious coryza
• an antigen selected from avian influenza, laryngotracheitis, infectious bronchitis, infection of the Fabrizio pouch (Gumboro), hepatitis, PRRS and circovirus is used.
• the exogenous nucleotide sequence consists of the gene that codes for the hemoagglutinin (HA) of the avian influenza virus, selected from the 16 subtypes of hemoagglutinin or immunogenic variant of the influenza virus, which more preferably it codes for at least one of the subtypes H1, H2, H3, H5, H6, H7 or H9 of said protein.
• HA hemoagglutinin
• said viral vector in the preferred embodiment in which said viral vector where the sequence of inserts is inserted exogenous nucleotides, is the Newcastle disease virus (rNDV), said viral vector is preferably selected from vaccine strains, such as strains LaSota, Ulster, QV4, B1, CA 2002, Roakin, Komarov, Clone 30, VGGA or strains of genetic groups I to V of Newcastle disease.
• the recombinant virus is from the LaSota strain (rNDV / LS).
• the adenovirus is selected from aviary and porcine adenoviruses, and more preferably from the avian adenovirus type 9 (rFAdV / 9) and the porcine adenovirus type 5 (rSAdV /5).
• the antigenic site when the disease of interest is influenza, it is preferred that it be that corresponding to the hemoagglutinin (HA) protein of avian influenza, the gene preferably being obtained from the avian influenza virus, and encodes for any of the 16 existing subtypes, preferably H5, H7 and H9, preferably encode for subtype H5, which is preferably obtained from the strains: Bive, 435 and Viet (VT) 1 described below.
• HA hemoagglutinin
• the gene preferably being obtained from the avian influenza virus, and encodes for any of the 16 existing subtypes, preferably H5, H7 and H9, preferably encode for subtype H5, which is preferably obtained from the strains: Bive, 435 and Viet (VT) 1 described below.
• the source strain of the gene coding for the HA of subtype H5 is not critical for the present invention since the experimental results show that any strain can provide the genetic material to achieve the objective of the present invention.
• H5 gene of the Bive strain corresponds to a VIABP-H5N2 isolated in Mexico in 1994 from biological samples of broilers and that has been identified by the Mexican government like (A / chicken / Mexico / 232 / CPA).
• Said virus strain is authorized by the Ministry of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA) for use in the preparation of inactivated emulsion vaccines, so that in this way recombining this virus with the gene of interest also ensures biosecurity in the recombinant vaccine of the present invention.
• SAGARPA Ministry of Agriculture, Livestock, Rural Development, Fisheries and Food
• the genetic material that is the H5-435 gene was obtained from an isolation of VIABP-H5N2 isolated in Mexico in 2005 from biological samples of broilers.
• the viral vector of the vaccine of the present invention can be prepared by amplifying by PCR the sequence of nucleotides of interest, from the identification of the antigenic sites of an isolation of the pathogen of origin, to be able to insert them later, amplified within the viral vector , preferably selected from adenovirus or paramyxovirus.
• the insertion is done using standard molecular biology techniques such as restriction enzymes and DNA ligases, among other.
• the infectious clone thus produced is introduced into a cell line for the generation of the recombinant virus according to the viral vector.
• the virus replicates in any system suitable for its growth, such as SPF chicken embryo, or commercial cell lines or expressly designed to grow viruses.
• the virus is inactivated.
• the inactivation is carried out by physical or chemical procedures well known in the state of the art, preferably by chemical inactivation with formaldehyde or beta-propiolactone.
• Pharmaceutically acceptable carriers for the vaccines of the present invention are preferably aqueous solutions or emulsions. More particularly, it is preferred that the vehicle used is a water-oil emulsion.
• the specific formulation of the vaccine will depend on the viral vector used, as well as on the exogenous nucleotide sequence that has been inserted. However, in the preferred embodiment in which the viral vector is a Newcastle disease virus, it is preferred that the dose be between 10 4 and 10 10 DIEP50% / ml. In the embodiment in which the viral vector is an adenovirus, it is preferred that the dose be between 10 2 and 10 8 DIEP50% / ml. As regards the application of the vaccine, it is preferably performed subcutaneously in the middle and posterior part of the neck of the birds.
• the vaccine of the present invention is applied in poultry, such as broilers, laying birds, breeding birds, turkeys, fighting roosters, guinea fowl, partridges, quails, ducks, geese, swans or ostriches.
• the vaccine is applied subcutaneously, although in certain species it can be intramuscularly in birds of any age.
• the vaccine When the vaccine is applied in chickens in Newcastle vector in emulsion, the vaccine preferably contains 10 8 to 10 9 DIEP50% / 0.5 ml per chicken, and more preferably the vaccine contains 10 85 DIEP50% / 0.5 ml per chicken. Vaccination in chickens can be performed more easily at 10 days of age.
• the present invention provides very important competitive advantages.
• the inactive recombinant vaccine of the present invention makes it possible to establish vaccination programs with the exclusive use of recombinant vaccines in viral vector and with insertion of genes of pathogens difficult to control, which gives rise to a method of identifying infected animals of animals that only received one vaccine (DIVA), useful in the control and eradication of diseases, characterized in that it comprises: a) Subject to a first method of detecting antibodies, at least a sample of at least one animal to which a recombinant vaccine was applied of an inactivated viral vector that has an exogenous nucleotide sequence inserted that codes for an antigen of a disease caused by a pathogen, to detect if there are antibodies present in said sample corresponding to said antigen; b) Submit a second antibody detection method, at least one sample of the same animal whose sample was subjected to the first antibody detection method, to detect if there are antibodies present in said sample that correspond to the pathogen causing the disease; c
• the pathogen has a difficult control, such as VIA, mainly H5 and H7, which are the cause of high mortality in poultry
• VIA mainly H5 and H7
• excellent protection is achieved at systemic level , which also offers a high degree of biosafety comparatively to the use of complete AI viruses that constitute a serious risk if they have not been properly inactivated. This risk is increased during the elaboration process in which the viruses are active.
• the present invention also allows the epidemiological differentiation of vaccinated birds with respect to other birds exposed to complete viruses (DIVA system) since when only the hemoagglutinin (HA) gene of the avian influenza virus is inserted, the laboratory test that is used to detect antibodies induced by the vaccine against avian influenza is the inhibition of hemagglutination (Hl).
• Current immunological tests such as ELISA and other tests such as agar gel diffusion, are negative for the detection of antibodies against avian influenza induced by the recombinant vaccine of the present invention, since they are designed to detect antibodies induced by another type of antigens contained in complete viruses.
• the birds vaccinated with the recombinant vaccine of the present invention are infected with the field virus, the latter tests are positive for the detection of antibodies against avian influenza, with which the infected birds can be distinguished.
• the present invention allows programs with exclusive use of recombinant vaccines to be established in an inactive and active manner, the first that will provide the systemic immunity already indicated and the recombinant active vaccine that it will complement the immunity at the level of the mucous membranes leading to protections equal to or close to 100% at the field level.
• This program also uses the DIVA system discussed above.
• the recombinant vector of the inactivated emulsion vaccine is Newcastle with an influenza gene inserted, both for challenges with WENC and VIAAP, it can be applied simultaneously with an active vaccine with the same vector and antigen, directly in the respiratory mucosa, either by the ocular route, by spraying, or in drinking water, so that the response is strongly stimulated at the local level (in the respiratory and digestive mucous membranes) generating the production of immunoglobulins Type A secretaries (IgA), with which the field virus replication is significantly reduced and its excretion and dissemination is significantly reduced.
• IgA immunoglobulins Type A secretaries
• the vaccine of the present invention allows to establish control programs and possible eradication by differentiation of vaccinated birds from infected, since when applying the inactivated recombinant vaccines of the present invention it is possible the differentiation of birds vaccinated from birds infected with virus field (DIVA System) since the recombinant vaccines only contain the hemoagglutinin of the VIA as an antigen making it possible to use diagnostic tests such as ELISA that detect antibodies induced by other virus antigens and not those induced solely by hemoagglutinin.
• DIVA System virus field
• pNDV / LS an intermediate vector called "pNDV / LS" was developed.
• the extraction of total viral RNA from Newcastle strain LaSota was carried out by the triazole method.
• the synthesis of cDNA (complementary DNA) of the viral genome was carried out, using as a template the total RNA purified above.
• all the Newcastle genome genes (15, 183 base pairs (bp)
• 7 fragments with "overlapping" ends and cohesive restriction sites were amplified by PCR.
• Fragment 1 comprises nucleotide (nt) 1-1755, F2 goes from nt 1-3321, F3 comprises from nt 1755-6580, F4 goes from 6,151-10, 210, F5 covers nt 7,381-11, 351, F6 ranges from 1 1, 351 -14,995 and F7 comprises nt 14,701-15, 186.
• the assembly of the 7 fragments was performed within a cloning vector called pGEM-T using techniques ligation standards, which allowed the reconstruction of the Newcastle LaSota genome, which after cloning contains a unique Sacll restriction site, between the P and M genes, which serves to clone any gene of interest in this viral region of the vector.
• Example 2 Cloning of the HA gene of the VIA subtype H5N2 strain 435.
• the extraction of total viral RNA was carried out by the Triazole method. This purified total RNA was subsequently used to synthesize cDNA (complementary DNA) and through the use of specific oligonucleotides with the PCR technique, the AI virus HA gene was amplified.
• the HA gene of 435 was subsequently inserted into the pGEM-T vector using standard cloning techniques and thus generating the plasmid: pGEMT-435.
• pSacllGE / GS was constructed, by initial PCR amplification of the GE / GS sequences taking as molds the genome of Newcaslte and the subsequent insertion of these sequences in pGEM-T.
• Plasmid pGEMT-435 was digested with Hpal-Ndel and subsequently cloned into pSacllGE / GS, to generate plasmid pSacllGE / GS-HA435.
• Hep-2 and A-549 cells were dressingalmente infected with MAV-7 virus at a multiplicity of infection (MOI) of 1. After Inora incubation at 37 0 C under CO 2 5%, the cells were transfected with 1 microgram ( ⁇ g) of DNA from the clone pNDVLS-435, together with 0.2 ⁇ g of DNA from the expression plasmids: pNP, pP and pL which code for the viral proteins P, NP and L, necessary for the generation of the recombinant in both cell types. 12 hours after the transfection, the recombinant virus generated in both cell types was harvested and injected into 10-day-old SPF chicken embryos to amplify the generated virus. The allantoic liquid harvested 48 hours later, was titrated by plaque assay in Vero cells, thus generating the final recombinant virus, used in the preparation of the vaccine.
• MOI multiplicity of infection
• the recombinant viruses containing the genes obtained from the Bive and Viet strains were generated in the manner described above.
• Example 5 Method of making an emulsified vaccine inactivated with recombinant Newcastle LaSota virus with H5 insert of the Avian Influenza virus. rNDV / LS-H5. Antigen elaboration
• the vaccine was prepared in an emulsion of the water-in-oil type.
• mineral oil and surfactants of the Span 80 and Tween 80 type were used.
• the aqueous phase the FAA was mixed with a conservative solution (thimerosal).
• the Ia was slowly added aqueous phase to the oil phase under constant stirring.
• a homogenizer or a colloid mill was used. Antigenic content.
• the vaccine was formulated to provide a minimum of 10 85 DIEP 50% /0.5 ml in order to use a dose per bird of 0.5 ml.
• H5-Bive gene which was obtained from the VIABP subtype H5N2 strain (A / chicken / Mexico / 232 / CPA), isolated in Mexico in 1994 from biological samples of broilers, and corresponding to the strain of virus authorized by SAGARPA for the preparation of inactivated emulsion vaccines.
• Gene H5-435 It was obtained from an isolation of VIABP subtype H5N2 isolated in Mexico in 2005 from biological samples of broilers. Strain 435 demonstrated to have differential antigenic characteristics in tests of inhibition of hemagglutination (Hl) with the Bive strain and in nucleotide sequencing studies showed important changes.
• H5-Vt gene This gene was isolated in Vietnam and corresponds to the H5 gene of an H5N1 subtype AI virus.
• Example 5 a recombinant experimental vector vaccine (rNDV / LS) with anchor (Rd) with inactivated and emulsified H5-Bive gene in pharmaceutical formula water / oil, which was called Emi Rd, was developed. -Bive.
• Example 5 a recombinant experimental vector vaccine (rNDV / LS) with anchor (Rd) with inactivated and emulsified H5-435 gene in a water / oil pharmaceutical formula, called Emi Rd-435, was prepared.
• rNDV / LS experimental vector vaccine
• Rd anchor
• Emi Rd-435 water / oil pharmaceutical formula
• Example 5 a recombinant experimental vector vaccine (rNDV / LS) was prepared with an anchor with inactivated and emulsified H5-Vt gene in water / oil pharmaceutical formula, which was called Emi Rd-Vt.
• rNDV / LS recombinant experimental vector vaccine
• rNDV / LS recombinant experimental vector vaccine without anchorage with inactivated and emulsified H5-Bive gene in pharmaceutical formula water / oil, which was called Emi Re-Bive, was prepared.
• rNDV / LS recombinant experimental vector vaccine without anchorage with inactivated and emulsified H5-435 gene in a water / oil pharmaceutical formula, called Emi Re-435
• rNDV / LS recombinant experimental vector vaccine without anchoring with inactivated and emulsified H5-Vt gene in the water / oil pharmaceutical formula, called Emi Re-Vt, was prepared.
• WENC virus Chimalhuacán strain containing 8 0 DIEP50% / ml, equivalent to 10 6 - 5 DIEP50% / 0.03ml / chicken.
• the challenges were made at 35 days of age of the birds (21 days post-vaccination -DPV-) in isolation units of the INIFAP-CENID-Microbiology, in level 3 biosecurity acrylic insulating cabinets. It was subdivided into two subgroups and each subgroup was placed in the corresponding isolation units following the pre-established biosecurity procedures.
• the VIAAP-H5N2 was diluted in a 1: 10 ratio with PBS pH 7.2 and 0.06 ml (2 drops) was applied to each chicken in each eye and 0.09 ml (3 drops) in each nostril, equivalent 0.3 ml or 100 DLP50%.
• the challenge with WENC virus was performed by applying to each chicken by eye 0.03 ml of a viral suspension containing 10 8 0 DIEP50% / mi, equivalent to 10 6 5 DIEP50% / bird.
• the PD assessment with WENC was carried out for 14 days, while the PD assessment with VIAAP-H5N2 was performed for 10 days according to the guidelines suggested by the OIE.
• the morbidity index (MI) of each group was calculated using an equation obtained by taking the data of the day in which it showed the greatest severity of clinical signology during the PD observation period.
• the three recombinant inactivated rNDV / LS-H5 vaccines with anchorage of the present invention are capable of conferring in SPF chickens 100% protection against mortality (M) induced by the WENC challenge virus ( Figure 1).
• the three inactivated recombinant vaccines also conferred 100% protection against mortality (M) induced by VIAAP-H5N2 ( Figure 2) as well as conventional vaccines.
• ENC and IA inactivated elaborated with complete viruses that are currently authorized worldwide for use in the control of ENC and IA, which are elaborated containing normally the LaSota strain Newcastle disease virus with a titre of 10 86 DIEP50% / ml and avian influenza virus of low pathogenicity with a titre of 10 80 DIEP50% / ml, chemically inactivated with formaldehyde and emulsified in oil.
• the protection results indicate that the rNDV / LS-H5 inactivated recombinant vaccines with anchorage comply with Mexican and international standards for use in the control of ENC and IA, which demonstrates that the present invention proved successful for This is the recombinant version with anchor.
• the three inactivated recombinant vaccines also conferred 100% protection against mortality (M) induced by VIAAP-H5N2 as well as the rNDV / LS-H5 inactivated recombinant vaccines with anchorage and Conventional emulsified vaccines made with complete inactivated ENC / IA-Bive and ENC / IA-435 viruses.
• examples 6A and 6B indicate that the inactivated recombinant vaccines made in vector with anchorage or without anchorage and with VIA H5 genes of different origin and antigenic characteristics in the Hl tests (H5N2 or H5N1), are capable of conferring a same protection towards the challenge with VIAAP-H5N2.
• the results suggest that the inactivated recombinant vaccines elaborated with any VIA H5 gene can confer protection towards the challenge with VIAAP with any of the influenza virus subtypes that contain hemoagglutinin H5, the type of neuraminidase not being relevant.
• ENC / IA-Bive as well as the inactivated vaccines of the present invention obtained according to Examples 5A (Emi Rd-Bive), 5B (Emi-Rd-435) and 5C (Emi- Rd- Vt). TABLE 4. Potency in commercial broilers with maternal immunity to ENC and IA, immunized with inactivated vaccines made with the recombinant rNDV / LS-H5 virus with anchorage (Rd)
• the three recombinant inactivated rNDV / LS-H5 vaccines with anchorage of the present invention are capable of conferring in commercial broilers with maternal immunity towards the ENC and IA viruses, protections equal to or greater than 90% a The mortality (M) induced by the WENC challenge virus ( Figure 5). Additionally, and independently of the H5 gene with which they were cloned, the three inactivated recombinant vaccines also conferred protections equal to or greater than 80% protection against mortality (M) induced by VIAAP-H5N2, as well as conventional emulsified vaccines made with complete inactivated viruses for use in the control of ENC and IA.
• inactivated vaccines are essential to achieve adequate protection at the field level to prevent the mortality generated by VIAAP and WENC virus, since under field conditions in industrial poultry farms only the use of conventional active vaccines against ENC or recombinant active against Ia
• AI may not be enough.

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