HK1164691A - Recombinant inactivated viral vector vaccine - Google Patents

Description

Recombinant inactivated viral vector vaccines

Technical Field

The present invention relates to a technique for preventing and treating diseases, especially avian diseases, and more particularly, to a recombinant vaccine comprising an inactivated viral vector having an inserted foreign nucleotide sequence encoding a protein having antigenic activity of a disease and a pharmaceutically acceptable vehicle, adjuvant or excipient.

Background

It is well known that vaccines against viral pathogens are formulated from the corresponding viruses, which are isolated for the production of vaccines, which are administered to animals or humans through a diverse formulation.

On the one hand, some vaccine preparations are made with whole live viruses that show low pathogenicity in the wild or with viruses that have been attenuated by laboratory attenuation and which, when administered, elicit an antigenic response sufficient to provide protection against highly pathogenic strains of the same species.

For example, newcastle disease (ENC) is caused by a virus, is highly contagious, and may be fatal. The disease infects poultry and wild birds, causing high morbidity and mortality. ENC is caused by a virus of the avian Paramyxoviridae (paraamyxoviridae) family Paramyxoviridae (Avulavirus). These strains are classified according to their pathogenicity and virulence: light (lentog nickas), moderate (mesogenic) and strong (velog nickas) virulence, respectively, are low, medium and high pathogenicity (Office International des epizooies (2008).

ENC viruses have a variety of transmission sources. For example, direct transmission via the liver or dead birds and their products or by-products (supbproducts), or indirect transmission via vectors such as infected insects or other animals, including humans. The latent time of highly virulent ENC virus (VVENC) causing high mortality is about 21 days, showing respiratory and/or neurological symptoms such as dyspnea (jadeo), sneezing (estornudo) and ataxia (incordinaci Lolo n), upright wings (alas erizadas), leg dragging (arastre de patas), head and neck contortion (cabeza, cuello tordots), tics (tics), central shift (delazamios) in circles, depression (depesi n), apheresis (apacia), and complete paralysis (par li complex). In addition, egg production is partially or completely interrupted, forming either unformed eggs or thin rough egg shells with aqueous albumin.

One of the strategies for the control and prevention of ENC is the use of live virus (virus activity) vaccines, usually made from light strains. Live vaccines against ENC induce protection at the mucosa of the respiratory tract and have been industrialized for more than 50 years. These live virus vaccines are mainly based on the use of light-virulent viruses from the Hitchner B1 and LaSota strains, the latter being the most commonly used vaccine (op.

However, the stability of the emulsified live vaccine is limited, as the live virus can be inactivated by some components of the emulsion. As a result, they are often used in other types of formulations or they are delivered by in situ mixing, which makes their use difficult in large-scale poultry farms.

The main problem with live viruses is that they have a high capacity for genetic variation, recombine with other live viruses, or may alter their pathogenic properties, such as influenza viruses, and are therefore not always useful as vaccines. Influenza is a respiratory disease that can infect mammals and birds. The emergence of influenza virus strains in particular populations can lead to serious consequences for individuals, including poultry, humans, or other mammals. When the virus infects domestic hens and mammals, they rapidly change to adapt themselves to this new population, and during this adaptive evolution, large biological changes may be induced in the same virus, with fatal consequences for the host and animal or human population.

Specifically, avian Influenza (IA) is a highly infectious viral epidemic caused by a type a virus of the orthomyxoviridae family. Most IA Viruses (VIA) have been isolated from wild birds, particularly waterfowl, and function to carry and accumulate low pathogenic IA Viruses (VIABP). When these viruses infect non-natural hosts such as poultry, mainly gallinaceae (i.e., hens, turkeys, and quails, etc.), they mutate to a highly pathogenic form (VIAAP) during adaptation.

VAIs can be classified according to two viral outer membrane proteins. The first is hemagglutinin, the most important protein, because it is responsible for reacting with neutralizing antibodies in infected or vaccinated birds, and 16 different subtypes or serotypes have been reported. The second protein is neuraminidase, and 9 different subtypes have been reported. In particular, the most important viruses for avians are those with hemagglutinin serotypes H5 and H7, which when mutated to highly pathogenic cause nearly 100% mortality.

Likewise, there are two clinical forms of IA disease in birds: the first is low pathogenic avian Influenza (IABP), which causes mild disease, sometimes manifested as feather damage and reduced egg production. However, IA is of predominant importance in avians because the virus has a high mutability, always causing a second clinical form, becoming highly pathogenic avian Influenza (IAAP), causing nearly 100% mortality.

In particular, clinical symptoms of IA are diverse, influenced by the subtype of the virus involved, the pathogenicity, immune status, and the avian species infected. The VIAAP incubation period is 21 days, with a variety of clinical symptoms: conjunctivitis (conjunctivitis), elevated temperature characterized by erect feather, depression, prostration (postrati Lou n) and death. The most commonly mentioned lesions are: pulmonary congestion (congestis and Lolo n pulmonar), bleeding (hemorragias) and edema (edemas).

Once VIA enters the poultry farm, it can be discharged to the environment VIA feces and respiratory droplets. The transmission and spread of the virus to other birds is primarily through direct contact with the secretions of the infected birds, especially contaminated manure, food, water, equipment and clothing. The susceptibility to and clinical manifestations of the disease are very diverse.

For such diseases which are difficult to control and where uncontrolled administration may result in a live virus vaccine which is dangerous for the health of the animal or even human, it is preferred to use a vaccine of inactivated virus, usually emulsified.

Several vaccines have been developed in the prior art to prevent a variety of viral diseases, such as the above-mentioned IA. For this latter disease, emulsified vaccines containing whole IA virus have been prepared from chicken embryos. The virus has been inactivated and emulsified in water-oil for subcutaneous or intramuscular administration to commercial birds (official International des epizootics (2008).

More specifically, vaccines made with inactivated IA virus stimulate a strong immune response systemically, which has a positive effect on the control of both forms of IA. The immunization is used to prevent the clinical symptoms of the disease and, possibly, to reduce the expulsion of infected birds into the environment. The reduction in viral shedding reduces the chance of transmission of the virus from vaccinated birds to uninfected susceptible birds (Swayne, D, y Kapczynski, D (2008), Vaccines, Vaccination and immunization for avianine viruses in poultrying. in Avian infection. Ed by David Swayne. Blackwell publishing, USA, p.407-451).

In addition, the emulsified inactivated virus vaccine has increased stability, better management of the vaccine, and prolonged shelf life. Thus, ENC vaccines have been formulated as emulsified inactivated viruses.

An important consideration is that one of the major differences between live and inactivated virus vaccines is the amount of virus required to achieve an antigenic response after administration.

Since live viruses have the complete ability to replicate themselves in cells, the amount of relevant virus required in the vaccine is lower than the dose that elicits the antigenic response, which avoids discomfort to the vaccinated individual given that the virus is naturally replicating in an amount that achieves the desired antigenic response once in the body.

On the other hand, inactivated viruses require a higher concentration of virus than live viruses, usually at least 10-fold higher, to achieve the same antigenic activity, because the virus is treated to remove its replicative capacity, so that the total amount of antigen necessary to elicit an immune response when the vaccine is administered is sufficient, because the body cannot normally replicate the virus and the amount of virus does not increase.

In addition, one of the most significant advances in the biotechnology field is the use of recombinant vaccines. The ability to isolate and splice (or recombine) specific DNA fragments of an organism at the genetic level, transfer the fragments into another organism using a vector or DNA plasmid, produce antigens, and thereby induce the production of protective antibodies, has led to the development of new vaccines. Unlike conventional vaccines, recombinant technology provides significant advantages for diseases such as IA, where live virus cannot be used due to high mutability, and where the use of whole inactivated virus always poses a risk due to improper inactivation procedures. The activated recombinant vaccine, after insertion of the essential nucleotides expressing the antigen against the target disease, can be safely administered in live viral vectors with low pathogenicity to induce local immunity of the respiratory mucosa, which is not possible in the case of non-recombinant live viruses because of the associated risks.

A further advantage of recombinant vaccines is that the viral vectors used do not generally correspond to the disease for which protection is sought, which facilitates their use in the field of veterinary diagnostics and prevention technology to distinguish vaccinated from infected animals, so-called DIVA (Capua, I et al, "Development of a DIVA (differentiated fed from transformed animals) targeted use a vaccinating a heterologous gene for the control of infection". Avian Pathology 32(1) pp.47-55).

However, current vaccines for the control of IA (emulsification in oil, intact inactivated virus) and other similar diseases prevent death by VIAAP, but do not avoid infection and replication of VIA in birds, and thus, only partially achieve a reduction in detoxification and viral spread.

Thus, viral vectors have been developed in the art from low pathogenic diseases such as Newcastle disease, into which genes encoding antigenic sites of diseases that are difficult to control such as avian influenza, e.g., activated recombinant vaccines disclosed by Ge, Deng, Tianet al, "new castle disease virus-based live infected vaccine completed vaccines and mice", j.vir.vol.81, No.1, p.150-158 ", are inserted. Specifically, this document discloses the results of clinical trials with the LaSota strain carrying the avian influenza subtype H5N1 gene.

Another document in the same field is Park, Man Seong et al, "Engineered visual vacutainec structures with dual specificity: avian Influenza and Newcastle disease ". PNASVol.103, No.21, May 12, 2006 p.8203-8208. This document relates to techniques for increasing the expression of avian influenza genes, such techniques being hereinafter referred to as "anchoring".

Although some recombinant vaccines have replaced live virus vaccines due to the above advantages, recombinant vaccines have not achieved the advantages of inactivated whole virus vaccines and, importantly, they have not been able to provide correct immunity against inserted foreign genes, primarily because recombinant vaccines such as newcastle disease plus influenza, described above, cause antigenic activity against both diseases but require more contact with foreign antigenic sites inserted into the vector. As a result, new technologies, such as anchoring technologies, which utilize genetic modification to achieve better antigen expression in the viral vector in the case of the influenza described above, continue to be developed. Such techniques have not been entirely successful.

Thus, recombinant vaccines from live viruses are typically formulated with approximately 10-fold higher concentrations of the viral vector used than non-recombinant vaccines from live viruses in order to achieve suitable exposure of the target microbial antigenic site.

Likewise, recombinant vaccines have not been used in inactivated form, as this may mean that the concentration of viral vectors is 100 times higher than that required for normal viruses (10 times higher than that of live recombinant viruses), which can be very complex in terms of industry. As a result, these recombinant live virus vaccines have never been used as emulsions in general because of their limited stability and because emulsions have no advantage in this respect due to the active character of the live virus vectors.

In view of the above, there is a great need for vaccines against diverse diseases that are provided by recombinant techniques in a safe and effective manner, with high stability, suitable control and efficacy outcomes.

Brief description of the invention

During the development of the present invention, it was unexpectedly discovered that when a vaccine comprises a recombinant inactivated viral vector into which is inserted a foreign nucleotide sequence encoding an antigenic site of a disease of interest; and the vaccine further comprises a pharmaceutically acceptable emulsified vehicle, adjuvant or excipient, with a viral vector titer similar to that required for a recombinant live virus vaccine based on the same viral vector, to provide the desired protection against the disease of interest.

In one embodiment of the invention, the exogenous nucleotide sequence is selected from the group consisting of anti-influenza (influenza), infectious laryngotracheitis (largnotraquisites infecciiosa), infectious bronchitis (bronquisifecciiosa), bursal infection (infli ease n de la bois de Fabricio, Gumboro), hepatitis (hepatitis), viral rhinotracheitis (rinotraque viral), influenza (coizainfecciosa), Mycoplasma hyopneumoniae (mycosphaerella hygieniae), pasteurellosis (pasteurelosis), porcine reproductive syndrome (sri nodorhodoro repiroratorino, porcelovirus (cirrus), bordetella (borteuresis), parainfluenza virus (parainfluenza virus), and other vectors having an antigen inserted into a corresponding site or any other antigen of a size. Preferably, an antigen selected from the group consisting of avian influenza (influenza aviar), laryngotracheitis, infectious bronchitis, bursal infection (Gumboro), hepatitis, PRRS, and circovirus is used.

In a particular embodiment of the invention, said foreign nucleotide sequence consists of a gene encoding avian influenza virus Hemagglutinin (HA) selected from the 16 hemagglutinin subtypes or immunogenic variants of this influenza virus, more preferably encoding at least one of the H1, H2, H3, H5, H6, H7 or H9 subtypes of said protein.

In a particular embodiment of the invention, the H5 gene is obtained from Mexican avian influenza virus subtype H5N2, or from asian origin subtype H5N1, very good protection against the two subtypes against mortality caused by the VIAAP subtype H5N2 being observed.

With respect to the viral vector of the present invention, in a preferred embodiment, newcastle disease virus (rNDV) corresponds to a viral vector having an inserted foreign nucleotide sequence, said viral vector is preferably selected from vaccine strains such as LaSota, Ulster, QV4, B1, CA 2002, Roakin, Komarov, Clone 30, or VGGA strains, or strains from newcastle disease genetic groups I to V. Preferably, the recombinant virus is a LaSota strain (rNDV/LS).

Likewise, in another embodiment, the viral vector is an adenovirus selected from the group consisting of avian and porcine adenoviruses, more preferably from avian adenovirus type 9 (rFAdV/9) and porcine adenovirus type 5 (rSAdV/5).

As will be seen from the results detailed below, with the present invention, recombinant inactivated viral vaccines can be produced in emulsion or in other pharmaceutically acceptable adjuvants using exogenous nucleotide sequences located in the viral vector that encode specific antigenic determinants of the disease of interest.

The results obtained with the vaccine of the invention (rNDV/LS-H5) are unexpected because it was traditionally thought that, in the case of recombinant vaccines in viral vectors, the replication of said viral vectors in host cells requires sufficient expression of the recombinant protein to stimulate a suitable immune response, but, in the present invention, the results obtained show that the antigenic protein of the disease of interest is expressed in sufficient quantity and correctly on the surface of the vector virus and that it is present only in inactive form, which enables the generation of a suitable antigenic and protective response against said disease of interest.

In particular, in highly pathogenic and difficult to control diseases such as avian influenza, one advantage of the recombinant vaccines of the present invention is that the whole virus is not used, thereby reducing the risk of outbreaks caused by improper inactivation of the vaccine virus. Moreover, the vaccine of the present invention achieves local immune responses in the respiratory mucosa of birds and also immune responses in the whole body, and enables differentiation of immune responses caused by exposure of birds to whole viruses (viruses used as vaccines or wild-type viruses) by specific laboratory tests, representing an important advance in the field of epidemiology.

The vaccine is formulated for subcutaneous administration; but any systemic route such as intramuscular or intradermal administration may also be successfully employed. Preferably, the vaccine is applied with a liquid vehicle, more preferably a water-in-oil emulsion, but other immune response adjuvants or modulators can also be successfully applied.

The recombinant vaccine of the invention can reduce the toxin expelling of wild viruses to the environment, thereby greatly reducing the spread of the viruses.

Drawings

The features of the invention believed to be characteristic of the various novel aspects of the invention are set forth with particularity in the appended claims. The vaccine of the present invention, however, as well as additional objects and advantages thereof, will be best understood from the following detailed description of certain embodiments when read in conjunction with the accompanying drawings. In the drawings:

FIG. 1 shows the mortality results (M) and the morbidity Index (IM) of example 6A, obtained with a challenge with a virulent ENC virus (VVEENC).

FIG. 2 shows the mortality results (M) and morbidity Index (IM) of example 6A, obtained with a highly pathogenic IA Virus (VIAAP) subtype H5N2 challenge.

FIG. 3 shows mortality results (M) and morbidity Index (IM) for example 6B, obtained with VVENC challenge.

FIG. 4 shows the mortality results (M) and morbidity Index (IM) of example 6B, obtained with a challenge with the VIAAP subtype H5N 2.

Fig. 5 shows mortality results (M) and morbidity Index (IM) for example 6C, obtained with VVENC challenge.

FIG. 6 shows the mortality results (M) and morbidity Index (IM) for example 6C, obtained with a challenge with the VIAAP subtype H5N 2.

Fig. 7 shows mortality results (M) and morbidity Index (IM) for example 6D, obtained with VVENC challenge.

FIG. 8 shows the mortality results (M) and morbidity Index (IM) of example 6D, obtained with a challenge with the VIAAP subtype H5N 2.

Detailed Description

During the development of the present invention it was surprisingly found that when the vaccine comprises an inactivated viral vector into which a nucleotide sequence encoding a disease of interest is inserted; and the vaccine further comprises a pharmaceutically acceptable vehicle, adjuvant or excipient, with a viral vector titer similar to that required for a live virus vaccine based on the same viral vector, to provide the desired protection against the disease of interest.

It is an essential point of the present invention that the viral vector is inactivated, by which is meant that the recombinant virus functions as a viral vector and contains a nucleotide sequence encoding an antigenic site of the disease of interest, which loses its replicative properties. Inactivation is achieved by physical or chemical methods known in the art, preferably chemical inactivation with formaldehyde or beta-propiolactone (Office International des epizootics (2008). In contrast, live virus refers to a virus that retains its replication ability.

The viral vector, preferably selected from those of adenovirus or of the Paamavirus (paramixovirus), has been inactivated and inserted with at least one foreign nucleotide sequence encoding at least one antigenic site of the disease of interest, preferably at least one foreign nucleotide sequence selected from influenza, infectious laryngotracheitis, infectious bronchitis, bursal infection (Gumboro), hepatitis, viral rhinotracheitis, influenza, Mycoplasma pneumoniae, Pasteurella disease, Porcine Respiratory and Reproductive Syndrome (PRRS), circovirus, Bordetella disease, parainfluenza virus, or any other antigen having a size capable of being inserted into a corresponding viral vector. More preferably, an antigen selected from the group consisting of avian influenza, laryngotracheitis, infectious bronchitis, bursal infection (Gumboro), hepatitis, PRRS, and circovirus may be used.

In a particular embodiment of the invention, said foreign nucleotide sequence consists of a gene encoding avian influenza virus Hemagglutinin (HA) selected from the 16 hemagglutinin subtypes or immunogenic variants of this influenza virus, more preferably encoding at least one of the H1, H2, H3, H5, H6, H7 or H9 subtypes of said protein.

With respect to the viral vector of the present invention, in a preferred embodiment, the newcastle disease virus (rNDV) corresponds to a viral vector having an inserted foreign nucleotide sequence, which is preferably selected from vaccine strains such as LaSota, ullster, QV4, B1, CA 2002, Roakin, Komarov, Clone 30, VGGA strains, or strains from newcastle disease genetic groups I to V. Preferably, the recombinant virus is a LaSota strain (rNDV/LS).

Likewise, in another embodiment, the viral vector is an adenovirus selected from the group consisting of avian and porcine adenoviruses, more preferably from avian adenovirus type 9 (rFAdV/9) and porcine adenovirus type 5 (rSAdV/5).

As far as antigenic sites are concerned, when influenza is the disease of interest, antigenic sites corresponding to the avian influenza Hemagglutinin (HA) protein are preferred, preferably from the gene of the avian influenza virus, and encoding any of the existing 16 subtypes, preferably H5, H7 and H9, preferably encoding subtype H5, which is preferably obtained from: live, 435 and Viet (VT) strains, as described below. In this connection it can be pointed out that the source strain of the gene encoding the HA subtype H5 is not critical to the present invention, since experiments have shown that any strain can provide the genetic material for the purposes of the present invention.

As a preferred gene source, it is worth mentioning that the H5 gene from the live strain corresponds to VIABP-H5N2 isolated from young chicken biological specimens in Mexico in 1994, which was identified by the Mexico government as (A/chicken/Mexico/232/CPA). This virus strain has been approved for large-scale production of emulsified inactivated vaccines by "Secretar ia de Agricultura, Ganader ia, Desarrolol Rural, Pesca y Alimentaci Lou (SAGARA)", and therefore, reconstitution of the virus with the target gene also ensures the safety of the recombinant vaccine of the invention.

A second preferred genetic material is the H5-435 gene from VIABP-H5N2, isolated in 2005 from young chicken biological specimens in Mexico.

The viral vector of the vaccine of the invention can be prepared as follows: the nucleotide sequence of interest is PCR amplified, an antigenic site is identified from an isolate of the starting pathogen, inserted into the viral vector (preferably selected from adenovirus or Baramella virus), and amplified in the vector. The insertion is effected using conventional molecular biology techniques, such as restriction enzymes and DNA ligases. The resulting infectious clone is introduced into a cell line used for the preparation of the recombinant virus according to the viral vector.

Depending on the nature of the viral vector, the virus is replicated in any suitable growth system, such as SFP chicken embryos, or commercially available cell lines, or systems specifically designed for culturing the virus.

The concentration of virus required to achieve an antigenic response is preferably 10²-10¹⁰DI 50%/ml, depending on the viral vector used, virus was inactivated once this concentration was reached. Preferably, the inactivation is performed by physical or chemical methods known in the art, preferably chemical inactivation with formaldehyde or beta-propiolactone.

The pharmaceutically acceptable vehicle for the vaccines of the present invention is preferably an aqueous solution or emulsion. More preferably, a water-in-oil emulsion vehicle is employed. The specific formulation of the vaccine depends on the viral vector used, as well as the foreign nucleotide sequence inserted. However, in a preferred embodiment, when the viral vector is Newcastle disease virus, a preferred dose is 10⁴-10¹⁰DIEP 50%/ml. In embodiments where an adenovirus is used as the viral vector, a preferred dose is 10²-10⁸DIEP50％/ml。

For vaccine administration, subcutaneous administration in the middle of the back of the neck of the bird is preferred. The vaccine of the present invention is administered to poultry, such as tender chicken, hen, breeding hen, turkey, chicken drumstick, guinea fowl, partridge, quail, duck, goose, swan or ostrich. Preferably, the vaccine is administered subcutaneously, but in some species, administration can be intramuscularly to avians of any age.

When the vaccine is administered to chickens emulsified in a newcastle disease carrier, the vaccine preferably contains 10⁸-10⁹DIEP 50%/0.5 ml/chicken, more preferably 10%^8.5DIEP 50%/0.5 ml/chicken. For small 10-day-oldThe chickens can be easily inoculated.

The present invention provides a very important competitive advantage. The recombinant inactivated vaccine of the present invention makes it possible to establish a vaccination plan with only a recombinant vaccine inserted with genes from a pathogenic organism that is difficult to control in a viral vector, which results in a method of differentiating infected animals from vaccinated animals alone (DIVA), which can be effectively used for the control and eradication of disease, comprising:

a) subjecting at least one animal receiving a recombinant vaccine inactivated with a viral vector having an inserted foreign nucleotide sequence encoding an antigen of a pathogenic pathogen to a first antibody detection method in at least one sample to detect the presence of antibodies corresponding to said antigen in said sample;

b) subjecting at least one sample of the same animal from which the first antibody test has been taken to a second antibody test method to detect the presence in said sample of antibodies corresponding to the pathogen causing the disease;

c) determining whether the animal is infected or vaccinated based on the results of the first and second antigen detection methods.

For example, when pathogens that are difficult to control, such as VIA, primarily H5 and H7, cause high mortality in poultry, very excellent systemic protection can be achieved with the recombinant inactivated vaccines of the present invention, with greater biosafety than if IA whole virus were used, but not properly inactivated, causing significant risk. This risk will increase during large scale production or virus production. The present invention also enables to distinguish vaccinated birds from other birds exposed to whole virus at the epidemiological level (DIVA system) since the laboratory test for detecting vaccine-induced antibodies against avian influenza virus is Hemagglutination Inhibition (HI) when only the avian influenza virus Hemagglutinin (HA) gene is inserted. Existing immunological tests, such as ELISA, as well as other tests such as agar gel diffusion, etc., are all negative in detecting anti-avian influenza antibodies induced by the recombinant vaccines of the present invention, as they are designed to detect antibodies induced by other antigens in whole virus. When birds vaccinated with the recombinant vaccine of the invention are infected with wild virus, these tests are positive for antibody detection against avian influenza, so that infected birds can be distinguished.

Accordingly, the present invention allows the establishment of a combined program using only inactivated and live recombinant vaccines, the former will produce the above mentioned systemic immunity, while the recombinant live vaccines will complement the immunity at the mucosal level, ultimately producing protection at or close to the 100% wild level. In this project, the above-mentioned DIVA system is also used.

In a preferred embodiment of the invention, the recombinant vector of the emulsion inactivated vaccine is newcastle disease inserted with influenza genes for both VVENC and VIAAP challenge, and live vaccines with the same vector and antigen can be administered simultaneously, directly to the respiratory mucosa, by ocular route, spray, or in drinking water, such that the local response level is greatly stimulated (at the respiratory and digestive mucosa) to produce secretory immunoglobulin a (iga), thereby significantly reducing wild-type virus replication and thus its detoxification and transmission.

On the other hand, the vaccine of the invention allows to establish a control program, making it possible to achieve eradication by differentiating between vaccinated and infected birds, since it is possible to differentiate vaccinated birds from birds infected with wild-type virus when administering the recombinant inactivated vaccine of the invention (DIVA system), since the recombinant vaccine contains only VIA hemagglutinin as antigen, allowing to apply diagnostic tests such as ELISA, which detect antibodies induced by other viral antigens, not only by hemagglutinin.

The recombinant vaccines against influenza of the present invention are more clearly illustrated by the following specific examples, which are provided for illustrative purposes only and are not intended to limit the present invention.

Examples

Example 1

Generation of Newcastle disease-LaSota vector

In order to clone the genome of the newcastle disease virus LaSota strain and thus prepare a viral vector, an intermediate vector "pNDV/LS" is first prepared. The total virus RNA of the Newcastle disease LaSota strain is extracted by a triazole method. cDNA (complementary DNA) was synthesized from the purified RNA of the viral genome using the previously purified total RNA as a template. To clone all genes of the newcastle disease genome (15, 183 base pairs (bp)), 7 fragments with "overlapping" ends and cohesive restriction sites were amplified by PCR. Fragment 1(F1) comprises nucleotides (nt)1-1755, F2 comprises nt 1-3321, F3 comprises nt 1755-6580, F4 comprises 6, 151-10, 210, F5 comprises nt 7, 381-11, 351, F6 comprises 11, 351-14, 995, F7 comprises nt 14, 701-15, 186. The 7 fragments were assembled by standard ligation techniques in the cloning vector pGEM-T to reconstitute the Newcastle disease LaSota genome, which after this cloning had only a single restriction site SacII, located between the P and M genes, and served to clone any gene of interest into the viral region of the vector.

Example 2

Cloning of HA Gene from VIA subtype H5N2435 Strain 435

Total viral RNA was extracted by triazole method to clone the HA gene of VIA 435 strain. cDNA (complementary DNA) was then synthesized from this purified total RNA and the HA gene of IA virus was amplified by PCR technique using specific oligonucleotides. The HA gene of 435 was then inserted into the pGEM-T vector using standard cloning techniques, resulting in a plasmid: p-GEMT-435.

Example 3

The IA 435HA gene carrying the SacII site of the pNDV/LS vector was cloned to make a plasmid: pNDV/LS-435

A. Preparing an intermediate vector pSacIIGE/GS:

the intermediate vector pSacIIGE/GS was constructed to introduce the transcriptional sequence GE/GS of Newcastle disease at the 5' end of the HA435 gene by initial PCR amplification of the GE/GS sequence using the Newcastle disease genome as a template and then inserting these sequences into pGEM-T.

B. Subcloning the HA gene into the pSacIIGE/GS vector:

the plasmid pGEMT-435 was digested with Hpal-Ndel and cloned into pSacIIGE/GS to give plasmid pSacIIGE/GS-HA 435.

C. Subcloning GE/GS-HA435 into pNDV-LS vector

Two plasmids: both pSacIIGE/GS-HA435 and pNDV/LS were digested with SacII, the digests were purified, the GE/GS-HA435 region was purified and inserted into the SacII site of pNDV/LS, resulting in infectious clones: pNDV/LS-435.

Example 4

Production of recombinant Virus rNDV/LS-HA435 in cell culture

Hep-2 and A-549 cells were initially infected with MAV-7 virus at a multiplicity of infection (MOI) of 1. 5% CO at 37 ℃₂After 1 hour of incubation in the environment of (1), cells were transfected with 1 microgram (. mu.g) of DNA cloned from pNDVLS-435 and 0.2. mu.g of DNA from the expression plasmids pNP, pP and pL (coding for the viral proteins P, NP and L, respectively, which are required for recombinant production in the two cells). 12 hours after transfection, recombinant viruses produced in these two cells were harvested and injected into 10-day-old SPF chick embryos to amplify the resulting viruses. After 48 hours, allantoic fluid was harvested and titrated with Vero cells on a plate, thereby producing the final recombinant virus for use in vaccine preparation.

Recombinant viruses with genes from the live and Viet strains were prepared as described above.

Example 5

A method for producing an emulsion inactivated vaccine by using a Newcastle disease LaSota recombinant virus rNDV/LS-H5 with an avian influenza virus H5 insert comprises the following steps:

generation of antigens

From production breeding, chick embryo zygotes without Specific Pathogen (SPF) are inoculated with a predetermined infection dose. Embryos were incubated at 37 ℃ for 72 hours and checked daily for mortality. After this period, the live embryos are refrigerated for 1 day, preferably 24 hours, and amniotic Fluid (FAA) is aseptically harvested. The FAA is clarified by centrifugation and inactivated with formaldehyde, although any other known inactivating agent commonly used in the preparation of such vaccines may be used. FAA was tested to confirm its inactivation, and to confirm its purity, sterility, DIEP titer and HA titer.

Preparation of the emulsion

The vaccine is made into a water-in-oil emulsion. Mineral oil and surfactants Span 80 and Tween 80 were used in the oil phase. To prepare the aqueous phase, FAA was mixed with a preservative solution (thimerosal). To prepare the emulsion, the aqueous phase was slowly added to the oil phase with constant stirring. A homogenizer or colloid mill is used to achieve a specific particle size.

Antigen content

Formulating the vaccine to produce a minimum of 10^8.5DIEP 50%/0.5 ml so that a dose of 0.5 ml/bird was used.

According to the method, 6 kinds of recombinant viruses are prepared: three with the newcastle disease LaSota strain vector (rNDV/LS) and with the HA anchor of the IA virus (called Rd), and three with the same vector but without the anchor (called Re); the Rd and Re genomes were cloned with three different HA genes, respectively, to obtain 6 vaccines:

h5-live gene: obtained from strain VIABP subtype H5N2 (A/chicken/Mexico/232/CPA) was isolated in 1994 from a biological specimen of a young chicken in Mexico, corresponding to the virus strain approved by SAGARPA for the production of an emulsion inactivated vaccine.

H5-435 gene: the isolation from the VIAAP subtype H5N2 was isolated in 2005 from young chicken biological specimens in Mexico. Strain 435 showed different antigenic properties from the live strain in Hemagglutination Inhibition (HI) experiments, as well as important changes in nucleotide sequencing.

H5-Vt gene: the H5 gene isolated from vietnam, corresponding to the IA virus subtype H5N 1.

Example 5A

An emulsion inactivated recombinant experimental vaccine with the anchor (Rd) and H5-live genes in the (rNDV/LS) vector was prepared as described in example 5 and formulated as a water/oil pharmaceutical formulation, designated EmiRd-live.

Example 5B

An emulsion inactivated recombinant experimental vaccine with the anchor (Rd) and H5-435 genes in the (rNDV/LS) vector was prepared as described in example 5 and formulated as a water/oil pharmaceutical formulation, designated EmiRd-435.

Example 5C

An emulsion inactivated recombinant experimental vaccine with an anchor and the H5-Vt gene in a (rNDV/LS) vector was prepared as described in example 5 and formulated in an aqueous/oil pharmaceutical formulation, designated Emi Rd-Vt.

Example 5D

An emulsion inactivated recombinant experimental vaccine without anchor (Rd) and H5-live genes in the (rNDV/LS) vector was prepared as described in example 5 and formulated as a water/oil pharmaceutical formulation, designated Emi Re-live.

Example 5E

An emulsion inactivated recombinant experimental vaccine without anchor (Rd) and H5-435 genes in the (rNDV/LS) vector was prepared as described in example 5 and formulated as a water/oil pharmaceutical formulation, designated EmiRe-435.

Example 5F

An emulsion inactivated recombinant experimental vaccine without anchor and H5-Vt gene in (rNDV/LS) vector was prepared as described in example 5 and formulated as an aqueous/oil pharmaceutical formulation, designated EmiRe-Vt.

Example 6

Evaluation of in vivo efficacy of recombinant vaccines in ENC-LaSota vectors with and without Anchor to IA Virus HA Gene

To determine the efficacy of the emulsified recombinant inactivated vaccines of the invention and to demonstrate that these efficacies can be achieved with different cloned hemagglutinin genes of different antigenic subtypes and variants of the IA virus, the efficacy of these vaccines to prevent death caused by IAAP virus subtypes H5N2 and VVENC in SPF birds, as well as in commercially available young chickens with maternal immunity against VIA and ENCV, was tested.

The strains used in the different challenge experiments to measure vaccine efficacy were as follows:

1. avian influenza (VIAAP-H5N 2):highly pathogenic virus subtype H5N2, A/chicken/Quer é taro/14588-19/95 strain, titer 10^8.0DIEP 50%/ml, equivalent to 100DLP 50%/0.3 ml/chicken.

VVENC virus:chimalhuacan strain contains 10^8.0DIEP 50%/ml, equivalent to 10^6.5DIEP 50%/0.03 ml/chicken.

The challenge was performed on 35 day old chicks (-21 days after DPV-inoculation) in an acrylic isolation unit of INIFAP-CENID-Microbiolia (biosafety level 3). To carry out the challenge, each experimental group was further divided into two subgroups, each of which was assigned to a respective isolation unit according to a pre-established biosafety procedure.

VIAAP-H5N2 was diluted 1: 10 with PBS pH 7.2, 0.06ml (2 drops) per chick was administered to each eye and 0.09ml (3 drops) was administered to each nostril, corresponding to 0.3ml or 100DLP 50%.

Each chicken was challenged with ocular administration of VVENC virus using 0.03ml of a virus suspension containing 10^8.5DIEP 50%/ml, equivalent to 10^6.5DLP 50%/birds.

For post-challenge (PD) evaluation, all groups were checked daily to record mortality and morbidity, including clinical symptom severity, and each chicken in each group was monitored on each PD day (DPD) and assigned a value according to the criteria of table 1:

TABLE 1 mortality and morbidity scores

Clinical symptoms	Light and slight	Severe severity of disease
			No obvious clinical manifestation	0
Conjunctivitis	1	2
			Conjunctivitis and feather uprightness	3	4
Conjunctivitis, feather uprightness, fatigue (exhaustion)	5	6
			Death was caused by death	7

PD evaluation was performed for 14 days with VVENC and 10 days with VIAAP-H5N2 as recommended by OIE.

The incidence Index (IM) of each group was calculated using the equation obtained from the data corresponding to the day of the most severe clinical symptoms during the observation period of PD

Wherein

A-sum of all lesion severity scores on the day of observation

B-the maximum possible severity score for the clinical disease during the day.

These experiments were performed as follows:

example 6A

Groups of SPF birds were challenged with VVENC and VIAAP-H5N2 at 21DPV after immunization with the inactivated vaccines of the invention, prepared as described in examples 5A (EmiRd-Bive), 5B (Emi Rd-435) and 5C (Emi Rd-Vt), as shown in Table 2. For comparison, two other groups were immunized with two emulsified commercial vaccines against avian influenza and newcastle disease, prepared from the emulsified and inactivated whole virus e.enc/AI-435 and e.enc/AI-live, respectively.

TABLE 2 efficacy in SPF birds immunized with inactivated vaccine prepared with recombinant virus rNDV/LS-H5 with Anchor (Rd)

The efficacy results for VVENC and VIAAP-H5N2 are shown in FIGS. 1 and 2, respectively.

The results show that all three recombinant inactivated vaccines rNDV/LS-H5 of the present invention with anchors provide 100% protection against mortality (M) caused by VVENC challenge virus in SPF chicks. Likewise, and regardless of which H5 gene they were cloned from, all three recombinant inactivated vaccines also provided 100% protection against mortality (M) due to VIAAP-H5N2 (FIG. 2), which is equivalent to conventional inactivated vaccines made with whole viruses currently approved worldwide for control of ENC and IA, which typically include 10^8.6DIEP 50%/ml Newcastle disease LaSota strain Virus, 10^8.0DIEP 50%/ml low pathogenic avian influenza virus, chemically inactivated with formaldehyde and emulsified with oil. The results of said protection show that the recombinant inactivated vaccine rNDV/LS-H5 with the anchor meets the criteria for use in Mexico and internationally for the control of ENC and IA, and that this recombinant form with the anchor according to the invention has proved successful.

Example 6B

The second experimental design was to determine the effect of the anchors, groups of SPF birds challenged with VVENC and via ap-H5N2 at 21DPV after immunization as shown in table 3 with the following vaccines: two emulsified commercial anti-influenza and newcastle disease vaccines made with emulsified and inactivated whole viruses e.enc/AI-435 and e.enc/AI-Bive, and three emulsified and inactivated vaccines of the invention without anchors made from examples 5D (Emi-Re-Bive), 5E (Emi-Re-435) and 5F (Emi-Re-Vt).

Table 3-efficacy in SPF birds immunized with inactivated vaccine made with recombinant virus rNDV/LS-H5 without anchor (Re).

The efficacy results for VVENC and VIAAP-H5N2 are shown in FIGS. 3 and 4, respectively.

Surprisingly, these results show that all three recombinant inactivated vaccines of the invention, rNDV/LS-H5, without anchor, also provide 100% protection against the mortality (M) caused by VVENC challenge virus in SPF-chickens. Likewise, and regardless of which H5 gene they were cloned from, all three recombinant inactivated vaccines also provided 100% protection against mortality (M) by VIAAP-H5N2, which is equivalent to recombinant inactivated vaccine rNDV/LS-H5 with anchors, and emulsified conventional vaccines made with whole virus inactivated vaccines ENC/AI-Bive and ENC/AI-435.

The results of examples 6A and 6B show that recombinant inactivated vaccines with or without anchors in the vector with VIA H5 genes (H5N2 or H5N1) of different origin and with different antigenic properties in the HI test both provide the same protection against the VIA ap-H5N2 challenge. These results suggest that recombinant inactivated vaccines made with any of the VIA H5 genes can provide protection against challenge with any of the influenza virus subtypes VIA ap with hemagglutinin H5, regardless of the type of neuraminidase.

This thus indicates that the present invention may effectively comprise different types of neuraminidases, consistent with the findings in a traditional Inactivated whole virus vaccine (Soto et al, Inactivated mexican H5N2 aversion in vaccine technologies from the antibacterial high strain genetic H5N1 aversion viruses. proceedings of the 56th Western strain association (WPDC), USA, p.79.. 2007 and Swayne, d.and Kapczynski, d.2008. Vaccines, Vaccination and immunization for expression viruses. in. avanza. b.e. bright. 407, USA).

Example 6C

The third experiment was to test the stimulation of wild-type disease in commercial avians by the vaccine of the invention, in which commercial young chickens with maternal immunity against ENC and IA were challenged with VVENC and via ap-H5N2 at 21DPV, which were first immunized with the following vaccines as shown in table 4: two emulsified commercial anti-influenza and newcastle disease vaccines made with emulsified and inactivated whole viruses E.ENC/AI-435 and E.ENC/AI-Bive, and inactivated vaccines of the invention made from examples 5A (Emi-Rd-Bive), 5B (Emi-Rd-435) and 5C (Emi-Rd-Vt).

TABLE 4 efficacy in commercial chickens with maternal immunity against ENC and IA that have been immunized with an inactivated vaccine made with recombinant virus rNDV/LS-H5 with Anchor (Rd)

The efficacy results for VVENC and via ap-H5N2 are shown in figures 5 and 6, respectively.

These results indicate that all three recombinant inactivated vaccines of the invention, rNDV/LS-H5, with anchors, are able to provide more than 90% protection against VVENC challenge virus-induced mortality (M) in commercial young chickens with maternal immunity against ENC and IA (FIG. 5). Furthermore, regardless of which H5 gene they were cloned from, all three recombinant inactivated vaccines also provided more than 80% protection against mortality (M) due to VIAAP-H5N2, which is equivalent to conventional emulsion vaccines made with inactivated whole virus for the control of ENC and IA.

These protection results indicate that the recombinant inactivated vaccine with anchor, rNDV/LS-H5, of the present invention, can be successfully used to control IAAP in commercial young chickens with maternal immunity against IA and ENC viruses, with protection similar to that provided by conventional vaccines made with inactivated whole virus IA, but with the additional advantage of complete biosafety due to the administration of only recombinant live and inactivated vaccines, and the ability to establish DIVA systems to both apply vaccination programs and eradicate IA.

Example 6D

To determine the effect of the anchors in the real wild environment, commercial young chickens with maternal immunity against ENC and IA were challenged with VVENC and via ap-H5N2 at 21DPV, which were first immunized with the following vaccines as shown in table 5: two emulsified commercial anti-influenza and newcastle disease vaccines made with emulsified and inactivated whole viruses e.enc/AI-435 and e.enc/AI-Bive, and three emulsified and inactivated vaccines of the invention without anchors made from examples 5D (Emi-Re-Bive), 5E (Emi-Re-435) and 5F (Emi-Re-Vt).

TABLE 5 efficacy in commercial broilers with maternal immunity against ENC and IA, immunized with an inactivated vaccine made with the recombinant virus rNDV/LS-H5 without Anchor (Re)

The efficacy results for VVENC and VIAAP-H5N2 are shown in FIGS. 7 and 8, respectively.

Surprisingly, these results show that all three recombinant inactivated vaccines of the invention, rNDV/LS-H5, without anchors, also provide more than 90% protection against mortality (M) from VVENC challenge virus in commercial young chickens with maternal immunity against ENC and IA (FIG. 7), and that, as well, and regardless of which H5 gene all three recombinant inactivated vaccines were cloned from, they also provide more than 80% protection against mortality (M) from VIAAP-H5N2, which is equivalent to recombinant inactivated vaccine rNDV/LS-H5 with anchors, and to conventional emulsion vaccines made with inactivated whole viruses ENC/AI-live and ENC/AI-435.

These studies confirm the success of the present invention in that it has been demonstrated that inactivated forms of recombinant vaccines against IA, when used in emulsion or in pharmaceutically acceptable vehicles, adjuvants or excipients in susceptible birds, produce very good immune responses, which are capable of providing 100% protection against via ap challenge in SPF chicks and greater than 80% protection in young chickens with maternal immunity against ENC and IA, which is completely contrary to the previously thought viewpoint that replication of recombinant viruses in immunized birds is necessary for adequate expression of the target protein for appropriate immune response in the bird, and is completely beyond this previous viewpoint

The use of inactivated vaccines is essential to provide adequate protection at the wild level against mortality caused by VIAAP and VVEENC, since in the poultry development industry under wild conditions, the use of only traditional live vaccines against ENC, or recombinant live vaccines against IA, may not be sufficient.

While specific embodiments of the present invention have been illustrated and described herein, it will be appreciated that many modifications may be made to them, such as the IA virus or adenovirus strain used, the type of emulsion used or the vehicle used. Accordingly, the invention is not to be construed as limited except by the prior art teachings and by the appended claims.

Claims (25)

1. A recombinant vaccine comprising a viral vector and a pharmaceutically acceptable vehicle, adjuvant or vehicle, characterised in that the viral vector is inactivated and has an inserted foreign nucleotide sequence encoding an antigen of a disease of interest.

2. The recombinant vaccine of claim 1 further characterized in that said foreign nucleotide sequence encodes an antigen selected from the group consisting of: influenza, infectious laryngotracheitis, infectious bronchitis, bursal infection (Gumboro), hepatitis, viral rhinotracheitis, influenza, Mycoplasma hyopneumoniae, Pasteurella disease, Porcine Reproductive Respiratory Syndrome (PRRS), circovirus, Bordetella disease or parainfluenza virus.

3. The recombinant vaccine of claim 2, further characterized in that said exogenous nucleotide sequence consists of a gene encoding avian influenza virus Hemagglutinin (HA).

4. The recombinant vaccine of claim 3, further characterized in that said gene encoding Hemagglutinin (HA) is selected from at least one of avian influenza virus Hemagglutinin (HA) subtype H1, H2, H3, H5, H6, H7, or H9.

5. The recombinant vaccine of claim 4 further characterized in that said gene encoding Hemagglutinin (HA) is subtype H5.

6. The recombinant vaccine of claim 1 further characterized in that said viral vector is selected from the group consisting of adenovirus or braamigo virus.

7. The recombinant vaccine of claim 6 further characterized in that said viral vector is selected from the group consisting of a Baramege virus.

8. The recombinant vaccine of claim 7, further characterized in that said rhamanovirus is a newcastle disease virus.

9. The recombinant vaccine of claim 8, further characterized in that said newcastle disease virus is selected from the group consisting of LaSota, ullster, QV4, B1, CA 2002, Roakin, Komarov, Clone 30, or VGGA strains, or strains from newcastle disease genetic groups I to V.

10. The recombinant vaccine of claim 6 further characterized in that said viral vector is selected from the group consisting of adenoviruses.

11. The recombinant vaccine of claim 10 further characterized in that said adenovirus is selected from the group consisting of avian or porcine adenovirus.

12. The recombinant vaccine of claim 11 further characterized in that said adenovirus is avian adenovirus type 9.

13. The recombinant vaccine of claim 11 further characterized in that said adenovirus is porcine adenovirus type 5.

14. The recombinant vaccine according to claim 1, further characterized in that the pharmaceutically acceptable vehicle of the vaccine is preferably an aqueous solution or emulsion.

15. The recombinant vaccine of claim 14 further characterized by the use of a water-oil emulsion as a vehicle.

16. The recombinant vaccine of claim 1, further characterized in that the desired titer of the viral vector is similar to the desired titer of a recombinant live virus vaccine.

17. The recombinant vaccine of claim 16 further characterized in that the concentration of virus required to achieve an antigenic response is 10²-10¹⁰DI50％/ml。

18. The recombinant vaccine of claim 7 further characterized in that the concentration of virus required to achieve an antigenic response is 10⁴-10¹⁰DIEP50％/ml。

19. The recombinant vaccine of claim 18 further characterized by a concentration of 10 of said virus when said vaccine is prepared for administration to chickens⁸-10⁹DIEP50％0.5 ml/chicken.

20. The recombinant vaccine of claim 19 further characterized in that said vaccine has a 10^8.5DIEP 50%/0.5 ml/chicken.

21. The recombinant vaccine of claim 10 further characterized in that the concentration of virus required to achieve an antigenic response is 10²-10⁸DIEP50％/ml。

22. The recombinant vaccine of claim 1 further characterized in that the concentration of virus required to achieve an antigenic response is that the vaccine is prepared as a subcutaneous or intramuscular vaccine.

23. A method of immunizing against a disease in an animal comprising administering to the animal a recombinant vaccine comprising an inactivated viral vector having inserted therein a foreign nucleotide sequence encoding an antigen of the disease.

24. The method of immunizing against disease in an animal according to claim 23 further characterized by also administering to said animal another recombinant vaccine comprising the same inactivated viral vector but an activated viral vector having an inserted foreign nucleotide sequence encoding an antigen of said disease.

25. A method for identifying infected and vaccinated animals useful for controlling and eradicating disease comprising:

a) subjecting at least one animal receiving a recombinant vaccine inactivated with a viral vector having an inserted foreign nucleotide sequence encoding an antigen of a pathogenic pathogen to a first antibody detection method in at least one sample to detect the presence of antibodies corresponding to said antigen in said sample;

b) subjecting at least one sample of the same animal from which the first antibody test has been taken to a second antibody test method to detect the presence in said sample of antibodies corresponding to the pathogen causing the disease;

c) determining whether the animal is infected or vaccinated based on the results of the first and second antibody detection methods.