HK1129570B - Avian influenza viruses, vaccines, compositions, formulations, and methods - Google Patents

Description

Avian influenza virus, vaccine, composition, preparation and method

Cross Reference to Related Applications

This application claims priority from U.S. patent application Ser. No. 11/737,104, applied on day 4, 18, 2007 and U.S. provisional patent application Ser. No. 60/794,054, applied on day 4, 21, 2006, the disclosures of both of which are incorporated herein by reference in their entirety.

Background

Field of the invention

The present invention relates generally to influenza vaccines, and more particularly to avian influenza vaccines and formulations thereof for vaccinating susceptible birds. The present invention also relates to novel methods for preventing or ameliorating avian influenza viral diseases in poultry.

Description of the Related Art

The most well-known of the influenza viruses are influenza a and influenza b, which are important causes of morbidity and mortality worldwide, causing outbreaks each year. Pandemics occur periodically, but at irregular intervals, causing particularly high morbidity and mortality. Historically, the pandemics were the result of new subtypes of influenza a virus, resulting from segmental genome reassortment (antigenic shift), while annual epidemics were generally the result of the evolution of surface antigens (antigenic drift) of influenza a and b viruses. Human influenza viruses usually originate from avian influenza virus strains, so influenza infection is based on zoonosis. There is also evidence that pigs can serve as intermediate hosts ("mixed-channel") for the production of novel avian strains pathogenic to humans (Scholtissek et al, Virology 1985, 147: 287). The H5N1 influenza A outbreak in hong Kong 1997 indicates that highly pathogenic influenza A viruses can also be transmitted directly from birds to humans (Claas et al, Lancet1998, 351: 472; Suarez et al, J.Virol.1998, 72: 6678; Subbarao et al, Science 1998, 279: 393; Shortridge, Vaccine 1999, 17 (supplement 1): S26-S29). In 2003, the H5N1 virus in southeast Asia contained different co-circulating genotypes, but in 2004, only one genotype (called the "Z-genotype") predominated (Li et al, Nature 2004, 430: 209).

Current evidence suggests that the human lethal event is due to direct transmission of the genotype from bird to human, and that it also infects cats, transmitted directly from cat to cat (Kuiken et al, Science 2004, 306: 241). These and other evidence for changes in the host range and spread distribution of the virus have been associated with the potential for the H5N1 virus to acquire properties that allow human-to-human spread. Humans are not immune to such novel H5N1 viruses and can therefore cause a catastrophic influenza pandemic (Fouchier et al, Nature 2005, 435: 419). The potential of influenza a viruses to generate new pathogenic strains from a large number of circulating strains in animal hosts suggests that disease control requires monitoring of these viruses and the development of improved antiviral therapies and vaccines. In such monitoring work, there is a need for vigilance of the speed of development of new strains of virus, including improved techniques for assessing the efficacy of vaccines against new strains.

Avian influenza, also known as "AI", is an acute, highly infectious viral infection of chickens and other poultry. Influenza viruses can be classified into different subtypes according to antigenic differences between hemagglutinin (HA; also abbreviated as H) and neuraminidase (NA; also abbreviated as N) molecules, which can be "reassorted" or "mutated" in different seasons. Because the virus is constantly mutated, it is not possible to predict which strains will appear in the next season, and thus vaccines are difficult to prepare. Vaccine preparation using strains under production conditions usually cannot propagate rapidly, so if waiting for a particular strain to appear and then reproducing the correct vaccine for protection against that strain, such a solution is not feasible. Typically, the prevalence of a particular strain will last for months and then may disappear for years.

Influenza viruses can be classified into a type a, a type b and a type c according to their group antigens (group antigens). Influenza a, b and c viruses differ by antigenic differences in the viral Nucleocapsid (NP) and matrix (M) proteins. Influenza a viruses are classified into different subtypes according to antigenic differences of Hemagglutinin (HA) and Neuraminidase (NA). 9 subtypes of neuraminidase NA protein (termed NA1 to NA9) and 15 different subtypes of serum hemagglutinin HA protein (termed HA1 to HA15) have been identified. In birds, viruses have been isolated that each carry a distinct HA (or H) and NA (or N) subtype.

Influenza a, b and c in the Orthomyxoviridae (Orthomyxoviridae) all have segmented negative strand RNA genomes, replicate in the nucleus of infected cells, have a combined coding capacity of about 13kb, and contain genetic information for 10 viral proteins. Specifically, Influenza Viruses have 8 negative sense RNA (nsrna) gene segments encoding at least 10 polypeptides, including RNA-guided RNA polymerase proteins (PB2, PB1, and PA), Nucleoprotein (NP), Neuraminidase (NA), hemagglutinin (HA, which is enzymatically associated with subunits HA1 and HA 2), matrix proteins (M1 and M2), and nonstructural proteins (NS1 and NS2) (Krug et al, The inflenza Viruses, r.m. Krug, Plenum Press, New York, 1989, pp 89-152).

A reverse genetics system developed to date allows manipulation of the influenza genome (Palese et al, Proc. Natl. Acad. Sci. USA 1996, 93: 11354; Neumann and Kawaoka, adv. Virus Res.1999, 53: 265; Neumann et al, Proc. Natl. Acad. Sci. USA 1999, 96: 9345; Fodor et al, J. Virol.1999, 73: 9679). For example, plasmid-driven expression of 8 influenza nsRNAs from the pol I promoter and co-expression of the polymerase complex protein has been shown to result in the formation of infectious influenza A virus (Hoffmann et al, Proc. Natl. Acad. Sci. USA 2000, 97: 6108).

The influenza virus has a virion size of about 125nm and consists of a negative sense viral RNA core associated with a nucleoprotein, and a viral envelope surrounding a lipid bilayer structure. The inner layer of the viral envelope is composed primarily of matrix proteins, and the outer layer contains most of the lipid material from the host. The so-called "surface proteins", Neuraminidase (NA) and Hemagglutinin (HA) are nailed on the surface of the virus. Infectivity of new influenza viruses depends on cleavage of HA by specific host proteases, while NA is involved in the release of progeny virions from the cell surface and can prevent aggregation of newly produced viruses.

The HA and NA proteins embedded in The viral envelope are The major antigenic determinants of Influenza viruses (Air et al, Structure, Function, and Genetics, 1989, 6: 341-356; Wharton et al, eds. The Influenza Virus, R.M. Krug, Plenum Press, New York, 1989, pp. 153-174). Because of influenza segmental genome reassortment (reassortment), new HA and NA variants are constantly being generated and newly infected organisms do not have a memory immune response to them. The HA glycoprotein is the primary antigen of neutralizing antibodies and is involved in the binding of viral particles to host cell receptors.

HA molecules of different virus strains show significant sequence similarity at both the nucleic acid and amino acid levels. This level of similarity is different when strains of different subtypes are compared, and some strains clearly show a higher level of similarity than others (Air, proc. natl. acad. sci. usa, 1981, 78: 7643). The level of amino acid similarity between strains of one subtype and strains of the other subtype differs (Air, proc. Natl.Acad.Sci.USA, 1981, 78: 7643). This variation is sufficient to establish an evolutionary lineage of discrete subtypes and different strains, but the DNA and amino acid sequences of different strains can still be readily aligned using conventional bioinformatics techniques (Air, proc. natl. acad. sci. usa, 1981, 78: 7643; Suzuki and Nei, mol. biol. evol.2002, 19: 501).

HA is a viral surface glycoprotein consisting of about 560 amino acids, representing 25% of the total viral protein. It is primarily responsible for adhesion of viral particles at the early stage of infection and penetration of host cells. Among viral proteins, hemagglutinin undergoes mainly post-translational rearrangement. After hemagglutinin synthesis is complete, the molecule follows the extracellular pathway of the host cell (HA folds along this pathway), assembles into trimers and glycosylates. Finally, HA is cleaved into two subunits, Hi and H2; this activates the molecule and increases the infectivity of the viral particle.

For many avians, differences in basic amino acid sequence within the cleavage site are correlated with the ability of avian influenza viruses to produce either locally symptomatic infections or systemic infections with lethal consequences. It is therefore proposed that this feature may be important in affecting the organ tropism, host specificity and pathogenicity of the virus. In terms of pathogenicity of the virus, strains with a multi-base site HA find proteases that cleave the HO molecule (Hi and H2 in active state) in several cell types, thus generating multiple infection cycles, producing large quantities of infectious viral particles and causing widespread infection in all areas in a short time (HPAI strain). Thus, the infection has an acute-over-acute course with a high mortality rate.

Neuraminidase (NA) is the second membrane glycoprotein of influenza a virus. NA is a protein of 413 amino acids, encoded by a gene of 1413 nucleotides. NA is involved in disrupting the cellular receptor of viral hemagglutinin, i.e., by cleaving the sialic acid molecule from the hemagglutinin itself. It is believed that this may facilitate release of viral progeny by preventing newly formed viral particles from accumulating in the cell membrane, and by facilitating transport of the virus through the mucus at mucosal surfaces. NA is an important antigenic determinant, which may undergo antigenic variation.

Currently the influenza vaccines approved by the public health authorities for use in the united states and europe are inactivated influenza vaccines as well as attenuated flu live vaccines in the united states. Epidemiologically important influenza a and influenza b virus strains are cultured in chicken embryos, and the virus particles are then purified and chemically inactivated to obtain a vaccine stock. The World Health Organization (WHO) selects the most likely popular subtype for vaccine development each year.

Although influenza vaccines have been used for vaccination of humans since the early 1940 s and horses since the late 1960 s, the development of new therapies against viruses has been prompted due to the presence of a wide range of animal hosts and the threat of new influenza viruses that can cause pandemics. Several important advances have been made in the influenza field over the last years (for reviews see Cox and Subbarao, Lancet1999, 354: 1277-82). For example, an experimental, intranasally administered trivalent attenuated live influenza vaccine has shown high efficacy in protecting young children from influenza a H3N2 and influenza b infection. Other methods of improving the efficacy of current (dead) influenza virus vaccines also include the generation of cold-adapted and genetically engineered influenza viruses containing specific attenuating mutations (for review see palette et al, j.infact.dis., 1997, supplement 176: S45-9). It is expected that these genetically altered viruses, in which the HA and NA genes from an epidemic strain are recombined, can be used as safe live influenza virus vaccines to induce a long-lasting protective immune response in humans. Although cold-adapted vaccines appear to be effective in children and young adults, they are too attenuated to stimulate the desired immune response in the elderly, and 20000-40000 die annually in the united states from influenza infection.

A readily available vaccine would provide the most effective tool for emerging pandemic influenza. After the 1997 hong kong outbreak of H5N1, vaccines produced by two different methods have been tested in the population. The subunit H5 vaccine conventionally produced by A/Duck/Singapore/3/97 (A/duck/Singapore/3/97) is poorly immunogenic to humans, even against antigenically closely related strains, and still poorly after multiple vaccinations (Nicholson et al, Lancet 2001, 357: 1937; Stephenson et al, Journal of infection diseases 2005, 191: 1210). The antibody titer of this H5 Vaccine was increased using adjuvant MF59 (Stephenson et al, Vaccine2003, 21: 1687). Vaccination with an inactivated "split" vaccine from the nonpathogenic A/duck/HK/836/80 (H3N1) virus and a modified H5 hemagglutinin from the A/HK/156/97(H5N1) virus induced only detectable neutralizing antibody titers (Takada et al, Journal of Virology1999, 73: 8303). Thus, although all of these H5N1 vaccines are well tolerated, they appear to be poorly immunogenic. There is currently a lack of effective vaccines against the H5N1 virus strain, so these viruses have the threat of causing pandemic disease.

The serum antibody titer method is an acceptable alternative to determine immunoprotection following vaccination or viral infection. The mainly used methods for serum antibody titer are virus neutralization titer determination and Hemagglutinin Inhibition (HI) titer determination. These assays are based on the ability of influenza antibodies in human serum to cross-react with antigen under in vitro conditions. In certain cases, the method of measurement is selected for each type of measurement based not only on its ability to provide stable and useful results, but also on its ease of use and the requirements placed on the equipment.

Briefly, the virus neutralization assay measures the ability of antibodies in a serum sample to block infection of cultured cells by influenza virus. The assay was performed as follows: serial dilutions (titers) of serum samples were prepared and each such dilution was mixed with a standard amount of infectious virus. Each diluted mixture was then mixed with a specific cell culture and the resulting infection rate was determined. Virus neutralization titer determination is considered to be a very useful and reliable test method for detecting the level of immunoprotective antibodies present in a particular individual. However, this relies on specialized cell culture equipment and is therefore not universally applicable. The method is also laborious and time consuming and is therefore not well suited for screening large numbers of samples.

The Hemagglutinin Inhibition (HI) assay also measures the ability of antibodies in serum samples to bind to a standardized reference virus. The assay is based on the fact that: influenza viruses bind to and agglutinate erythrocytes. In the HI assay, serial dilutions of serum samples are mixed with a standard amount of reference virus and added to red blood cells after a set incubation period. The reference virus was then visually associated with the red blood cells to form a complex. The highest dilution of serum that inhibits hemagglutinin is the hemagglutinin inhibition titer. Although vaccine immunogenicity is less sensitive than other assays, the HI assay is widely used because of its relative simplicity in technology and laboratory requirements.

Asian H5N1 highly pathogenic avian influenza is now compromised in most parts of asia and enters europe and africa. It affects both rural and commercial chickens in many southeast Asia countries, unlike H5 avian influenza, which historically has been pathogenic and lethal in other poultry (e.g., ducks and turkeys) and wildlife. There is a need for effective vaccines for field use that can prevent infection and disease and are used in a variety of birds.

Traditionally, an important control strategy for outbreaks of Highly Pathogenic Avian Influenza (HPAI) in poultry has been eradication by restricting movement and fighting infected and dangerous birds. However, since asian H5N1 virus is now widely present in rural poultry (including ducks and turkeys) as well as in wild species (especially migrating birds), changes in control strategies must be considered, where vaccines appear to be a key factor.

The current commercial vaccine for avian influenza is an oil emulsion killed virus vaccine which is used primarily to control the epidemic Low Pathogenicity Avian Influenza (LPAI) of chickens and turkeys, or the HPAI outbreaks of pakistan and mexico. Halvorson, Avian Path.31: 5-12 (2002); naem, Proceedingsof the 4th International Symposium on Avian Influenza 31-35 (Athes, Georgia, USA, 1998); swayne and surez, rev.sci.tech.off.int.epiz.19: 463-482(2000). Both killed and recombinant fowlpox vaccines are currently used to control low-pathogenic avian influenza in mexico. As above. The european union has approved the use of inactivated oil emulsion vaccines for italy as long as they are able to distinguish between vaccinated and infected birds. Capua et al, Avian Path.32: 47-55(2002).

Although it has been demonstrated that inactivated oil emulsion vaccines are able to block the transmission of H7N7(Van der Goot et al, Proc. Natl. Acad. Sci. U.S. A.102: 18141-18146(2005)) or the current H5N1HPAI (Ellis et al, Avian Path.33: 405-412(2004)), it is still considered that these vaccines may not be 100% effective in the field and may not prevent the release of the virus at all. In addition, its use also fails to distinguish between vaccinated and infected birds, which interferes with the monitoring of disease status within the flock and in the region; nor can they be formulated or tested for vaccination efficacy in ducks.

Asian H5N1 virus cannot be cultured to high titers in chicken embryos, which is a traditional method of virus production for human and avian influenza vaccines. Therefore, alternative approaches to the viroid vaccines are under development. Live vector vaccines provide additional safety and distinguish between infected and vaccinated birds.

Vectors expressing H5 fowlpox virus (Qiao et al, Avian Path.32: 25-31(2003), infectious laryngotracheitis virus (infectious arynggothecitis virus) (Luschow et al, Vaccine 19: 4249-.

Reverse genetics has been used to generate influenza reassortants with hemagglutinin and neuraminidase genes from either the human H5N1 isolate, A/HK/491/97(Subbarao et al, Virol.305: 192-200(2003)) or the avian H5N1 isolate, A/Goose/Guangdong/96 (A/Goose/Guangdong/96) (Tian et al, Virol.341: 153-162 (2005)), both of which are not pathogenic to chickens, and reassortants with H5 and N1 from A/Goose/Guangdong/96 have been formulated into formalin inactivated preparations for testing protective efficacy against the parent HPAI H5N1 in pathogen-free (SPF) chickens, as well as non-SPF geese and ducks.

The inclusion of different neuraminidase subtypes in the reconstituted vaccine allows for the differentiation of infected and vaccinated birds. This principle has been demonstrated using reassortants of the hemagglutinin gene and the remaining genes from H5 and H7 LPAI viruses, including N1 from A/WSN/33 (Lee et al, Vaccine 22: 3175-3181 (2004)). Oil emulsion vaccines with these reassortants reduced replication of the parent H5 and H7 LPAI strains in SPF chickens. Reassortant viruses with H5 from A/Goose/Hong Kong/437-4/99 (A/Goose/Hong Kong/437-4/99) and N3 from A/Duck/Germany/1215/73 (A/Duck/Germany/1215/73) have been constructed (Liu et al, Virol.314: 580-590 (2003)). When formulated as an oil emulsion, the vaccine protects SPF chickens from death after challenge with HPAIH5N1 virus. At the appropriate vaccine dose, no challenge virus was detected in the birds.

It is hypothesized that reverse genetics influenza vaccines can be used for these "DIVA" approaches, where vaccines are administered that have N's that are different from the strain of virus to which the birds are vaccinated, thus facilitating the differentiation between vaccinated animals and infected birds. Published PCT WO 03/086453 (incorporated herein by reference in its entirety) describes the DIVA technique and representative vaccines that can be used in these methods.

Reverse genetics vaccines offer a number of distinct advantages over conventional vaccines prepared from naturally occurring viral strains. By reverse genetics techniques, a particular gene of virus a can be replaced with the corresponding gene of virus B. In addition, these genes can be modified to reduce viral pathogenicity while retaining the protective properties of the resulting vaccine.

There is an urgent need for vaccines for various poultry based on the existing asian H5N1 strain and overcoming these difficulties to provide an alternative means of eradicating the infected population. The requirements for such avian influenza virus vaccines are: they (a) induce a rapid immune response in vaccinated birds, and (b) distinguish vaccinated birds from infected birds. Thus, there is a need for improved avian influenza vaccines that not only induce a rapid immune response and a higher titer response, but also produce a killing effect (sterizing effect) that prevents the growth, release and transmission of the challenge virus to other susceptible species.

Summary of The Invention

These and other related needs are met by the present invention, which provides reassortant avian influenza viruses and avian influenza vaccines, compositions comprising one or more avian viruses and/or vaccines, formulations thereof, and methods of use of the avian viruses and/or vaccines, compositions, and/or formulations of the present invention, wherein the viruses and vaccines comprise an HA gene from a highly pathogenic avian influenza virus, an NA gene from a low pathogenic avian influenza virus, and a viral backbone comprising the remaining avian influenza virus genes from the low pathogenic avian influenza virus.

The low pathogenic avian influenza viruses and vaccines disclosed herein are effective in preventing or ameliorating avian influenza and provide the added benefit that they prevent the growth, release and/or spread of challenge influenza viruses to other species. Thus, the low pathogenic avian influenza viruses and vaccines of the present invention comprise: an HA moiety from a first highly pathogenic strain of H5 avian influenza; a NA moiety from a second low pathogenic strain, wherein the second low pathogenic strain HAs a different N subtype than the virus from which the HA moiety was derived; and the remainder of the viral genome selected from the group consisting of low-pathogenic viruses, wherein the low-pathogenic viruses are the same as or different from the virus from which part N is derived.

In certain embodiments, the HA moiety is derived from a highly pathogenic strain of H5 avian influenza, which is exemplified herein as the H5N1 asian strain, designated a/chicken/Vietnam/C58/04 (a/chicken/Vietnam/C58/04). In other embodiments, the NA portion is derived from a second, less pathogenic strain having a different NA subtype than the virus from which the HA portion is derived. Typically, the NA subtype is from a european or american lineage strain with subtypes N3, N5 or N9. An example herein is a reassortant H5N3 avian influenza virus in which the N3 gene is derived from a low pathogenic H2N3 avian influenza virus strain, designated a/DK/germany/1215/73. In other embodiments, the remaining viral genome is selected from low-pathogenic viruses, an example of which herein is low-pathogenic avian influenza virus, designated a/Puerto Rico/8/34H 1N1(a/Puerto Rico/8/34H 1N 1).

Thus, examples herein are reassortant H5N3 avian influenza viruses and vaccines comprising: the HA H5 gene, which is from the current pathogenic Asian outbreak A/Ck/Vietnam/C58/04 (H5N 1); NA N3 gene from the low pathogenic strain a/DK/germany/1215/73 (H2N3), which facilitates differentiation from wild type infection (N1); and an avian influenza backbone from a low pathogenic strain a/puerto rico/8/34 (H1N1) that is well characterized and is a safe virus that is nonpathogenic to humans or animals. The construction of H5N3 reverse genetics virus is described in detail in published PCT WO01/083794 (incorporated herein by reference in its entirety).

One unexpected property of the avian H5N3 vaccine construct described herein is: when formulated into a composition with a suitable adjuvant, the pathogenic influenza virus provides a killing effect when it attacks an vaccinated individual, thereby preventing the pathogenic virus from growing in susceptible tissues and flowing into the environment of the individual. This surprising feature of the present invention is particularly advantageous over existing vaccines available in the art because it reduces or eliminates the spread of disease by pathogenic viruses that could otherwise be transmitted from an immunized individual to an unimmunized susceptible individual.

Thus, in one aspect, the present invention relates to vaccine compositions that are effective in preventing and/or ameliorating avian influenza and that prevent the growth, release and transmission of pathogenic challenge influenza viruses from infected individuals to uninfected individuals, for example from infected birds to uninfected birds. More specifically, the present invention relates to a vaccine composition comprising a reverse genetics virus comprising: (i) an HA moiety from a first highly pathogenic H5 avian influenza virus strain, (ii) an N moiety from a second low pathogenic avian influenza virus strain having an N subtype different from the N subtype of the first highly pathogenic H5 avian influenza virus strain, and (iii) a backbone avian influenza virus genome from a third low pathogenic virus.

In certain embodiments, the second low-pathogenic strain and the third low-pathogenic strain are both from the same avian influenza virus isolate. In an alternative embodiment, the second low pathogenic strain and the third low pathogenic strain are from different isolates of avian influenza virus.

Other aspects of the invention provide avian influenza vaccine compositions and formulations effective in preventing or ameliorating avian influenza virus infection. Such formulations of the invention comprise reverse genetics strains of avian influenza virus (especially inactivated forms of reverse genetics strains of avian influenza virus) and optionally comprise one or more surfactants (including sorbitan oleate). Typically, avian influenza virus vaccines comprise a total Hemagglutinin (HA) of at least about 75 HA/dose of the vaccine formulation.

A vaccine composition effective in preventing or ameliorating avian influenza virus infection comprising a reverse genetics virus formulated in a biologically acceptable adjuvant material, the composition of the virus being as follows: an HA moiety from a highly pathogenic strain of H5 avian influenza; an N moiety from a second low pathogenic strain, wherein the second low pathogenic strain HAs a different N subtype than the virus from which the HA moiety was derived; and the remainder of the viral genome selected from the group consisting of low-pathogenic viruses, wherein the low-pathogenic viruses are the same or different from the virus from which part N is derived; the vaccine composition wherein the total amount of Hemagglutinin (HA) is at least about 75 HA/dose, or at least about 125 HA/dose, or about 250 HA/dose.

In certain aspects, the vaccine compositions and/or formulations may optionally further comprise one or more surfactants, for example one or more sorbitan oleates and/or one or more sorbitan oleatesVarious ethylene oxide/propylene oxide block copolymers. In certain such embodiments, the sorbitan oleate is tween 8080) And/or sorbitan sesquioleate. In other aspects, vaccine compositions and/or formulations may comprise an avian influenza virus of the present invention formulated in a water-in-oil emulsion.

Examples herein are vaccine compositions wherein the backbone virus genome is derived from H1N1 avian influenza virus i.e. a/puerto rico/8/34 (aka PR 8). The low pathogenic strains are particularly advantageous in applications requiring an influenza vaccine that is safe across two or more different species.

The invention also provides vaccine compositions comprising at least two strains of avian influenza virus, wherein the HA/dose is typically greater than about 75 HA/dose, more typically greater than about 128 HA/dose, or greater than about 200 HA/dose, or greater than about 250 HA/dose and 300 HA/dose.

It will be appreciated that the selection of a particular strain of avian influenza virus from which the reverse genetics influenza virus vaccine of the invention is derived will depend on the particular strain being pandemic in a particular geographical region, with the proviso that the vaccine is administered with (a) its HA subtype generally being the same as that of the pandemic or challenge strain, and (b) its NA subtype being different from that of the pandemic or challenge strain, so that it can rely on DIVA technology.

Other aspects of the invention provide methods for preventing or ameliorating an outbreak of an avian influenza virus infection, said methods comprising the step of administering to poultry a vaccine composition comprising an inactivated reverse genetics avian influenza virus, wherein the total amount of Hemagglutinin (HA) is at least about 75 HA/dose of the vaccine composition.

For example, the present invention provides a method of preventing or ameliorating an outbreak of an avian influenza virus infection, said method comprising the step of administering to poultry a virus or vaccine composition as disclosed herein. The virus or vaccine composition may be administered, for example, via drinking water or spray. Generally, a suitable dose range is between about 1ng and about 1 μ g, or between about 5ng and about 250ng, or between about 20ng and about 125ng, or between about 50ng and about 100 ng. The effective dose administered is generally about 0.25ml to 2.0ml per poultry. The virus and/or vaccine may be administered in a single dose, or may be administered in duplicate in two or more doses.

These and other embodiments, features and advantages of the present invention will be apparent from the detailed description of the invention given herein below and the appended claims.

Brief description of the drawings and sequence identification

FIG. 1 is an alignment of goose HK 437H 5 and chicken VN C58H 5.

SEQ ID NO: 1 is the amino acid sequence of goose HK 437H 5

(dqicigyhannsteqvdtimeknvtvthaqdilekthngklcdldgvkplilrdcsvagwllgnpmcdefinvpewsyivekaspan

dlcypgdfnnyeelkhllsrtnhfekiqiipksswsnhdassgvssacpyhgkssffrnvvwlikknsayptikrsynntnqedllvlw

gihhpndaaeqtklyqnpttyisvgtstlnqrlvpeiatrpkvngqsgrmeffwtilkpndainfesngnfiapeyaykivkkgdsaim

kseleygncntkcqtpmgainssmpfhnihpltigecpkyvksnrlvlatglrntpqietrglfgaiagfieggwqgmvdgwygyhh

sneqgsgyaadkestqkaidgvtnkvnsiidkmntqfeavgrefnnlerrienlnkkmedgfldvwtynaellvlmenertldfhdsn

aknlydkvrlqlrdnakelgngcfefyhkcdnecmesvkngtydypqyseearlnreeisgvklesmgtyqilsiystvasslalaimv

aglslwmcsngslqcrici)。

SEQ ID NO: 2 is the amino acid sequence of chicken VN C58H 5

(DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAG

WLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS

SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIH

HPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRLATRSKVNGQSGRMEFFWTILRPNDAI

NFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNAKCQTPMGAINSSMPFHNIHPLTI

GECPKYVKSNRLVLATGLRNSPQIETRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQG

SGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLD

VWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNE

CMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLVLAIMVAGLSL

WMCSNGSLQCRICI)。

Detailed Description

The present invention is based on the unexpected discovery that reverse genetics techniques can be applied to the production of low-pathogenicity avian influenza viruses from highly pathogenic viruses while maintaining protection from highly pathogenic viruses. The monovalent low pathogenic avian influenza virus vaccines disclosed herein provide effective protection without evidence of challenge virus release.

The invention will be better understood with reference to the following definitions.

Definition of

The term "influenza virus" as used herein is used to define the viral species: pathogenic strains of the virus can cause a disease known as influenza.

The term "stock strain virus" refers to a virus strain that provides a backbone for constructing low pathogenic influenza virus vaccine strains by reverse genetics methods as described herein. These parental strains typically provide 6 or 7 viral segments (PB1, PB2, PA, NP, M, NS, and optionally NA) to the vaccine virus. That is, the mother strain virus may be optionally used as a source of the NA gene. In the specific case of the H5N3 avian influenza virus vaccine described herein, the "mother strain virus" giving the backbone genes is an isolate of avian H1N1 virus, referred to as a/puerto rico/8/34.

The term "polypeptide" refers to a polymer of amino acids, rather than to a product of a particular length; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. The term also does not refer to or include post-translational polypeptide modifications, such as glycosylation, acetylation, phosphorylation, and the like.

As used herein, "infectivity" refers to the ability of a virus to replicate in a cell and produce viral particles. Infectivity can be assessed by measuring the virus (i.e., viral load) or by observing the disease progression of the animal.

As used herein, "subject" or "patient" or "animal" refers to a vertebrate animal that supports negative strand RNA virus infection, particularly influenza virus infection, including, but not limited to, birds (e.g., waterfowl and chicken) and members of mammals (e.g., dogs, cats, wolves, weasels, rodents (racines and mice, etc.), horses, cows, sheep, goats, pigs, and primates, the latter including humans).

The term "immunogenic" as used herein means that the virus or polypeptide is capable of inducing a humoral or cellular immune response, preferably both immune responses. The immunogenic entity is also antigenic. An immunogenic composition is a composition that induces a humoral or cellular immune response, or both, when administered to an animal.

A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains an "epitope" of at least about 5, preferably at least about 10 amino acids. The antigenic portion of the polypeptide, also referred to herein as an "epitope," may be a portion that is immunodominant for antibody or T cell receptor recognition, or it may be a portion used to raise antibodies to the molecule, i.e., for immunization, by conjugating the antigenic portion to a carrier polypeptide. Without a carrier, an antigenic molecule need not be immunogenic by itself (i.e., the ability to induce an immune response).

The term "amino acid substitution" as used herein refers to the presence of an amino acid at a particular position in the amino acid sequence of a molecule. Amino acid substitutions may occur with respect to any other amino acid occupying that position. Polypeptides derived from amino acid sequence changes may include changes in post-translational modifications, such as glycosylation, acetylation, phosphorylation, or any other amino acid modification as well as amino acid substitutions.

The term "reverse genetics system" as used herein refers to a method of producing influenza virions, polypeptides, virions or nucleic acids, i.e., by genetic engineering methods. These methods include, but are not limited to, the "plasmid system" described by Hoffmann in the following references (Hoffmann et al, Vaccine 20: 3165 (2002); U.S. patent publication No. 2002/0164770A1, App. 2002, 11/7/2002, which are all incorporated herein by reference). In general, reverse genetics systems can allow the production of viral particles, polypeptides and/or nucleic acids having specific sequences by genetic engineering methods known to those skilled in the art. These systems are described in more detail below.

The term "receptor binding site" as used herein refers to the portion of the HA molecule to which a receptor of interest (e.g., a sialic acid receptor on an erythrocyte) binds. The H5 molecular structures of goose HK 437H 5 and chicken VN C58H 5 are disclosed herein as SEQ ID NOs: 1 and 2, and can be seen in the sequence diversity alignment shown in figure 1. The molecular structure of H5 of a/duck/Singapore (a/duck/Singapore) and the location of the receptor binding site of hemagglutinin of this H5 subtype are described in I Ha et al, proc.natl.acad.sci.u.s.a.98: 11181(2001).

The term "diagnostic reference virus" refers to a virus with high HA antigenicity. Such diagnostic reference viruses are useful in immunoassays, such as hemagglutinin inhibition assays.

The term "exposed virus" refers to a virus that a single animal has been exposed to. Such exposure may be during daily activities, such as contacting an infected individual, for example, exposing a person to an infectious influenza virus. The exposure may also be due to a specific clinical attack, for example in the case of laboratory tests where the laboratory animal is intentionally exposed to the virus. Such exposure can be deliberately created by immunization with an influenza vaccine.

The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar untoward reactions (e.g., gastric upset, dizziness, etc.) when administered to a human. Preferably, the term "pharmaceutically acceptable" as used herein refers to materials approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, particularly humans.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including petroleum, animal or vegetable oils, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solutions, saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly in the form of injectable solutions. Suitable pharmaceutical carriers are described in e.w. martin, "Remington's pharmaceutical Sciences", 18 th edition.

The term "adjuvant" as used herein refers to a compound or mixture that enhances the immune response against an antigen. Adjuvants are used as tissue-sustained release preparations for the slow release of antigens, and also as activators of the lymphatic system, to non-specifically enhance immune responses (Hood et al, Immunology, second edition, Menlo Park, CA: Benjamin/Cummings, 1984, page 384). Typically, initial challenge with antigen alone in the absence of adjuvant will not induce a humoral or cellular immune response. Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gel (e.g., aluminum hydroxide), surfactant (e.g., lysolecithin), pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpetBlood blue eggWhite and potentially useful human adjuvants (e.g. N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-N-muramyl-L-alanyl-D-isoglutamine, N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine, BCG (bacillus calmette-guerin) and Corynebacterium parvum preferred adjuvants are pharmaceutically acceptable adjuvants.

The term "isolated" as used herein refers to the removal of material of interest from a natural environment (e.g., a cell or virus). Thus, the isolated biological material may be free of some or all of the cellular components, i.e., cellular components of naturally occurring natural material (e.g., cytoplasmic components or membrane components). A material is considered isolated if it is present in a cell extract or supernatant. In the case of nucleic acid molecules, isolated nucleic acids include PCR products, isolated mRNA, cDNA, or restriction fragments. In another embodiment, the isolated nucleic acid is preferably excised from the chromosome from which it is found to be derived, and more preferably is no longer linked or adjacent to a non-coding region of the isolated nucleic acid molecule (but may be linked to its native regulatory region or a portion thereof) of the chromosome from which it is derived, or is no longer linked or adjacent to other genes located upstream or downstream of the gene contained in the isolated nucleic acid molecule. In yet another embodiment, the isolated nucleic acid lacks one or more introns. An isolated nucleic acid molecule includes a sequence that is inserted into a plasmid, cosmid, artificial chromosome, or the like, i.e., when it forms part of a chimeric recombinant nucleic acid construct. Thus, in a specific embodiment, the recombinant nucleic acid is an isolated nucleic acid. The isolated protein may be associated with other proteins or nucleic acids or both that are associated with the cell or cell membrane (if a membrane bound protein). Removing the isolated organelle, cell, or tissue from the anatomical location of the source organism. The isolated material may be purified, but need not be.

The term "purified" as used herein refers to a material that is isolated under conditions that reduce or eliminate the presence of extraneous material (i.e., contaminants), including native material from the material obtained. For example, the purified virions are preferably substantially free of host cell or culture medium components, including tissue culture or chicken embryo proteins, non-specific pathogens, and the like. The term "substantially free" as used herein is operatively used in the context of analytical testing of a material. Preferably, the purified material that is substantially free of contaminants is at least 50% pure; more preferably at least 90% pure, still more preferably at least 99% pure. Purity can be determined by chromatography, gel electrophoresis, immunoassay, compositional analysis, biological assays, and other methods known in the art.

Purification methods are well known in the art. The viral particles can be purified by ultrafiltration or ultracentrifugation, preferably continuous centrifugation (see Furminger, supra). Other purification methods are also possible and encompassed by the present invention. The purification material may contain less than about 50%, preferably less than about 75%, most preferably less than about 90% of the cellular components, culture medium, proteins or other unwanted components or impurities (as desired) with which it was originally associated. The term "substantially purified" refers to the highest purity that can be achieved using conventional purification techniques known in the art.

In a particular embodiment, the term "about" or "approximately" means within a numerical range of statistical significance. Such a range may be in the range of orders of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10% and even more preferably within 5% of a given value or range. The allowable error covered by the term "about" or "approximately" depends on the particular system under study, as one of ordinary skill in the art can readily appreciate.

Reverse genetics method

As mentioned above, the basis of the present invention is the generation of avian influenza viruses and their vaccines, which are generated by using the reverse genetics approach detailed in the working examples presented herein. Briefly, a low-pathogenic reassortant avian influenza virus is constructed by combining the HA gene from a first highly pathogenic avian influenza virus and the NA gene from a second low-pathogenic avian influenza virus into the backbone (which contains the remaining avian influenza virus genes) from a second or third low-pathogenic avian influenza virus. As noted above, the construction of exemplary low pathogenic reassortant avian influenza viruses disclosed and exemplified herein is as follows: the H5N3 virus was generated by incorporating the HA gene from the a/Ck/vietnam/C58/04 isolate (H5N1) and the NA gene from the a/DK/germany/1215/73 isolate (H2N3) into the a/puerto rico/8/34 backbone.

The reverse genetics systems currently being developed have enabled manipulation of influenza virus genomes (Palese et al, Proc. Natl. Acad. Sci. U.S.A.93: 1354 (1996); Neumann and Kawaoka, Adv. Virus Res.53: 265 (1999); Neumann et al, Proc. Natl. Acad. Sci. U.S.A.96: 9345 (1999); Fodor et al, J.Virol.73: 9679 (1999); U.S. patent application 20040029251). For example, plasmid-driven expression of 8 influenza vrnas from the pol I promoter and expression of all mrnas from the polII promoter has been shown to result in the production of infectious influenza a virus (Hoffmann et al, proc. natl. acad. sci. usa 2000, 97: 6108; U.S. patent publication No. 20020164770, the description of the minimal plasmid reverse genetics system and genetic engineering methods of which are incorporated herein by reference).

These recombinant methods can allow the production of specific influenza virus types with specific changes in the polypeptide amino acid sequence. The HA molecule containing the desired substitution may be part of a recombinant influenza virus. Recombinant influenza viruses can be prepared by any method known to those skilled in the art, including by genetic engineering methods, such as the "plasmid-only" system (Hoffmann et al, Vaccine2002, 20: 3165). The recombinant influenza virus may be derived from a H5N1 virus. The recombinant virus may have the genetic background of the H1N1 virus used for vaccine development, for example the A/PR/8/34 virus or any influenza A virus, including cold-adapted strains of A/Raninggler/134/17/57 (A/Leningrad/134/17/57), A/Raninggler/134/47/57 and A/Arab/6/60 (A/Ann Arbor/6/60). Nucleic acids corresponding to the sequence of the HA molecule can be isolated from the virus and sequenced.

Techniques for isolating and modifying specific nucleic acids and proteins are well known to those skilled in the art. According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art may be used. These techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & manitis, Molecular Cloning: a laboratory Manual, 2 nd edition, Cold Spring Harbor, NY: cold Spring harbor laboratory Press, 1989 (herein "Sambrook et al, 1989"); DNA Cloning: APractcal Appliach, volumes I and II (D.N. glover, 1985); oligonucleotide Synthesis (MJ. Gait eds., 1984); nucleic acid hybridization [ B.D.Hames & S.J.Higgins eds (1985) ]; TranscriptionnND transformation [ B.D. Hames & S.J. Higgins, eds (1984) ]; animal CellCulture [ R.I. Freshney, eds (1986) ]; immobilized Cells And Enzymes [ IRL Press, (1986) ]; B.Perbal, A Practical Guide To Molecular Cloning (1984); ausubel, f.m. et al (eds.); current Protocols in Molecular biology, John Wiley & Sons, Inc., 1994. These techniques include site-directed mutagenesis using oligonucleotides with altered nucleotides for generating PCR products with mutations (e.g., the "Quikchange" kit produced by Stratagene).

Low pathogenicity avian influenza virus vaccine

An example of an existing low-pathogenic avian influenza virus vaccine herein is the reassortant H5N3 virus produced by incorporating the H5 gene from the highly pathogenic asian outbreak a/Ck/vietnam/C58/04 (H5N1), the N3 gene from the low-pathogenic avian influenza strain a/DK/germany/1215/73 (H2N3) into the low-pathogenic avian influenza virus backbone a/puerto rico/8/34 (H1N 1; PR 8). The low pathogenic avian influenza virus of the present invention ensures optimal protection against otherwise highly pathogenic avian influenza viruses. The N1 gene from a/puerto rico/8/34 was replaced with the N3 gene from a/DK/germany/1215/73 in order to use the DIVA program to determine whether anti-neuraminidase antibody titers in individual birds were the result of vaccination with a highly pathogenic virus against a less pathogenic virus.

Avian influenza isolates useful for deriving the vaccines of the present invention can be isolated using techniques available in the art. For example, tissue or serum from infected chickens may be obtained from commercial broiler breeders (broilers cock). The virus can then be passaged in tissue or other suitable medium to create a mother seed virus. The skilled person can also perform further characterization using feasible methods. The virus can be inactivated by any feasible method, such as heat and chemical treatment.

Formulations comprising low pathogenic avian influenza virus vaccines

Also provided herein are vaccine formulations comprising one or more low pathogenic avian influenza virus vaccines of the present invention and an adjuvant and/or emulsion formulation. Such formulations disclosed herein exhibit improved efficacy at reduced HA unit concentrations compared to the aforementioned HA unit concentrations. More specifically, in the formulations of the present invention, a low pathogenic avian influenza virus vaccine is effective when the HA unit is between about 10ng and about 1 μ g, more typically between about 20ng and about 500ng, still more typically between about 50ng and about 250ng, or between about 75ng and about 200ng, most typically about 100ng, about 125ng, about 150ng, or about 175 ng.

The vaccine compositions of the present invention can be formulated using available techniques, preferably using a pharmacologically acceptable carrier. For example, in one embodiment, an aqueous formulation is included. Such formulations use water, saline or phosphate buffer or other suitable buffers. In a further embodiment, the vaccine composition is preferably a water-in-oil or oil-in-water emulsion. Also included are double emulsions, which are typically characterized as water-in-oil-in-water emulsions. The oil helps to stabilize the formulation and may also act as an adjuvant or enhancer. Suitable oils include, but are not limited to, white oil, Drakeoil, squalane or squalene, and other animal and vegetable oils or mineral oils, whether of natural or synthetic origin.

As described above, modified viruses that inherently contain HA molecules that enhance antigenicity are more immunogenic, which in turn can provide a stronger immune response and better vaccine potential.

Strategies to increase the effectiveness of influenza vaccines include the use of adjuvants (Wood and Williams, supra), co-administration of immunostimulatory molecules (salaller and Lodge, j.surg.oncol.1998, 68: 122) and mucosal vaccination strategies. Mucosal vaccination strategies include encapsulation of the virus in microcapsules (U.S. Pat. nos. 5,075,109, 5,820,883 and 5,853,763) and the use of immunopotentiating membrane carriers (WO 98/0558). In addition, the immunogenicity of orally administered immunogens can be enhanced by the use of red blood cells (rbc) or rbc ghost cells (U.S. patent No. 5,643,577) or by the use of blue tongue antigen (U.S. patent No. 5,690,938). While these methods hold promise for improving future vaccination strategies, their use in certain circumstances requires validation and monitoring to ensure vaccine effectiveness. It is contemplated that the invention described herein will enhance these strategies, including by increasing the ability to detect their immunogenic effects.

In addition, the vaccine composition may contain other suitable adjuvants available in the art. These may include, for example, aluminum hydroxide and aluminum phosphate, as well as other metal salts.

Additional excipients, such as surfactants or other wetting agents or formulation adjuvants may also be included in the vaccine composition. The surfactant may comprise sorbitan monooleate (f)Series), and ethylene oxide/propylene oxide block copolymer(s) ((ii)Series), and other materials available in the art. Other compounds may also be included in the vaccine as stabilizers or preservatives. These compounds include, but are not limited to, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, dextrin, or glucose, etc.) and preservatives (e.g., formalin).

The vaccine composition may also be formulated as a dry powder substantially free of extraneous moisture, such powder being reconstituted by the end user immediately prior to use. Optionally, the vaccine composition is formulated with killed or inactivated virus.

The vaccine compositions of the invention preferably contain a minimum of about 200HA in total from their influenza virus components. In one embodiment of the invention, the vaccine contains about 128 HA/dose from each strain, even more preferably about 192 HA/dose from each strain.

The vaccine composition of the invention may also comprise other poultry antigens against other diseases or be administered together with the latter. For example, vaccine antigens directed against herpes virus of chicken, anemia virus of Chicken (CAV), newcastle disease virus and Infectious Bronchitis (IB) virus, as well as reovirus antigens may be included as part of the vaccine composition of the invention. It is especially preferred that one or more reovirus antigens are part of the vaccine composition of the present invention.

Method for inducing protective immune response

The invention also provides methods of inducing protection against avian influenza virus infection. The methods disclosed herein comprise administering to a poultry animal a vaccine and/or formulation thereof comprising one or more low-pathogenic avian virus vaccines as described above, wherein the one or more low-pathogenic avian virus vaccines comprise a combined HA content of greater than about 75 HA/dose, more typically greater than about 125 HA/dose, or greater than about 200 HA/dose, or between about 250 HA/dose and about 300 HA/dose.

The skilled artisan can select the mode of administration, which will depend on the specific application considerations. For example, the vaccine composition may be administered to hatched chicks (several days to several weeks of age) by drinking water, spraying or eye drops. In ovo administration is also included herein. For example, chick embryos may be inoculated, typically for about 18-19 days. Other methods of administering the vaccine compositions of the present invention parenterally, subcutaneously, peritoneally, orally, intranasally or by other feasible methods (preferably parenterally, more preferably intramuscularly) according to effective dosages according to a schedule determined by the time of expected potential exposure to the vector of the pathogenic avian influenza virus are also within the scope of the present invention.

The dosage will generally range from about 0.25ml to about 2.0ml per poultry animal, more preferably from about 0.5ml to about 1.0ml per animal. Thus, 1,2 or more doses are included herein, wherein it is particularly preferred that the number of doses is as small as possible.

As previously mentioned, the present invention relates to novel avian influenza vaccine compositions and methods of use thereof in poultry. The term "poultry" includes, but is not limited to, all commercially farmed poultry animals including chickens, ducks, geese, turkeys, peacocks, bantams, and the like.

Immunoassay method

According to the present invention, various methods known in the art for detecting immunospecific binding of an antibody to an antigen can be used to detect binding and increased antigenicity. Early methods of detecting antigen-antibody interactions included detection and analysis of complexes by precipitation in gels. Another method of detecting an antibody binding pair of analyte-detector includes the use of a radioiodinated detection antibody or radioiodinated protein that can react with IgG (e.g., protein a). These early Methods are well known to those skilled in the art and are reviewed in Methods in enzymology 1980, 70: 166-198.

Late-stage methods for detecting the presence of an analyte in a sample using only one antibody include competitive binding assays. In this technique, an antibody, which is typically immobilized on a solid support, is contacted with a sample suspected of containing the analyte and a known amount of the labeled analyte. The two analytes (labeled analyte and analyte in the sample) will then compete for the binding site on the antibody. The amount of competing analyte in the sample is known from the measurement of free labeled analyte or bound labeled analyte. A more complete description of this method can be found in "Basic Principles of Antigen-Antibody Reaction" (Labat, Methods in enzymology, 70, 3-70, 1980). In this example, the labeled analyte may be labeled with a radioisotope or with an enzymatic label.

More recent immunoassays use a dual antibody approach for determining the presence of an analyte. For a review of these techniques, see Methods in Enzymology, supra, in the same volume. Thus, according to one embodiment of the invention, the presence of a single label is detected using a pair of antibodies directed against each label to be detected. One of the pair of antibodies is referred to herein as a "detection antibody", and the other of the pair of antibodies is referred to herein as a "capture antibody". Accordingly, one embodiment of the present invention uses a dual antibody sandwich method for the determination of an analyte in a biological fluid sample. In this method, the analyte is sandwiched between a detection antibody and a capture antibody, which is irreversibly immobilized on a solid support. The detection antibody contains a detectable label to recognize the presence of the antibody-analyte sandwich and thus the analyte.

Common early forms of solid supports include polystyrene plates, tubes or beads, which are well known in the fields of radioimmunoassay and enzyme immunoassay. Recently, various porous materials such as nylon, nitrocellulose, cellulose acetate, glass fiber, and other porous polymers have been used as solid supports.

Various different techniques and corresponding sensor devices may be used. The automated assay device comprises a continuous/random access assay. Examples of such systems include OPUS from PB Diagnostic System, Inc^TMAnd IMX introduced by Abbott Laboratories (North Chicago, I11)^TMAn analyzer. Automated meters by PB Diagnostic Systems, inc. can be found in U.S. patent No. 5,051,237; 5,138,868, respectively; 5,141,871, and 5,147,609.

A further type of immunochemical analytical system that may be used in the practice of the present invention is an optical immunosensor system. In general, an optical immunosensor is a device that quantitatively converts chemical or biochemical concentration or activity of a target into an electrical signal using optical principles. These systems can be divided into 4 main types: reflection technology; surface plasmon resonance; fiber optic technology and Integrated optical devices (Integrated optical devices). Reflection techniques include ellipsometry, multiple integral reflectance spectroscopy (multiple integral reflectance spectroscopy), and fluorescent capillary fill devices (fluorescent capillary device). Fiber optic technologies include evanescent field fluorescence (evanescentfield fluorescence), fiber optic capillary, and fiber optic fluorescence sensors. The integrated optical device includes planar evanescent field fluorescence (planar evanescent field fluorescence), an input stepped coupler immunosensor (input stepped coupler immunosensor), a Mach-Zehnder interferometer (Mach-Zehnder interferometer), a hartmann interferometer (Hartman interferometer), and a differential interferometer sensor. The holographic detection of the binding reaction is accomplished by detecting the presence of a holographic image produced at the predetermined image location when one reactant of the binding pair binds to the second reactant of the immobilized binding pair (see U.S. Pat. No. 5,352,582, Lichtenwalter et al, 10.4.1994). A general review article of examples of optical immunosensors can be found in g.a. robins, Advances in Biosensors 1991, 1: 229-256. More specific descriptions of these devices can be found in, for example, U.S. patent nos. 4,810,658, 4,978,503, and 5,186,897; R.A. Brady et al (Phil. Transs.R.Soc.Land.B.1987, 316: 143-160) and G.A. Robinson et al (Sensors and actors, Elsevier 1992).

Serological assays are widely used to detect influenza diagnosis and to study the epidemiology and antigenicity of viral strains. In particular, the Hemagglutinin Inhibition (HI) assay is widely used due to its low laboratory requirements and ease of use. It is contemplated that the present invention will improve the utility of HI assays by increasing their sensitivity. The HI assay can also be used to show the antigenicity of a modified HA molecule and is helpful in characterizing a modified HA molecule because of the reduction or increase in antigenicity compared to a non-modified molecule.

The HI assay can detect the ability of an antibody in a serum sample to bind to a standardized reference. In the HI assay, serial dilutions (titers) of serum samples are mixed with standard amounts of red blood cells and their association into complexes is visualized. The lowest level of serum tested that caused a visible complex was the assay result.

As described above, the present invention provides improved methods of production and validation of vaccines for treating or preventing influenza virus infection. In particular, the present invention can use reverse genetics techniques for vaccine preparation. It is contemplated that the present invention can be used to confirm and verify immune responses after vaccination. In particular, but not exclusively, the invention provides enhanced antibody detection methods in which antibody detection is performed after exposure of an individual to influenza virus, due to the enhanced antigenicity of the modified HA molecule. This enhanced antigenicity results in an increased sensitivity of the assay used to detect the immune response (e.g., the HI assay).

Examples

The invention will be better understood by reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Example 1

Construction of H5N3 avian influenza vaccine

Virus

Influenza viruses A/PR/8/34(H1N1), A/Chicken/Vietnam/C58/04 (A/Chicken/Vietnam/C58/04) (H5N1) and A/DK/Germany/1215/73 (A/DK/Germany/1215/73) (H2N3) are available from the depository of St.Jude Children's research Hospital. A/Muscovy Duck/Vietnam/453/2004 (A/MuscovyDuck/Vietnam/453/2004) H5N1 is available from the Regional Animal Health center in Callicarpa Vietnam (Regional Animal Health center, Ho Chi Minh City, Vietnam).

RT-PCR and plasmid construction

RNA was isolated from influenza viruses A/chicken/Vietnam/c58/04 (H5N1), A/PR/8/34(H1N1) and A/DK, Germany/1215/73 (H2N3) using the Rneasy kit (Qiagen). The RNA was reverse transcribed into cDNA using the Uni 12-primer (AGC AAA AGC AGG; SEQ ID NO: 3).

Then, the mixture was prepared according to Hoffmann et al (2001) Arch.Virol.146: 2275 As described in 2289 (incorporated herein by reference), the resulting cDNA is amplified using segment-specific primers. In particular, segment-specific primers can be used as described in table 1 below:

TABLE 1

Primers useful for amplifying influenza virus segments

Gene	Forward primer	Reverse primer
			PB2	Ba-PB2-1：TATTGGTCTCAGGGAGCGAAAGCAGGTCSEQ ID NO：4	Ba-PB2-2341R：ATATGGTCTCGTATTAGTAGAAACAAGGTCGTTTSEQ ID NO：5
PB1	Bm-PB1-1：TATTCGTCTCAGGGAGCGAAAGCAGGCASEQ ID NO：6	Bm-PB1-2341R：ATATCGTCTCGTATTAGTAGAAACAAGGCATTTSEQ ID NO：7
			PA	Bm-PA-1：TATTCGTCTCAGGGAGCGAAAGCAGGTACSEQ ID NO：8	Bm-PA-2233R：ATATCGTCTCGTATTAGTAGAAACAAGGTACTTSEQ ID NO：9
HA	Bm-HA-1：TATTCGTCTCAGGGAGCAAAAGCAGGGGSEQ ID NO：10	Bm-NS-890R：ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTTSEQ ID NO：11
			Modified HA (1)	Bm-HA-1：TATTCGTCTCAGGGAGCAAAAGCAGGGGSEQ ID NO：10	Bm-H5-1025R：ATTACGTCTCTCCTCTTGTCTCAATTTGAGGGGTATTSEQ ID NO：12
Modified HA (2)	Bm-H5-1020：ATTACGTCTCAGAGGACTATTTGGGGCTATAGCAGGSEQ ID NO：13	Bm-NS-890R：ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTTSEQ ID NO：11

Gene	Forward primer	Reverse primer
			NP	Bm-NP-1：TATTCGTCTCAGGGAGCAAAAGCAGGGTASEQ ID NO：14	Bm-NP-1565R：ATATCGTCTCGTATTAGTAGAAACAAGGGTATTTTTSEQ ID NO：15
NA	Ba-NA-1：TATTGGTCTCAGGGAGCAAAAGCAGGAGTSEQ ID NO：16	Ba-NA-1413R：ATATGGTCTCGTATTAGTAGAAACAAGGAGTTTTTTSEQ ID NO：17
			M	Bm-M-1：TATTCGTCTCAGGGAGCAAAAGCAGGTAGSEQ ID NO：18	Bm-M-1027R：ATATCGTCTCGTATTAGTAGAAACAAGGTAGTTTTTSEQ ID NO：19
NS	Bm-NS-1：TATTCGTCTCAGGGAGCAAAAGCAGGGTGSEQ ID NO：20	Bm-NS-890R：ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTTSEQ ID NO：21

The 5' end of the primer has a recognition sequence of restriction endonuclease BsmBI (Bm) or BsaI (Ba).

The ratio of antigen to protein can be determined according to Hoffmann et al (2002) Vaccine 20: 3165 plasmids encoding the PB1, PB2, PA, NP, M and NS genes of influenza A/PR/8/34 were constructed as described (incorporated herein by reference). Specifically, influenza genes for PB2, PB1, PA, NP, M, and NS can be amplified from the A/PR/8/34(H1N1) cDNA using the primers listed in Table 1. The PB1, PB2, PA, NP, M and NS genes were cloned by digesting the PCR fragments with BsmBI (PB1, PA, NP, M and NS) or BsaI (PB2) and ligating them to the cloning vector pHW2000 (also cleaved with BsmBI or BsaI). These plasmids were designated pHW191-PB2, pHW192-PB1, pHW193-PA, pHW195-NP, pHW197-M and PHW 198-NS.

The NA gene was amplified from A/DK/Germany/1215/73 (H2N3) cDNA. The NA genes were amplified by PCR using the NA-specific primers listed in Table 1(SEQ ID NO: 16 and SEQ ID NO: 17). The PCR fragment was digested with BsaI and ligated into vector pHW 2000.

The HA gene was amplified by PCR from the A/chicken/Vietnam/c58/04 (H5N1) cDNA with the HA-specific primers listed in Table 1(SEQ ID NO: 10 and SEQ ID NO: 11). The PCR fragment was digested with BsmBI and ligated into vector pHW 2000. The HA gene from a/chicken/vietnam/C58/04 (H5N1) was then modified by deletion of the poly-basic amino acid region at the cleavage site between HA1 and HA 2. Plasmids containing the modified HA gene were obtained by PCR amplification of two fragments of the plasmid encoding unmodified HA. The primers used for amplification of both fragments are shown in Table 1(SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 11). The fragments were digested with BsmBI and ligated to pHW2000-BsmBI by a three-fragment ligation reaction.

To ensure that there are no unwanted mutations in the gene, the cloned viral cDNA was sequenced.

Generation of recombinant viruses

According to Hoffman et al (2002) Vaccine 20: 3165-3170 (incorporated herein by reference), recombinant viruses are produced by DNA transfection. In particular, 293T and MDCK cells (each containing 0.2-1X 10) can be co-cultured⁶Cells) and used for transfection experiments. The co-cultured cells were transfected with 1ml of OPTIMEM-I (Gibco, NY) final volume of DNA-lipid complexes containing 1. mu.g each of the plasmids and 18ul of transformed (transit) LT1(Panvera, Wis.). Transfection may be performed for 6 hours, at which time the DNA-lipid complex may be removed and replaced with fresh medium. The cells were incubated for an additional 24 hours and 0.5. mu.g/ml TPCK treated trypsin (Worthington) was added. After 72 hours, the supernatant was removed from the cell fluid and 100ul was injected into 10 day old embryonated chicken eggs.

Preparation of vaccines

Virus was propagated in the allantoic cavity of 10 to 11 day old embryonated eggs at 35 ℃ for 48 hours. Allantoic fluid was harvested, added with beta-propiolactone (BPL) at a ratio of 1:2000 (by volume), and allowed to stand at room temperature for 4 hours and then at 4 ℃ for 24 hours to inactivate viruses. The treated allantoic fluid was not infected after two blind passages in embryonated eggs, and was confirmed to be inactivated.

Concentration of viruses by Amicon ultrafiltration

The inactivated virus in the allantoic fluid was clarified by centrifugation at 5000rpm for 15 minutes. The supernatant allantoic fluid was concentrated to 1/10 in its original volume using an Amicon concentration ultrafiltration unit.

Purification of viruses

The concentrated virus was purified by ultracentrifugation through 25% and 70% sucrose pads, and the pellet was centrifuged at 27,000rpm at 4 degrees Celsius for 1 hour. The pellet was resuspended in STE buffer, sonicated for 2 minutes, and then centrifuged with a SW28 rotor at 24,000rpm for 2.5 hours in a continuous gradient of 25% -70% sucrose. The viral bands were removed with a syringe, diluted in STE and then pelleted as described above. The pellet was resuspended in a suitable volume of STE and sonicated, and sodium azide was added to a final concentration of 200 ppm.

Standardization of vaccines

By Wood et al (1985) Avian Dis.29: 867-872 (incorporated herein by reference), the hemagglutinin protein content of allantoic fluid, Amicon-concentrated vaccine and purified vaccine can be standardized.

Example 2

Efficacy of inactivated H5N3 avian influenza vaccine in SPF chickens

Vaccine composition

The efficacy of different doses of inactivated avian influenza H5N3 vaccine was tested. An avian influenza H5N3 vaccine was prepared essentially as described in example 1.4 vaccines were prepared, one of which was a placebo vaccine containing virus-free allantoic fluid. The remaining 3 vaccines contained the inactivated avian influenza virus stock solution, and the resulting final preparation contained 1.2 μ g of HA protein (307 HAU)/0.6ml volume dose, 0.5 μ g of HA protein (128 HAU)/0.5ml volume dose, or 0.25 μ g of HA protein (64HAU)/0.5ml volume dose of antigen, based on radioimmunodiffusion (or hemagglutination inhibition) assays of the formulated virus stock solution. The vaccine-related dose levels were 64, 128 and 307 HAU/dose, respectively, in terms of hemagglutination units (HAU). The inactivated antigen stock solution was prepared by passaging once in Vero cells (Vero cells) during the construction of reassortant virus, followed by 6 passages of SPF chick embryos.

The vaccine was formulated as a water-in-oil emulsion (oil: water ratio of 60: 40) using mineral oil as the carrier and tween 80 and span 83 as emulsifiers. The tween and antigen components were mixed separately from Drakeol and span 83 and the aqueous phase was slowly added to the oil phase while stirring to form a pre-emulsion. The pre-emulsion was then mechanically homogenized using a fixed head Silverson L4R homogenizer.

Vaccination

The vaccine was administered intramuscularly in the pectoral muscle to 25 groups of SPF white-skinned chickens (Galluss domimesticacus; from Charles River/SPAFAS). Table 2 summarizes the vaccination protocol. Groups 1-3 were vaccinated with 0.5ml of vaccine and group 4 with 0.6ml of vaccine (see Table 2), using a 3ml sterile disposable syringe with a 20 gauge 1/2 "-3/4" needle. The chickens in group 5 were not vaccinated at week 2 and week 5. The placebo vaccine without antigen was also given intramuscularly to a group of 25 littermates (hashmate) and an additional 25 littermates served as uninoculated controls. Primary inoculations were performed when chickens were 2 weeks old and booster inoculations were performed in the same manner at 5 weeks old.

TABLE 2

Treatment table

Blood samples of the chickens were taken just before the first vaccination and 3 weeks after vaccination for detection of avian influenza antibody titers by Hemagglutination Inhibition (HI) assay. The serum samples before blood collection were all negative.

Attack and sampling

Challenge was performed when the chickens reached 8 weeks of age. The chickens were given challenge virus A/chicken/Vietnam/c58/04 at a dilution of 1:1054 to reach 30 CLD₅₀Chicken, administered by intranasal/intratracheal instillation in a volume of 1.0 ml. Mortality was observed daily for all chickens within 14 days after challenge.

Blood samples were taken from all chickens at 2,5, 8 and 10-11 weeks of age. The blood samples were left at 37 degrees celsius for 30 minutes and then moved to 4 degrees celsius overnight to allow clotting. Serum was aseptically transferred into a single sterile tube for serological analysis by hemagglutination inhibition assay. Serum samples were stored at-30 ℃ or lower for testing.

Live chicken tracheas and broilers were collected 3,5, 7,10 and 14 days after challengeCloacal swabs were virus re-isolated in SPF chick embryos. The swab was placed in a container containing 1ml of virus transport medium (1:1 PBS/glycerol mixture with 2X 10)⁶unit/L penicillin, 2X10⁶Unit/L polymyxin B, 250mg/L gentamicin, 0.5x10⁶unit/L nystatin, 60mg/L ofloxacin HCl, and 0.2gm/L sulfamethoxazole). The swab is frozen at-70 ℃ or lower for testing.

Serology

The results of the serological tests are reported in table 3 below.

TABLE 3

Sera pre-seed, post-primary and post-booster inoculations were assayed for hemagglutination inhibition Serological analysis

^*Chicken (#6, Pen 9) was excluded from consideration due to wrong inoculation

Serum samples taken from all chickens prior to inoculation did not contain detectable avian influenza-specific antibodies by hemagglutination inhibition assays. At 21 days after primary vaccination, placebo-vaccinated control chickens (group 1) and naive chickens (group 5) were still free of antibodies, while the geometric mean titer levels of each group vaccinated with the inactivated prototype (groups 2-4) were 254, 320 and 446, respectively.

At 21 days after the second vaccination, the unvaccinated control chickens (group 5) still had no detectable antibodies against avian influenza. One chicken (#6, Pen 9) in the placebo vaccinated group was found to have a titer of 1280, while all other placebo vaccinated chickens remained seronegative. Since the unvaccinated chickens of the same cohort of this chicken (#6) (pen-mate) were still seronegative, this cohort of chickens improperly exposed to avian influenza was excluded. The only possible explanation for this chicken (#6) antibody response was that it received an incorrect dose of inactivated prototype vaccine during the booster vaccination, rather than placebo vaccine. In this case, it is most appropriate to exclude the chicken from consideration when calculating serological or challenge results.

The geometric mean titer of all groups of chickens receiving the inactivated prototype vaccine reacted was 3671, 4101 and 4335, respectively.

Protection against death

Mortality data after challenge are summarized in table 4 below.

TABLE 4

Mortality following challenge with A/Chicken/Vietnam/c58/04

Treatment group	Death/attack number	Mortality (%)/protection (%)
			1*	24/24*	100/0*
2	0/25	0/100
			3	0/25	0/100
4	0/25	0/100
			5	25/25	100/0

^*Chicken (#6, Pen 9) was excluded from consideration due to wrong inoculation

All chickens not inoculated with the control group died entirely only 3 days after challenge. All chickens in the placebo vaccinated group (except one) died all 4 days after challenge. The chickens that survived the challenge (wing labeled No. 6 (#6, Pen 9)) were excluded from consideration as described above, and both the ungrouped and placebo-vaccinated groups were 100% dead. This met the established validation criteria for the control group, i.e. a mortality of at least 90% was required after challenge.

All chickens of all 3 groups survived the challenge. The protection rate was considered to be 100% (95% CI 86, 100). This meets the criteria for the claimed protection against death, i.e. at least 70% vaccine efficacy.

Re-isolation of viruses

Virus re-isolation data are summarized in table 5 below (tracheal swab) and table 6 below (cloaca swab).

TABLE 5

Virus re-isolation from trachea following challenge with A/chicken/Vietnam/c58/04

^*Chicken (#6, Pen 9) was excluded from consideration due to wrong inoculation

TABLE 6

Re-isolation of virus from the cloaca following challenge with A/Chicken/Vietnam/c58/04

^*Chicken (#6, Pen 9) was excluded from consideration due to wrong inoculation

In the unvaccinated control group, all chickens died 3 days after challenge, before the first sampling, so that no virus re-isolation data was possible in this group.

For the placebo vaccinated group, all chickens (except 2) died before the first sampling day. As described above, chicken #6 (#6, Pen 9) was mistakenly vaccinated with one dose of the test vaccine, and was therefore antibody positive in challenge and therefore survived. Virus could not be re-isolated in swabs taken from the trachea or cloaca of the chicken at any time after challenge. At 3 days post challenge, one day prior to death, virus was re-isolated from the trachea and cloaca of another placebo-vaccinated chicken (#113, Pen 10).

For group 2, the lowest antigen level vaccine tested in the study (0.25 μ g, 64 HAU/dose) was given and virus was re-isolated from the trachea of one of the 25 chickens, 3 and 5 days after challenge. Thereafter, all other chickens in group 2 did not re-isolate virus from tracheal or cloaca swabs until the end of the study.

For group 3, vaccines formulated to contain moderate antigen levels (0.5 μ g, 128 HAU/dose) were administered and the virus was re-isolated from 2 of the 25 tested chickens, one at each sampling point (one 3 days post challenge and the other 5 days post challenge). All other samples from both chickens, as well as all samples from all other chickens in group 3, were negative for virus re-isolation.

For group 4, the vaccine formulated to the highest antigen level measured in the study (1.2 μ g, 307 HAU/dose) was administered and no virus was re-isolated from the trachea or cloaca of any of the 25 chickens tested at any time.

Since the non-treated group and placebo-controlled group died almost all before sampling, these groups lacked re-segregation data and therefore no statistical inference could be made as to re-segregation rates.

Vaccine efficacy

After inoculation of SPF chickens with the prototype at 2 and 5 weeks of age, protection against challenge mortality was observed by detecting high levels of antibody by hemagglutination inhibition assay. Very few viruses were re-isolated from the vaccinated chickens after challenge, so it could not be said that there was a significant decrease in re-isolation rate compared to the controls (since all controls that died before re-isolation could be evaluated in these groups), and it is clear that the vaccine resulted in a significant reduction in release. For such clear evidence, it is necessary to modify the challenge protocol so that naive or placebo-vaccinated chickens do not die immediately after challenge. This is difficult to do because the H5N1 challenge strain is very pathogenic to chickens.

The levels of 3 different antigens of such whole virus inactivated vaccines were determined. The results were essentially identical for all 3 antigen levels, so the lowest protective dose was not determined in this study. Further studies were performed with vaccines formulated to contain less than 0.25 μ g (64HAU) of antigen per dose, which is necessary to determine the minimum dose. At present, it can be concluded that two inoculations with an adjuvanted water-in-oil emulsion containing not less than 0.25 μ g (64 HAU)/dose of H5N3 reassortant virus are highly effective in preventing death caused by the highly virulent field isolate of vietnam H5N1, and also in preventing the release of highly virulent H5N 1.

The vaccine provides 100% protection against death. The re-isolation of challenge virus from all live chicken tracheal swabs and cloaca swabs at different time points was minimal. It was concluded that two inoculations with an adjuvanted water-in-oil emulsion containing no less than 0.25 μ g (64HAU) per dose of H5N3 reassortant virus were highly effective in preventing death induced by the highly virulent field isolate of Vietnam H5N1(H5N1 Vietnam) and also in preventing the release of highly virulent H5N 1.

Example 3

Inactivated H5N3 avian influenza vaccine in SPF chickens

5 vaccines were prepared essentially as described in example 1. The vaccines were formulated to contain the following Table 7 (treatment Table)) Viral content/dose listed. HAU and EID₅₀The concentration of virus used in the assay was based on the titer of the virus mother liquor prior to inactivation. The indicated concentration of H5 protein antigen (μ g) is based on post-inactivation antigen stock titers measured using standardized single-radiation immunodiffusion (SRID). The indicated amount of antigen per dose was calculated from the measurement of these stock antigens and the prepared% antigen solution.

The vaccine was formulated as a water-in-oil emulsion (oil: water ratio of 60: 40) using mineral oil as the carrier and tween 80 and span 83 as emulsifiers. The tween and antigen components were mixed separately from Drakeol and span 83 and the aqueous phase was slowly added to the oil phase while stirring to form a pre-emulsion. The pre-emulsion was then mechanically homogenized using a fixed head Silverson L4R homogenizer.

TABLE 7

Treatment table

Treatment group	Vaccine	Number of chickens	Blood sampling
				1	64 HAU/107.0 EID500.09 μ g H5 (production batch No. 2285-26-06JUL05)	25	21dpv
2	128 HAU/107.3 EID500.18 μ g H5 (production batch No. 2285-21-28JUN05)	25	21dpv
				3	256 HAU/107.6 EID500.35 μ g H5 (production run 2250-55-09MAR05)	25	21dpv
4	512 HAU/107.9 EID500.71 μ g H5 (production run 2250-56-09MAR05)	25	21dpv
				5	1024 HAU/108.2 EID50/1.42 μ g H5 (production run 2250-57-09MAR05)	25	21dpv
6	Non-inoculated control	25	Same day as the inoculation group

Inoculation of

For each vaccine, 25 chickens were inoculated subcutaneously once in a total volume of 0.5 ml. 25 littermates were used as uninoculated controls. All chickens are fed with leg hoops and mixed with the chickens in the same column until blood sampling and finishing or sending to attack. Blood samples were taken from all chickens 3 weeks after inoculation.

Attack of

For groups 1-4 and 6, optionally 20 chickens per group, challenge with highly pathogenic avian influenza a/chicken/vietnam/c58/04 strain (H5N 1). A lethal dose (CLD) of 30 chickens of virus was administered to each chicken by intranasal/intratracheal instillation. As described in example 2, this viral dose has previously been shown to be effective in causing 100% mortality in uninoculated control chickens.

The mortality of the chickens was observed after challenge for a total of 14 days. Additionally, tracheal and cloaca swabs from all surviving chickens were re-isolated 4 days after challenge.

Serological reaction

Back titration of H5N3 antigen stock/dilution was performed for HI assay, confirming the use of 8HA units of antigen per 50 uL. A homologous H5N3 serum pool was established to serve as a positive control sample, demonstrating dilution of HI activity at 1:320 or 1: 640. PBS control wells had no HI activity, while 25 non-inoculated control sera had no HI activity at 1:10 dilution. These results indicate that, in general terms, the HI determination and potency test have been reasonably completed according to the criteria set forth in SO # 309.

The antibody responses of the groups vaccinated with the experimental prototype vaccine (and the unvaccinated control group) by the effective HI assay are summarized in table 8 below, and the results for each chicken are reported in tables 9-14 below.

TABLE 8

Summary of serological response to vaccination with inactivated prototype H5N3 vaccine as determined by hemagglutination inhibition

H5N3(BPL inactivated) antigen stock 2228-90-15FEB05 for all HAI assays

^*Removing non-responders

TABLE 9

Administration series 2285-26(64 HAU/10) ^7.0 EID ₅₀ Per 0.09. mu. g H5/dose) of each chicken

Response and protection against death

Chicken number #	HI potency	Death (+/-)	Heavy separation
				401	20	－	+
402	<10	+	n.d.
				403	<10	+	n.d.
404	<10	+	n.d.
				405	20	n.d.	n.d.
406	40	n.d.	n.d.
				407	≥640	－	－
408	320	－	－
				409	160	－	－
410	≥640	－	－
				411	80	－	－
412	≥640	－	－
				413	≥640	－	－
414	≥640	－	－
				415	≥640	n.d.	n.d.
416	40	－	+
				417	≥640	－	－
418	20	n.d.	n.d.
				419	80	－	－
420	≥640	－	－
				421	40	－	－
422	≥640	－	－
				423	≥640	n.d.	n.d.
424	≥640	－	－
				425	<10	+	n.d.

n.d. ═ not determined, chickens did not challenge or died before swabs were taken

Watch 10

Administration series 2285-21(128 HAU/10) ^7.3 EID ₅₀ Per 0.18. mu. g H5/dose) of each chicken serum

Chemical reactions and protection against death

Chicken number #	HI potency	Death (+/-)	Heavy separation
				226	320	-	-
227	≥640	-	-
				228	320	-	-
229	160	-	-
				230	≥640	-	-
231	≥640	-	-
				232	<10	+	n.d.
233	<10	n.d.	n.d.
				234	<10	+	n.d.
235	≥640	n.d.	n.d.
				236	≥640	-	-
237	<10	n.d.	n.d.
				238	≥640	-	-
239	≥640	-	-
				240	<10	-	-
241	≥640	n.d.	n.d.
				242	≥640	-	+
243	≥640	-	-
				244	<10	+	n.d.
245	≥640	n.d.	n.d.
				246	≥640	-	-
247	≥640	-	-
				248	≥640	-	-
249	≥640	-	-
				250	320	-	-

n.d. ═ not determined, chickens did not challenge or died before swabs were taken

TABLE 11

Administration series2250-55(256 HAU/10 ^7.6 EID ₅₀ Per 0.35. mu. g H5/dose) of each chicken serum

Chemical reactions and protection against death

Chicken number #	HI potency	Death (+/-)	Heavy separation
				301	≥640	-	-
302	≥640	-	-
				303	10	-	+
304	<10	+	n.d.
				305	≥640	-	-
306	<10	+	n.d.
				307	≥640	n.d.	n.d.
308	320	-	-
				309	≥640	n.d.	n.d.
310	≥640	-	-
				311	≥640	-	-
312	≥640	-	-
				313	≥640	-	-
314	≥640	n.d.	n.d.
				315	≥640	-	-
316	40	-	+
				317	<10	+	n.d.
318	<10	+	n.d.
				319	≥640	-	-
320	≥640	n.d.	n.d.
				321	80	n.d.	n.d.
322	≥640	-	-
				323	≥640	-	-
324	≥640	-	-
				325	≥640	-	-

n.d. ═ not determined, chickens did not challenge or died before swabs were taken

TABLE 12

Administration series 2250-56(512 HAU/10) ^7.9 EID ₅₀ Per 0.71. mu. g H5 dose) of blood from each chicken

Clearing response and protection against death

Chicken number #	HI potency	Death (+/-)	Heavy separation
				276	≥640	-	-
277	≥640	-	-
				278	≥640	-	-
279	≥640	n.d.	n.d.
				280	≥640	-	-
281	≥640	-	-
				282	≥640	-	-
283	≥640	-	-
				284	<10	+	n.d.
285	≥640	n.d.	n.d.
				286	≥640	-	-
287	≥640	-	-
				288	≥640	-	-
289	≥640	-	-
				290	≥640	n.d.	n.d.
291	≥640	-	-
				292	≥640	-	-
293	≥640	n.d.	n.d.
				294	≥640	-	-
295	≥640	-	-
				296	≥640	-	-
297	≥640	-	-
				298	≥640	n.d.	n.d.
299	≥640	-	-
				300	≥640	-	-

n.d. ═ not determined, chickens did not challenge or died before swabs were taken

Watch 13

Administration series 2250-57(1024 HAU/10) ^8.2 EID ₅₀ Per 1.42. mu. g H5/dose) of the blood of each chicken

Clearing response and protection against death

Chicken number #	HI potency	Death (+/-)	Heavy separation
				201	≥640	n.d.	n.d.
202	≥640	n.d.	n.d.
				203	≥640	n.d.	n.d.
204	≥640	n.d.	n.d.
				205	≥640	n.d.	n.d.
206	≥640	n.d.	n.d.
				207	≥640	n.d.	n.d.
208	≥640	n.d.	n.d.
				209	≥640	n.d.	n.d.
210	<10	n.d.	n.d.
				211	<10	n.d.	n.d.
212	≥640	n.d.	n.d.
				213	≥640	n.d.	n.d.
214	≥640	n.d.	n.d.
				215	≥640	n.d.	n.d.
216	≥640	n.d.	n.d.
				217	≥640	n.d.	n.d.
218	≥640	n.d.	n.d.
				219	≥640	n.d.	n.d.
220	≥640	n.d.	n.d.
				221	<10	n.d.	n.d.
222	≥640	n.d.	n.d.
				223	≥640	n.d.	n.d.
224	≥640	n.d.	n.d.
				225	≥640	n.d.	n.d.

n.d. ═ not determined, chickens did not challenge or died before swabs were taken

TABLE 14

Susceptibility to attack-induced death and lack of serological response in chickens not vaccinated with the control group

Chicken number #	HI potency	Death (+/-)	Heavy separation
				326	<10	+	n.d.
327	<10	+	n.d.
				328	<10	+	n.d.
329	<10	+	n.d.
				330	<10	+	n.d.
331	<10	+	n.d.
				332	<10	+	n.d.
333	<10	+	n.d.
				334	<10	+	n.d.
335	<10	n.d.	n.d.
				336	<10	+	n.d.
337	<10	n.d.	n.d.
				338	<10	+	n.d.
339	<10	n.d.	n.d.
				340	<10	+	n.d.
341	<10	+	n.d.
				342	<10	n.d.	n.d.
343	<10	+	n.d.
				344	<10	+	n.d.
345	<10	+	n.d.
				346	<10	n.d.	n.d.
347	<10	+	n.d.
				348	<10	+	n.d.
349	<10	+	n.d.
				350	<10	+	n.d

n.d. ═ not determined, chickens did not challenge or died before swabs were taken

The geometric mean titer levels of the reactions of each group (groups 1-5) inoculated with the inactivation prototype were 109, 174, 199, 527, and 328, respectively, when considering the results of each chicken tested. A large number of chickens had no detectable response indicating that they were incorrectly vaccinated or that they were non-responsive to vaccination. If these chickens were excluded from consideration only, the geometric mean titers of groups 1-5 were 195, 533, 403, 640 and 640, respectively. Note that: in the calculation of the geometric mean titer, one chicken had no detectable reaction (<1:10, lowest dilution measured), and the titer was considered to be 5. For the chickens with a reaction record of 640 (highest dilution measured), the actual endpoint must be higher than the test performed, but these chickens are still counted as a titer of 640. Thus, the geometric mean titer is the limiting value for evaluating the effectiveness of a vaccine.

For serological responses, increasing dose levels of group 1-5 seeds were 18/25 (72%), 19/25 (76%), 20/25 (80%), 24/25 (96%), and 22/25 (88%), respectively, with titers of 1:40 or greater.

Thus, the serological test results were further evaluated on a per chicken basis, and it was observed that some chickens had no detectable post-vaccination antibody titers. Considering the results of previous efficacy studies in which the same vaccine was tested and found to elicit reliable antibody responses, it is likely that mis-vaccination of these chickens is a significant factor. Further explanation is provided in the following discussion of attack results.

Protection against attacks

The results of this study are believed to demonstrate efficacy against death caused by a virulent H5-type avian influenza challenge. As the summary data shown in table 15 below clearly indicates, the highly pathogenic avian influenza virus strain a/chicken/vietnam/c58/04 (H5N1 type) caused 100% of the deaths of the unvaccinated control chickens within 2 days post challenge. At the same time, all vaccinated groups were protective against death after the same challenge, with a level of protection of 80% or more. However, both vaccines that were considered unsatisfactory in efficacy in this study were also protective against challenge. This is believed to indicate that the efficacy test method is stringent, with conservative criteria for demonstrating the ability of batches to provide protection under the test conditions.

Watch 15

Prevention of infection by avian influenza A/chicken/Vietnam/c58/04 (H5N1) with inactivated rgH5N3 vaccine

Death of (1)

Treatment group	Antigen/agent	Death/attack number	Mortality (%)	Prevention rate (95% CI)
					1(2285-26)	64 HAU107.0 EID500.09μg H5	4/20	20％	80％(56，94.3)
2(2285-21)	128 HAU107.3 EID500.18μg H5	3/20	15％	85％(62.1，96.8)
					3(2250-55)	256 HAU107.6 EID500.35μg H5	4/20	20％	80％(56.3，94.3)
4(2250-56)	512 HAU107.9 EID500.71μg H5	1/20	5％	95％(75.1，99.9)
					6(N/A)	Nonvac	20/20	100	0

Note that: processing group 5 not under attack

Swabs taken from trachea and cloaca of surviving chickens were also virus re-isolated 4 days after challenge. As previously seen, the mortality rate of the non-vaccinated chickens after challenge was 100%, and thus the re-segregation of the control group could not be determined, and thus a statistical evaluation of the protection against the re-segregation could not be performed.

Because each chicken had leg hoop designations, each chicken could track both serological and protective responses as reported in tables 9-14. Of particular interest are HAI titers determined by standardized potency assays and comparisons between mortality and re-isolation following viral challenge. As shown in table 16, mortality after challenge was virtually established for negative antibody-reactive (<10) chickens. In the case of chickens with a low antibody response (10-40), death was completely prevented, but virus release was not prevented. For chickens with an antibody response of 80 or higher, death and release from the trachea and/or cloaca is substantially prevented.

TABLE 16

Potency assay potency (HAI) vs. challenge in vaccinated chickens regardless of vaccine dose

Relationship between protection against highly toxic H5N1 avian influenza

Potency panel	Number of chickens in the titer group	Mortality rate	Positive rate of re-separation
				<10	13	12/13	0/1
10-40	5	0/5	4/5
				>40	62	0/62	1/62

The prevention rate of mortality was 100% (95% CI 94.6, 100) with detectable titers (> 1:10) compared to chickens with no detectable titers (<1: 10). The prevention rate of mortality was 100% for chickens with titers >1:40 compared to chickens with titers <1:10 (95% CI 94.2, 100). The number of chickens with intermediate titer levels (1:10-1:40) is not sufficient, and no statistical inference can be made about the protective nature of such titers. In addition, a threshold of 1:40 or greater appears to be a suitable location in the vaccination/serological release assay to ensure proper confirmation of batch efficacy.

These results indicate that proper vaccine administration is critical. Even the best vaccines, if the administration is not optimal and the chicken receive a less than optimal antibody response, this will also cause the surviving chicken to release the virus. If this is the case only, a two-dose regimen should be carefully considered for vaccination programs where it is desirable not only to prevent death of chickens exposed to highly pathogenic H5N1 avian influenza, but also to reduce viral release. Therefore, a field safety study (field safety study) performed with the support of conditional permission will involve the use of two inoculations.

Conclusion

For the contained dosage of 64 HAU/10^7.0 EID₅₀The efficacy against death caused by highly pathogenic H5-type avian influenza has been demonstrated for inactivated vaccines of H5N3 strain at doses of 0.09. mu. g H5 or higher.

Example 4

Efficacy of H5N3 inactivated influenza vaccine on ducks

Vaccine

Several vaccines were prepared, one of which was a placebo vaccine containing virus-free allantoic fluid. All other vaccines contained inactivated avian influenza virus stock solutions prepared essentially as described in example 1 and table 17. The inactivated antigen stock solution was prepared by passaging once in Vero cells (Vero cells) during the construction of reassortant virus, followed by 6 passages of SPF chick embryos.

The vaccine was formulated as a water-in-oil emulsion (oil: water ratio of 60: 40) using mineral oil as the carrier and tween 80 and span 83 as emulsifiers. The tween and antigen components were mixed separately from Drakeol and span 83 and the aqueous phase was slowly added to the oil phase while stirring to form a pre-emulsion. The pre-emulsion was then mechanically homogenized using a fixed head Silverson L4R homogenizer.

TABLE 17

Vaccines for use

Inoculation of

Prior to inoculation, ducks (Anas platyrhynchos; from Ideal Poultry, Cameron, Texas) were fitted with leg cuffs with records of treatment groups and columns. Ducks were inoculated as shown in tables 18-20 below. The ducks of experiment 1 were inoculated intramuscularly at the breast of the duck with 0.5ml (groups 1-3) or 0.6ml (group 4) using a 3ml sterile disposable syringe with a 20 gauge 1/2 "-3/4" needle. Group 5 ducks were not vaccinated. Primary vaccination was performed when ducks were 2 weeks old and booster vaccination was performed in the same way at 5 weeks old.

The duck of experiment 2 was inoculated intramuscularly at 0.5ml to the breast of the duck using a 3ml sterile disposable syringe with a 20 gauge 1/2 "-3/4" needle. Primary vaccination was performed when ducks were 2 weeks old and booster vaccination was performed in the same way at 5 weeks old.

The ducks of experiment 3 were also vaccinated intramuscularly at the breast of the duck with 0.5ml, except that group 8 received 0.25ml series # 2228-51. The ducks were vaccinated when they were 1 week old, not given booster vaccination.

Watch 18

Treatment Table- #1

Watch 19

Treatment Table- #2

Watch 20

Treatment Table- #3

Attack of

In experiment 1 (see table 18), the challenge was performed when the duck reached 8 weeks of age. The challenge virus A/chicken/Vietnam/c58/04 (30 CLD)₅₀(10^4.5 EID₅₀) Duck) was administered via intranasal instillation in a volume of 1.0 ml. All ducks were observed daily for mortality for at least 14 days after challenge.

In experiment 2 (see table 19). Challenge was performed when the ducks reached 8 weeks of age. The challenge virus A/chicken/Vietnam/c58/04 (30 CLD)₅₀(10^4.5 EID₅₀) Duck) was administered via intranasal instillation in a volume of 1.0 ml. All ducks were observed daily for mortality for at least 14 days after challenge.

In experiment 3 (see table 20), the challenge was performed when the ducks reached 8-9 weeks of age (59 days). Attack virus A/duck/Thailand/D4AT/04 (A/duck/Thailand/D4AT/04) (100 DLD)₅₀(10^5.0 EID₅₀) Duck) was administered via intranasal instillation in a volume of 1.0 ml. All ducks were observed daily for mortality for at least 14 days after challenge.

Sampling

Blood samples were taken from all ducks at different time points after inoculation and after challenge. The blood samples were left at 37 degrees celsius for 30 minutes and then moved to 4 degrees celsius overnight to allow clotting. Serum was aseptically transferred into a single sterile tube for serological analysis by hemagglutination inhibition assay. Serum samples were stored at-30 ℃ or lower for testing.

Swabs taken from live duck trachea and cloaca were virus re-isolated in SPF chick embryos at various time points after challenge. The swab was placed in a container containing 1ml of virus transport medium (1:1 PBS/glycerol mixture with 2X 10)⁶unit/L penicillin, 2X10⁶Unit/L polymyxin B, 250mg/L gentamicin, 0.5x10⁶unit/L nystatin, 60mg/L ofloxacin HCl, and 0.2gm/L sulfamethoxazole). The swab is frozen at-70 ℃ or lower for testing.

Vaccine efficacy of experiment 1

The results of experiment 1 are summarized in table 21 below.

TABLE 21

Efficacy of inactivated H5N3 influenza vaccine on Duck (experiment 1)

dpv equals days post inoculation

dpb days after boost inoculation

dpc is the number of days after challenge

Various doses of vaccine (1.2 μ g, 0.5 μ g and 0.25 μ g) induced detectable levels of antibody by HI assay; geometric mean titers at 21 days post primary vaccination were 52, 45 and 72, respectively. After re-inoculation, titers increased to 220, 151 and 290, respectively. At the same time, none of the placebo groups had detectable antibodies after the initial or re-inoculation. After challenge, the antibody titers of the placebo-vaccinated groups increased much, while those of the antigen-vaccinated groups did not change.

There were no signs of death or disease in placebo (or vaccinated) ducks challenged with A/chicken/Vietnam/C58/04 (H5N1) virus. However, virus was reisolated 3 days after challenge from all ducks in the placebo vaccinated group, but not from any of the vaccinated groups.

The vaccine efficacy against virus isolation from tracheal swabs was 100% (95% CI, 76.43% -100%), 100% (95% CI, 73.06% -100%) and 100% (95% CI, 74.86% -100%), respectively, for vaccine doses of 0.25, 0.5 and 1.2. The vaccine efficacy against virus isolation from cloacal swabs was 100% (95% CI, 75.07% -100%), 100% (95% CI, 71.47% -100%) and 100% (95% CI, 73.39% -100%), respectively, for vaccine doses of 0.25, 0.5 and 1.2.

Vaccine efficacy of experiment 2

The results of experiment 2 are summarized in table 22 below.

TABLE 22

Efficacy of inactivated H5N3 influenza vaccine on Duck (experiment 2)

dpv equals days post inoculation

dpb days after boost inoculation

dpc is the number of days after challenge

In this experiment, ducks receiving vaccine formulations with lower antigen content (0.0313, 0.0625, and 0.125 μ g HA) than in the first experiment had geometric mean HI titers of <10, 21, and 15, respectively. The lowest dose of vaccine (0.0313) induced a detectable HI response in only 5 out of 10 ducks after the initial vaccination. After inoculation with 0.0625 or 0.125 μ g HA, 8/10 groups of ducks had detectable HI antibodies. In the 0.25 μ g group, all 10 ducks responded to the primary vaccination. Upon re-inoculation, HI titers were significantly increased for all groups ranging from 92(0.0313) to 435(0.25), while placebo-inoculated groups were still negative. After challenge, the antibody titers of the placebo-vaccinated groups increased much, while those of the antigen-vaccinated groups were essentially unchanged.

After challenge, the challenge virus replicated only in 10/10 ducks that were not vaccinated with the control group. No viral replication was detected in ducks receiving 0.0313 or 0.0625 μ g HA at both doses. In contrast, the ducks with 2/10 in the 0.125 μ g group and 1/20 in the 0.25 μ g group released the lowest detected level of virus for 1 day. Compared to the control, the efficacy of the vaccine against tracheal release was 100% (95% CI, 68.05% -100%) after two doses of 0.0313 to 0.0625 μ g, while the efficacy of the vaccine against tracheal release was 94.44% (95% CI, 73.5% -9.82%) after two doses of 0.25 μ g. The efficacy of the vaccine to prevent tracheal release was 88.89% (95% CI, 51.87% -99.65%) after two doses of 0.125 μ g compared to control ducks raised in mixed pens.

Vaccine efficacy of experiment 3

Both initial experiments used a prime-boost protocol and all antigen levels tested induced protection against viral release. Thus, in a third experiment, a single vaccination with a lower antigen content vaccine was tested.

TABLE 23

Efficacy of inactivated H5N3 influenza vaccine on Duck (experiment 3)

dpc is the number of days after challenge

The pre-challenge antibody titers ranged from <10(0.015 μ g HA) to 101(0.5 μ g HA). In the vaccinated ducks, there was no increase in HI antibody titers against the challenge virus after challenge, but in the placebo-control group, indicating that the challenge virus did not replicate in the vaccinated ducks.

No signs of death or disease were detected in any vaccinated ducks. In contrast, 8/12 died in the placebo group, with the remainder having severe signs including neurological abnormalities and/or ocular cloudiness. The ducks were binned into treatment groups so that the binning effect was not excluded in the statistical evaluation of the protective differences provided by the tested vaccines. Nevertheless, p-values were calculated with Bonferroni adjustments, based on protective statistical analysis of individual ducks. In this case, the mortality rate was significantly prevented (p 0.0329) in the duck stalls vaccinated with 1.2, 0.0625, 0.0313 or 0.015 μ g vaccine dose compared to the duck stalls in the control stalls, and the vaccine efficacy was 100% in these groups (95% CI, 34.94% -100%). The difference in mortality between ducks vaccinated at 0.125, 0.5 and 0.25 μ g vaccine doses in each panel was not statistically significant compared to the control panel (p-0.0897). This may reflect a low statistical rate of the study, since the number of ducks that can be raised in the BSL3+ facility is limited, and the vaccine is not necessarily efficacious at these doses. The efficacy of the vaccine to prevent mortality in the duck cages receiving 0.125 μ g and 0.25 μ g vaccine doses was 100% (95% CI, 38.63% -100%) compared to the control. The efficacy of the vaccine to prevent mortality in duck cages receiving a vaccine dose of 0.5 μ g was 100% (95% CI, 22.03% -100%) compared to the control.

Conclusion

All measured dose levels and/or vaccination protocols induce a protective response, whether from the standpoint of preventing death and/or preventing release of the virus from the trachea or cloaca. For formulation, the use of a mineral oil based adjuvant system (an emulsion providing an oil to water ratio of 60/40 with span 83 and tween 80 as emulsifiers) proved to be particularly effective for ducks. Also, the emulsion proved to be very effective on formulations for chicken and related inactivated bivalent avian influenza products (Poulvac i-AI H5N9, H7N1, produced by FDAH in the netherlands).

In experiment 1, all dose levels were protective after two vaccinations, whereas experiment 2 evaluated the use of lower dose levels to define the lowest dose required for protection. However, even two vaccinations with a lower antigen content vaccine are completely protective against post challenge virus release. This resulted in the study design of experiment 3, in which a single dose vaccine (including a lower antigen content vaccine) was evaluated and challenged with a thailand isolate known to cause duck death.

Thus, experiment 3 represents the most rigorous trial performed in which ducks received a single dose of vaccine only at one week of age and were challenged with isolates highly pathogenic to waterfowl at 8-9 weeks of age. In this model, a single inoculation of 4HAU of avian influenza antigen (1/2 volumes of series 2228-51 doses) was demonstrated to be effective in preventing death and in preventing virus release from the trachea and cloaca. This experiment also demonstrated that the duration of immunity after administration of a single dose of vaccine was at least 7 weeks.

Taken together, these 3 experiments demonstrated the effectiveness of the vaccine on ducks in two different situations. The first two experiments prove that the vaccine can prevent the carrying state of the duck, because the inoculated duck does not release virus after being attacked by the isolate which can not cause obvious clinical symptoms or death of waterfowl but has high pathogenicity to chickens. The third experiment demonstrates that the vaccine can prevent the clinical symptoms, death and virus release of ducks only after challenge with known waterfowl pathogenic isolates.

For this vaccine, the recommended vaccination protocol will be combined with the results report of experiments performed in CSIRO/AAHL (Australia), and appropriate consideration of the epidemiology of avian influenza and conventional feeding methods for commercial production of ducks. The recommended regimen is a vaccine containing 256HAU/0.5ml (antigen content greater than that demonstrated to be protective in the current report and much less than that used in the safety study), a smaller volume day-of-age (day-of-age) dose administered subcutaneously, followed by a full volume booster dose at 3 weeks of age (one regimen demonstrated to be effective in the reference CSIRO study). According to this protocol, ducks can receive the benefit of early vaccination, booster vaccination given to shift any birds that are mistakenly vaccinated at day age (day of age) and/or boost immune responses to very high levels.

Example 4

Effect of commercial duck before immunization and inactivated avian influenza vaccine on attack of highly pathogenic avian influenza virus

Should be evaluated

Vaccine preparation

Infected allantoic fluid (passaged 2 times in SPF eggs) was diluted in sterile PBSA to give about 10 per 0.5ml^1.5Duck infection dose₅₀(DID₅₀) Or 10^4.7Infection dose of chicken₅₀(EID₅₀) The dosage of (a). Back-titration of the inoculum in SPF eggs confirmed the virus titer in the challenge.

Inoculation of

Commercial ducklings (Beijing ducks) of 60 days old were randomly divided into 3 groups of 20 ducks each. Before immunization, 10 ducklings were screened for avian influenza antibodies by c-ELISA. Each duckling tested is negative for avian influenza antibody.

3 groups were treated as follows:

group A: inoculation (inactivated H5 vaccine Poulvac i-AI H5N9, H7N1) at 1 day and 3 weeks of age, followed by intranasal (0.2ml), intraocular (0.1ml) and oral (0.2ml) routes at 6 weeks of age, with 0.5ml of viral suspension H5N1 (containing 10 of^1.5 DID₅₀(10^4.7 EID₅₀) ) attack.

Group B: inoculation (inactivated H5 vaccine Poulvac i-AI H5N3) at 1 day and 3 weeks of age, followed by intranasal (0.2ml), intraocular (0.1ml) and oral (0.2ml) routes at 6 weeks of age, with 0.5ml of viral suspension H5N1 (containing 10 of 10)^1.5 DID₅₀(10^4.7 EID₅₀) ) attack.

Group C: at 6 weeks of age by intranasal (0.2ml), ocularInternal (0.1ml) and oral (0.2ml) routes, 0.5ml virus suspension H5N1 (containing 10)^1.5 DID₅₀(10^4.7 EID₅₀) ) attack.

Day-old vaccination was given at a dose of 0.2ml subcutaneously in the upper part of the neck. A 0.5ml dose was re-inoculated subcutaneously in the upper part of the neck at 3 weeks of age.

Before challenge, the number of groups was reduced to 15 per group (14 controls), and all ducks were individually identified with a numbered leg cuff. Blood samples were taken from each duck just prior to challenge for serological studies, and then from each surviving duck 11 and 14 days after challenge for serological studies. The challenge was performed with a 1ml graduated syringe. The inoculum is carefully added to the open eyes and mouth through one or both nostrils.

The duck was observed daily for clinical symptoms throughout the study and twice daily for acute phase. To monitor virus release following challenge, tracheal swabs and cloaca swabs were collected from each challenged duck at 4, 5,6 and 7 days post challenge. The selected samples are then used for virus isolation in SPF eggs or commercial eggs (if SPF eggs are not feasible).

Mortality and morbidity

4 days after challenge, all control ducks (group C) were inactive and anorexia accompanied by green diarrhea and significant weight loss. On day 10, 4 control ducks died without clinical symptoms, another 5 had neurological symptoms (twitching, head bending, head swinging, wing paralysis) and the remaining 5 of 14 of this group.

In each of the two groups (group a and group B), all 15 performed clinically well throughout the study period.

Serology

Sufficient blood was collected by jugular vein or wingpuncture for all serological experiments. Serum was harvested and stored at-20 ℃ or lower for testing. All non-vaccinated ducks were HI-active negative for vietnam H5 antigen before challenge with H5N1 virus. Interestingly, there were differences in post-vaccination, pre-challenge HI titers between the two vaccinated groups, with ducks of group B H5N3 (N15) having higher titers (1:8 to 1:64) compared to group a (N15) (negative to 1:8) given bivalent vaccine.

Obviously, sera from 5 surviving control ducks in group C were switched to challenge with titers ranging from 1:32 to 1:256 on day 11 and 1:64 to 1:128 on day 14.

The sera of most ducks in group a (bivalent vaccine) were not converted to those against vietnam H5 before challenge, but 6 of them were seroconverted only on day 11 and most on day 14 (1:32 and 1: 128). 2 ducks with detectable HI titer before challenge were still single double diluted to escape seroconversion by day 14 (Two birds white had detected double-challenge HI titer trees one double dilution restriction from seroconversion by day 14). This suggests that virus replication in these 2 ducks may be inhibited to a slightly greater extent than in other members of the same group, and seroconversion of these 2 ducks may occur several days later than in the non-immunized ducks. Serological studies were necessary to confirm this at a later time after challenge.

In group B (reverse genetics vaccine), all duck sera were converted to those directed against vietnam H5 prior to challenge. However, no ducks showed a titer increase after challenge, indicating that no viral infection was established in this group of ducks.

Viral delivery

Tracheal swabs and cloaca swabs were collected from each challenged duck 4, 5,6 and 7 days after challenge. After the tracheal and cloaca samples were collected, the samples were placed in isotonic phosphate buffered saline (PBS, pH7.0-7.4) containing antibiotics. The suspension was stored at-80 ℃ prior to egg inoculation.

Virus isolation was performed in SPF embryonated poultry eggs, unless otherwise indicated. Supernatants from samples collected 4 and 7 days post challenge were inoculated into the allantois of at least 3 embryonated eggs incubated for 9-11 days. Eggs were incubated at 35-37 degrees celsius for up to 5 days. The Hemagglutination (HA) activity of allantoic fluid from eggs from dead or dying embryos, and all eggs remaining at the end of the incubation, was determined. All allantoic fluids with hemagglutinating activity were considered positive for the AI virus administered.

Group C: tracheal swabs from each control duck were re-isolated for avian influenza virus at 4 or 7 days post challenge, and cloacal swabs from 2 of these ducks were re-isolated for avian influenza virus at 4 days. These observations were consistent with those observed in ducks given the same challenge dose in a previous titer study.

Group A: re-isolation of avian influenza virus was performed from the trachea of 2 bivalently vaccinated ducks on day 4, and from cloacal swabs of 1 of these ducks on day 4. No virus was reisolated from any of the ducks in this group 7 days after challenge.

Group B: avian influenza virus was not reisolated from tracheal swabs and cloaca swabs from any duck taken with the reverse genetics H5N3 vaccine 4 or 7 days post challenge.

Conclusion

High levels of morbidity (100%) and mortality (65%) were observed in the control ducks. From the results of the combined release of the virus and seroconversion of survivors, this indicates that when the challenge virus is administered to commercial ducks that have never been exposed to the challenge virus, it is able to infect and cause disease.

Vaccination with bivalent inactivated H5 vaccine Poulvac i-AI H5N9, H7N1 did not result in the conversion of the serum of most ducks of this group to vietnam H5, but protected the ducks from developing symptoms of avian influenza disease. After challenge, the sera of most ducks were turned against H5, indicating that viral replication occurred in this group of ducks. The re-isolation of AI viruses from these ducks supports such serological interpretation.

Vaccination with the inactivated H5 vaccine Poulvac i-AI H5N3 resulted in all ducks' sera being converted to be directed against vietnam H5 and being protective against disease after challenge with H5N1 virus. The group of ducks did not seroconvert after virus challenge, indicating that virus replication was inhibited in these ducks. This was supported by the absence of re-isolation of AI virus from tracheal or cloaca swabs taken from day 4 or day 7.

Example 5

Vaccine composition

Adjuvants are selected based on the well-known immunostimulatory effect of mineral oil emulsions and formulated as water-in-oil (W/O) emulsions. Pharmaceutical grade light mineral oil (NF) Drakeoil 5 may be used in the formulation.

To obtain a stable water-in-oil emulsion (W/O), a surfactant may be used. The surfactants sorbitan sesquioleate (vegetable), hydrophobic surfactants and polysorbate 80 (vegetable), hydrophilic surfactants are chosen because of their emulsifying properties. It is known to use combinations of these surfactants to obtain stable emulsions.

Span 83V (Arlacel 83V) ═ sorbitan sesquioleate, an equimolar mixture of mono-and diesters;

CAS No. 8007-43-9, for the preparation of creams, milks and ointments.

Tween 80V (Tween 80V) ═ polysorbate 80 ═ polyoxyethylene 20 sorbitan monooleate;

CAS number 9005-65-6, was used to prepare stable oil-in-water emulsions.

Sorbitan esters (e.g., span 83V) produce stable W/O emulsions, but are often used in combination with varying proportions of polysorbates (e.g., tween 80V) to produce W/O emulsions.

Span 83V and Tween 80V used for formulating the product are both derived from plants.

Adjuvants are selected based on the well-known immunostimulatory effect of mineral oil emulsions and formulated as water-in-oil (W/O) emulsions. Pharmaceutical grade light mineral oil (NF) Drakeoil 5 may be used in the formulation.

In order to obtain a stable water-in-oil emulsion (W/O), it is necessary to use a surfactant. The surfactants sorbitan sesquioleate (vegetable), hydrophobic surfactants and polysorbate 80 (vegetable), hydrophilic surfactants are chosen because of their emulsifying properties. It is known to use combinations of these surfactants to obtain stable emulsions.

Span 83V (Arlacel 83V) ═ sorbitan sesquioleate, an equimolar mixture of mono-and diesters;

CAS No. 8007-43-9, for the preparation of creams, milks and ointments.

Tween 80V (Tween 80V) ═ polysorbate 80 ═ polyoxyethylene 20 sorbitan monooleate;

CAS number 9005-65-6, was used to prepare stable oil-in-water emulsions.

Sorbitan esters (e.g., span 83V) produce stable W/O emulsions, but are often used in combination with varying proportions of polysorbates (e.g., tween 80V) to produce W/O emulsions.

Span 83V and Tween 80V used for formulating the product are both derived from plants.

Dosage volumes of 0.5ml are commonly used in the poultry industry.

Watch 24

Product composition (dosage)

Ingredient name	Number of	Function(s)
			Active ingredientInactivation of avian influenza virus H5N3 RGAdjuvant compositionsSorbitan sesquioleate (vegetable) polysorbate 80 (vegetable) as light mineral oilComposition of auxiliary materialsThimerosal phosphate buffered saline solution	Adding more than or equal to 128HA230mg22.5mg4.3mg0.02mg into 0.5ml	Active ingredient adjuvant emulsifier preservative diluent

Example 6

The efficacy of different doses of inactivated avian influenza vaccine was tested. Formalin inactivated reassortant H5N3 virus was formulated as a prototype water-in-oil emulsion containing 0.25 μ g Hemagglutinin (HA) protein/agent, 0.5 μ g HA protein/agent, or 1.2 μ g HA protein/agent. These vaccine-related dose levels were 64, 128 and 307 HAU/dose, respectively, in terms of hemagglutination units (HAU). At 2 and 5 weeks of age, the vaccine prototype was administered intramuscularly to 25 SPF white-leghorn chickens of each group. The placebo vaccine without antigen was also given intramuscularly to a group of 25 littermates and another 25 littermates served as uninoculated controls.

Blood samples of the chickens were taken just before the first inoculation and 3 weeks after inoculation for detection of avian influenza antibody titers by Hemagglutination Inhibition (HI) assay. The serum samples before blood collection were all negative. Serum samples from all vaccinated groups contained high levels of antibodies 21 days after the primary vaccination and 21 days after the booster vaccination.

At 3 weeks after the booster vaccination, the chickens were challenged with highly pathogenic H5N1 avian influenza virus isolated from vietnam. The vaccine provides 100% protection against death. The re-isolation of challenge virus was minimal in tracheal swabs and cloaca swabs collected from all live chickens at different time points after challenge.

The wings were marked prior to inoculation and the wing markers were randomly assigned to treatment groups and classified. The inoculation was intramuscular at the breast of the chicken. The initial inoculation was given at 2 weeks of age of the chickens and the booster inoculation was given in the same manner at 5 weeks of age of the chickens.

Challenge was performed when the chickens reached 8 weeks of age. The chickens were given challenge virus a/chicken/vietnam/c58/04 by instillation intranasally/intratracheally in a volume of 1.0 ml.

Blood samples were taken from all chickens at 2,5, 8 and 10-11 weeks of age. Tracheal swabs and cloaca swabs from live chickens were used for virus re-isolation in SPF eggs 3,5, 7,10 and 14 days after challenge.

Serum antibody quantification and virus re-isolation methods are standard methods.

For the claimed prevention of death, the originally designed study was the basic outcome for assessing mortality, testing the null hypothesis that there was no difference in mortality among the groups. Mortality was compared for each group using the universal evaluation equation model, with mortality (0 or 1) as the two-dimensional dependent variable and the treatments included as independent variables. If desired, the positions of the columns may be included as covariates (covariates) of the model. Since the mortality rate was 100% in the non-vaccinated group, and no mortality in the vaccinated group, the chi-square analysis was used to calculate the prevention rate and its associated confidence interval.

For the purported prevention of viral release, the designed study was the basic result used to evaluate the rate of viral segregation, testing the null hypothesis that there is no difference in the rate of viral segregation among groups. As described below, the total mortality of the non-vaccinated and placebo-vaccinated control groups made statistical analysis of the re-segregation results impossible and therefore not performed.

After inoculation of SPF chickens of 2 and 5 weeks of age with the prototype tested, high levels of antibody were detected by hemagglutination inhibition assay and complete protection against challenge death was observed. After challenge, there was minimal virus re-isolation from the vaccinated chickens, although it cannot be assumed here that there was a significant reduction in re-isolation rate compared to controls (since all controls that died before re-isolation could be evaluated in these groups), and clearly the vaccine allowed a significant reduction in virus release. For clear evidence, it was necessary to modify the challenge protocol so that unvaccinated chickens or placebo-vaccinated chickens did not die immediately after challenge. This proved difficult because the H5N1 challenge strain was very pathogenic to chickens.

Such whole virus inactivated vaccines were tested for 3 different antigen levels. The results were essentially identical for all 3 antigen levels, so that the lowest protective dose could not be determined in this study. It is necessary to conduct further studies with vaccines formulated to contain less than 0.25 μ g (64 HAU)/dose of antigen to determine the minimum amount. It is presently concluded that two inoculations with an adjuvanted water-in-oil emulsion containing not less than 0.25 μ g (64 HAU)/dose of H5N3 reassortant virus are highly effective in preventing death caused by the highly toxic field isolate of vietnam H5N1, and also in preventing the release of highly toxic H5N 1.

While the present invention has been described in various embodiments, it is intended that certain modifications thereof will be apparent to those skilled in the art without departing from the true spirit and scope of the invention as described in the specification and as particularly claimed in the appended claims. The specific embodiments described herein are not intended to limit the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the specification and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It should also be understood that all numbers are approximate and are provided for descriptive purposes. All patents and patent applications cited herein are incorporated by reference in their entirety.

Sequence listing

<110> Huishi company (Wyeth)

St.Jude Children’s Research Hospital

Hoffmann，Erich

Krauss，Scott L.

Kumar，Mahesh

Webby，Richard J.

Webster，Robert G.

<120> avian influenza viruses, vaccines, compositions, formulations and methods

<130>00630/2204339-WO0

<150>60/794,054

<151>2006-04-21

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<170>PatentIn version 3.3

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Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu

100 105 110

Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser

115 120 125

Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe

130 135 140

Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile

145 150 155 160

Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp

165 170 175

Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln

180 185 190

Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg

195 200 205

Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly

210 215 220

Arg Met Glu Phe Phe Trp Thr Ile Leu Arg Pro Asn Asp Ala Ile Asn

225 230 235 240

Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile

245 250 255

Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly

260 265 270

Asn Cys Asn Ala Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser

275 280 285

Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys

290 295 300

Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser

305 310 315 320

Pro Gln Ile Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile

325 330 335

Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His

340 345 350

Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln

355 360 365

Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys

370 375 380

Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu

385 390 395 400

Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp

405 410 415

Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg

420 425 430

Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val

435 440 445

Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe

450 455 460

Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn

465 470 475 480

Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg

485 490 495

Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile Tyr Gln Ile

500 505 510

Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Ala Ile Met

515 520 525

Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln Cys

530 535 540

Arg Ile Cys Ile

545

<210>3

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>3

agcaaaagca gg 12

<210>4

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>4

tattggtctc agggagcgaa agcaggtc 28

<210>5

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>5

atatggtctc gtattagtag aaacaaggtc gttt 34

<210>6

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>6

tattcgtctc agggagcgaa agcaggca 28

<210>7

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>7

atatcgtctc gtattagtag aaacaaggca ttt 33

<210>8

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>8

tattcgtctc agggagcgaa agcaggtac 29

<210>9

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>9

atatcgtctc gtattagtag aaacaaggta ctt 33

<210>10

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>10

tattcgtctc agggagcaaa agcagggg 28

<210>11

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>11

atatcgtctc gtattagtag aaacaagggt gtttt 35

<210>12

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>12

attacgtctc tcctcttgtc tcaatttgag gggtatt 37

<210>13

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>13

attacgtctc agaggactat ttggggctat agcagg 36

<210>14

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>14

tattcgtctc agggagcaaa agcagggta 29

<210>15

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>15

atatcgtctc gtattagtag aaacaagggt attttt 36

<210>16

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>16

tattggtctc agggagcaaa agcaggagt 29

<210>17

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>17

atatggtctc gtattagtag aaacaaggag tttttt 36

<210>18

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>18

tattcgtctc agggagcaaa agcaggtag 29

<210>19

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>19

atatcgtctc gtattagtag aaacaaggta gttttt 36

<210>20

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>20

tattcgtctc agggagcaaa agcagggtg 29

<210>21

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>21

atatcgtctc gtattagtag aaacaagggt gtttt 35

Claims (12)

1. A vaccine composition effective in preventing or ameliorating avian influenza and preventing the growth, release and transmission of challenge influenza virus to other species; the composition comprises a reverse genetics virus formulated in a water-in-oil emulsion adjuvant material containing one or more sorbitan oleate esters, the virus consisting of: HA gene amplified from a/chicken/vietnam/C58/04H 5N 1; the N gene amplified from A/DK/Germany/1215/73H 2N 3; and PB2, PB1, PA, NP, M, and NS genes amplified from a/puerto rico/8/34H 1N 1.

2. The vaccine composition of claim 1, wherein the virus is an inactivated virus.

3. The vaccine composition of claim 1, wherein the emulsion further comprises one or more surfactants selected from ethylene oxide/propylene oxide block copolymers.

4. The vaccine composition of claim 1, wherein the sorbitan oleate ester is tween 80 and sorbitan sesquioleate ester.

5. Use of a vaccine composition according to any one of claims 1 to 4 in the manufacture of a medicament for preventing or ameliorating an outbreak of an avian influenza virus infection.

6. The use of claim 5, wherein the vaccine composition is administered via drinking water or spray.

7. The use of claim 5, wherein the dose ranges from 0.25 ml/poultry to 2.0 ml/poultry.

8. The use of claim 7, wherein no more than one dose of the vaccine is administered.

9. A vaccine composition effective for preventing or ameliorating avian influenza virus infection, said composition comprising a reverse genetics virus formulated in a biologically acceptable adjuvant material, said virus consisting of: HA gene amplified from a/chicken/vietnam/C58/04H 5N 1; the N gene amplified from A/DK/Germany/1215/73H 2N 3; and PB2, PB1, PA, NP, M and NS genes amplified from a/puerto rico/8/34H 1N 1; said vaccine composition wherein the total amount of Hemagglutinin (HA) is at least 250 HA/dose, and wherein said composition comprises two surfactants consisting of sorbitan oleate esters.

10. The composition of claim 9, wherein the surfactants are tween 80 and sorbitan sesquioleate.

11. The composition of claim 9, wherein the reverse genetics virus is an inactivated virus.

12. The composition of claim 9, further comprising at least one additional poultry antigen.