Modified influenza virus for monitoring and increasing vaccine efficacy

FIELD: medicine.

SUBSTANCE: it is described that immunogenicity of hemagglutinin (HA) molecule of influenza virus can be increased by substituting amino acids in the HA sequence. Substituting specific residues in HA such as asparagine introduction in position 223 in HA H5 allows ensuring more sensitive hemagglutination inhibition (HI) test provided by changing receptor specificity and/or ability to antibody-antigen linkage. The HA molecules having such substitutions can find application in creating diagnostic prototype viruses.

EFFECT: improved influenza virus vaccines.

9 cl, 3 dwg, 7 tbl, 7 ex

 

Cross-reference to related applications

This application claims the priority of provisional patent application U.S. No.60/705808, registered on August 4, 2005, which is included in its entirety in the present description by reference.

The statement relating to research or development, subsidized with Federal funds

The study, which was created by the present invention was carried out under the grant AI95357 National Institute of Allergy and infectious diseases (National Institute of Allergy and Infectious Disease) and with the support of the Center for cancer research (Cancer Center Support (CORE)grant CA the National institutes of health (National Institute of Health). In accordance with this, the U.S. government has certain rights in the invention.

The link to the app, recorded on compact disc.

Not applicable.

The technical field to which the invention relates.

In General, the present invention relates to increasing the antigenicity and/or immunogenicity of certain subtypes of the group of influenza viruses.

Background of invention

Influenza viruses

Influenza viruses, among which, first of all, it should be noted specific strains of viruses a and b, are a major cause of morbidity and mortality worldwide in the annual outbreaks for which Alemania. Periodically, but at irregular intervals occur pandemic, leading to particularly high levels of morbidity and mortality. Historically, pandemic was the result of the emergence of new influenza virus subtypes A, which are formed by rearrangement of the segmented genome (antigenic variation), while annual epidemics, as a rule, are the result of the evolution of the surface antigens of influenza virus a and b (antigenic drift). The human influenza virus often occur from avian influenza virus strains, so that the basis of influenza infection is zoonotic. It is also clear that pigs can serve as the intermediate host ("mixing vessels") in the formation of new originating from birds strains that are pathogenic for humans (Scholtissek and other Virology, 147, 1985, page 287). The outbreak in Hong Kong in 1997, caused by the H5N1 strain of influenza virus And showed that the high pathogenicity of influenza a viruses can also be transmitted directly from birds to humans (Claas and others, Lancet, 351, 1998, p.472; Suarez and others, J.Virol., 72, 1998, s; Subbarao and others, Science, 279, 1998, s; Shortridge, Vaccine, 17 (app. 1), 1999, SS. 26-29). In 2003, the strains of H5N1 viruses in Southeast Asia was a different coexisting genotypes, but in 2004 became one of the dominant genotype, known as "Z-genotype (Li and others Natre, 430, 2004, s). Modern evidence suggests that the human deaths were due to direct transmission of this genotype from poultry to humans, and that this genotype also infects cats by direct transmission from cat to cat (Kuiken and others, Science 2004, 306:241). This and other evidence of change of range of the hosts and the wide spread of this virus has led to the understanding that H5N1 viruses may acquire properties that allow you to migrate from person to person. People can not acquire immunity to such H5N1-virus that can cause catastrophic pandemic influenza (Fouchier and others, Nature, 435, 2005, s). The ability of influenza a viruses to create new pathogenic strains from a huge number of strains circulating in animals-carriers of pathogens of viral infection, means that the control of this disease requires monitoring of these viruses and the development of improved antiviral therapies and vaccines. The speed with which developing new strains of the virus, requires special attention in the implementation of such monitoring, including the use of improved methods for evaluating the effectiveness of vaccines against new strains.

Influenza viruses a, b and C of the family Orthomyxoviridae, all have a segmented genome with negative chain of RNA that replicates in the nucleus of infected the cells, has the total coding capacity of approximately 13 TPN, and contains the genetic information for ten viral proteins. Specifically influenza viruses have eight gene segments antisense RNA (nsPHK)that encode at least 10 polypeptides, including proteins RNA polymerase (RW, RW and RA), the target is RNA, nucleoprotein (NP), neuraminidase (NA), hemagglutinin (which after enzymatic cleavage forms the Association of subunits H1 and H2), matrix proteins (M1 and M2) and non-structural proteins (NS1 and NS2) (Krug and others, in: The Influenza Viruses, Ed. by R. Krug, published by Plenum Press, New York, 1989, SS. 89-152).

The recently developed reverse genetic system should provide the ability to perform manipulation of the genome of influenza virus (Palese and others, Proc. Natl. Acad. Sci. USA, 93, 1996, s; Neumann and Kawaoka, Adv. Influenza Res., 53, 1999, s; Neumann and others, Proc. Natl. Acad. Sci. USA, 96, 1999, s; Fodor and others, J. Virol., 73, 1999, s). It was demonstrated, for example, that the expression of eight nsPHK carried out by the plasmid under the control of pol I promoter, and coexpressed polymerase complex proteins leads to the formation of infectious influenza A (Hoffmann and others, Proc. Natl. Acad. Sci. USA, 97, 2000, s).

The viral particle of the influenza virus has a size of about 125 nm and consists of a nucleus, including antisense viral RNA associated with a nuclear protein, which is surrounded by the viral Obolo is Oh, having a lipid bilayer structure. The inner layer viral envelope consists mainly of matrix proteins, and the outer layer consists mainly of lipid material originating from the host. The so-called "surface proteins, neuraminidase (NA) and hemagglutinin () are in the form of peaks on the surface of the body of the virus. Infectivity of new influenza viruses depends on cleavage BY specific proteases of the host, while NA is involved in the Department of virion-descendants from the cell surface and prevents agglutination formed a new virus.

Proteins and NA, immersed in the viral envelope are the main antigenic determinants of influenza virus (Air and others, Structure, Function, and Genetics, 6, 1989, cs-356; Wharton and others: The Influenza Viruses, Ed. by R.M.Krug, published by Plenum Press, New York, 1989, SS-174). Due to the rearrangement of the segmented genome of influenza virus is constantly creating new ways and NA in respect of which the infected organism is not produced anamnestic response (secondary immune response). Glycoprotein is a major antigen for neutralizing antibodies and it is involved in the binding of viral particles to receptors on cells of the host.

The sequence of molecules of different strains of the virus have significant similarities, as ur the outside of nucleic acids, and at the level of amino acids. The degree of similarity varies when comparing strains of different subtypes, with some strains characterized by a higher degree of similarity than others (Air, Proc. Natl. Acad. Sci. USA, 78, 1981, s). The degree of similarity between the amino acids vary between viral strains of the same subtype and viral strains other subtypes (Air, Proc. Natl. Acad. Sci. USA, 78, 1981, s). This variation is sufficient to establish a separate subtypes and direction of evolutionary differentiation of different strains, however, can be quite easy to conduct a comparative analysis of the primary structure of DNA and amino acid sequences of different strains using a standard bioinformatics methods (Air, Proc. Natl. Acad. Sci. USA, 78, 1981, s; Suzuki and Nei, Mol. Biol. Evol., 19, 2002, p.501).

Influenza vaccine

Influenza vaccine permitted currently, health authorities for use in the United States and Europe, represent inactivated influenza vaccine and live attenuated vaccine called FluMist used in the United States. Viruses, which are important in epidemiological against strains of influenza virus a and influenza virus, grown in fertilized chicken eggs, and then viral particles are purified and inactivate by chemical means to gaining the biomass of the vaccine. Each year who selects subtypes, which forecast should meet with the greatest probability to create vaccines.

Although influenza vaccines used for vaccination of humans since the beginning of 1940-ies and for vaccination of pigs with the late 1960's, the existence of numerous animals-carriers of pathogens of viral infection, in combination with the threat of a new influenza virus that can cause a pandemic, forces conduct research to develop new therapeutic methods for combating the virus. Over the last few years in the field of influenza has been achieved a number of important successes (see review Sokh and Subbarao, Lancet, 354, 1999, SS-1282). For example, it was experimentally found that intranasal introduction of live attenuated trivalent influenza vaccine may have a high efficiency in protecting young children from the strain of influenza A H3N2 and influenza virus C. Other approaches to improving the effectiveness of existing (killed) vaccines on the basis of influenza virus include creating tailored to protect against cold and constructed by genetic influenza viruses that contain debilitating mutations (see review in Palese, etc., J. Infect. Dis., 176 Appendix. 1, 1997. SS-49). There is hope that such modified by genetic viruses, which by paragroup the programme were introduced genes ON and NA strains in circulation, can be used as a safe live vaccine on the basis of influenza virus for the induction of a prolonged protective immune response in humans. Although, apparently, adapted to protect against cold vaccines are effective for children and adolescents, they can be loosened sufficiently to promote the ideal of the immune response in the elderly, who constitute a major group of 20000-40000 individuals dying each year in the U.S. as a result of influenza infection.

Readily available vaccines have become the most effective tool to combat suddenly emerging influenza pandemic. After the outbreak of H5N1 in Hong Kong in 1997 people were tested vaccines produced using two different approaches. Conventional vaccine based subunit (subtype) H5 derived from A/duck/Singapore/3/97, had weak immunogenicity to humans even against closely related on the antigenic properties of the strains and even after multiple vaccinations (Nicholson and others, Lancet, 357, 2001, s; Stephenson and others, Journal of Infectious Disease, 191, 2005, s). The use of adjuvant MF59 increased antibody titer to the specified vaccine based on the N5 (Stephenson and others, Vaccine, 21, 2003, s). Vaccination using inactivated "split" vaccines derived from non-pathogenic virus A/duck/HK/836/80 (H3N1), and a modified hemagglutinin of H5 in the Rus And/HK/156/97 (H5N1), induced weakly detectable titers of neutralizing antibodies (Takada and others the Journal of Virology, 73, 1999, s). Thus, although the vaccine based on the H5N1 virus was well tolerated, it turned out that they had a weak immunogenicity. The current lack of effective vaccines against strains of the H5N1 virus increases the risk that these viruses can cause pandemic disease.

Immunogenicity of influenza vaccine

Methods for determination of antibody titers in serum are surrogate methods for assessment of immune protection after vaccination or viral infection. As methods for the determination of antibody titers in serum is mainly used tests titers of virus-neutralizing antibodies and assays titles with the use of response inhibition of haemagglutination (HI). These analyses are based on the ability of antibodies to influenza virus from human serum to engage in cross-react with antigens in vitro. Methods of analysis for the specific situation are chosen not only on the basis of their ability to provide consistent and applicable results, but also on the basis of the simplicity of their application and hardware requirements necessary to implement each type of analysis.

In General, the analysis based on the neutralization of viruses is that evaluate the ability of antibodies from about the of ASCA serum to block infection of cultured cells by influenza virus. This analysis is carried out by preparation of serial dilution (titer) serum sample and associations of each of these dilutions with a standard quantity of infectious virus. Then each mixture obtained using these dilutions, making certain cell culture and analyze the odds of infection. It is believed that the analysis of titers of neutralizing antibodies is extremely valuable and reliable test to assess the level of immunoprotective antibodies present in the body of a particular individual. However, it depends on the characteristics of specialized cell culture and therefore is not completely universal. In addition, the methodology is very time consuming and requires a large amount of time, which makes it ill-suited for screening large number of samples.

In the analysis using the response inhibition of haemagglutination (HI) also assess the ability of antibodies from the serum sample to contact with the standardised reference virus. This analysis is based on the fact that influenza viruses should contact the red blood cells and cause their agglutination. In HI-analysis of serial dilution of the serum sample is mixed with a standard reference quantities of the virus and after a series of incubation add to erythrocytes. After that, Viswa is Ino reveal leading to the formation of Association complexes between the reference virus and erythrocytes. The highest serum dilution that inhibited hemagglutination, take titer inhibition of haemagglutination. Although HI-analysis does not have the same sensitivity in terms of immunogenicity of the vaccine, as other tests, it finds wide application due to the relatively simple technology and low requirements for laboratory equipment.

Taking into account the above limitations of the present methods that are suitable for the development and evaluation of influenza vaccines, there is a need to improve methods for assessing the immunogenicity to determine the immune response after infection, and vaccine efficacy.

Summary of the invention

In the present invention proposed amino acid substitutions in the hemagglutinin molecule OF influenza a, which may alter the antigenicity and immunogenicity. These changes can cause changes in the areas of determinants by changing the specificity of the receptor and/or binding of the antibody-antigen. In various embodiments of the invention increased antigenicity caused by the replacement, can be used to increase the sensitivity analysis using the response inhibition of haemagglutination (HI) in serum samples taken from infected animals. Such information is important I was to create intended for diagnostic reference viruses and new influenza vaccines. Preferably the amino acid substitution leads to the formation of molecules that are immunogenic characteristics of amino acid substitution at the asparagine at position 223 in H5-subtype.

Thus, specific objects of the present invention relate to the hemagglutinin molecule OF influenza virus having one or more amino acid substitutions in the receptor binding site, which increases the antigenicity of the molecule IN respect to antibodies having specificity to the molecule, in which there is no amino acid substitution in its receptor binding site. Molecule with high antigenicity of the influenza virus can contain the amino acid asparagine at the position corresponding to position 223 in H5, with the inclusion of asparagine leads to increased reactivity against antisera derived from an animal's body that are infected with influenza virus carrying molecule IN the wild type. The increased antigenicity of the molecule ON the influenza virus may be due to the presence of the amino acid asparagine at the position corresponding to position 223 in H5, where the molecule is not derived from an isolate of human H5 And a/HK/213/03, and the inclusion of asparagine at position 223 leads to increased reactivity against antisera derived from an animal's body, infected in the Rus flu. In some embodiments the invention, the amino acid substitution leads to a change of the site of glycosylation. In some embodiments of the invention, the influenza virus is an influenza virus And human. Influenza virus a person may be, for example, a representative of subtype H5. Influenza virus a person may be a virus influenza A/Vietnam/1203/04 (H5N1). In some embodiments of the invention, the influenza virus is an influenza a Century Under the scope of the invention fall molecules ON influenza virus with high antigenicity, derived from a virus of bird flu.

Other objects of the invention relate to recombinant influenza virus with a hemagglutinin molecule OF influenza virus that contains one or more amino acid substitutions in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitutions in its receptor binding site. Recombinant influenza virus can include a modified molecule, derived from influenza virus H5N1 in the genetic environment of influenza virus A. influenza Virus And can be a source of vaccine strain of influenza. Recombinant influenza virus can be used as a diagnostic reference VIR the sa analysis using response inhibition of haemagglutination (HI). Recombinant influenza virus can be included in the set for analysis using the response inhibition of haemagglutination (HI).

Other objects of the invention relate to a reverse genetic systems, intended for the production of virus containing a molecule of hemagglutinin () influenza virus, which has an amino acid substitution in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitutions in its receptor binding site.

The following objects are presented in the present description the invention relates to a method of creating computer viruses containing the hemagglutinin molecule OF influenza virus, which has an amino acid substitution in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitutions in its receptor-binding site. In some embodiments of the invention the method is that being introduced recombinant vector that expresses a molecule with high antigenicity, reverse genetic system.

In addition, related objects of the invention are methods of determining the effectiveness of the vaccine on the basis of influenza virus to the animal is. In some embodiments of the invention the method is that anticigarette obtained from the body of the vaccinated animal, is subjected to the interaction with the molecules of hemagglutinin () influenza virus, which has one or more amino acid substitutions in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitution in the receptor binding site, present in the vaccine-based influenza virus. In some embodiments of the invention the molecule with high antigenicity obtained from the isolate And/HK/213/03 human H5N1, and the inclusion of asparagine at a position that corresponds to position 223 in ON of the H5N1 virus, leads to increased reactivity against the antisera obtained from the body of the vaccinated animal. In some embodiments of the invention, the animal is a human. In other embodiments of the invention, the animal is a ferret.

Other related objects of the present invention are methods of creating the influenza virus, which contains the hemagglutinin molecule (ON), which are cultivated using reverse genetic process of reverse genetic system, the which encodes DNA encodes one or more amino acid substitutions in the receptor binding site, which makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitution in the receptor binding site.

In addition, the invention relates to methods for increasing the sensitivity analysis using the response inhibition of haemagglutination (HI), which is that anticigarette obtained from the body vaccinated or infected animal, is subjected to the interaction with the molecules of hemagglutinin () influenza virus, which has one or more amino acid substitutions in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitution in the receptor binding site. In some embodiments of the invention a method of increasing the sensitivity of HI-analysis leads to at least 2-fold increase in the sensitivity analysis using the response inhibition of haemagglutination (HI), IN some embodiments of the invention a method of increasing the sensitivity of HI-analysis leads to at least 4-fold increase in the sensitivity analysis using the response inhibition of haemagglutination (HI).

Another object of the invention is how to determine whether infected l is an animal influenza virus. In some embodiments of the invention the method is that anticigarette obtained from an animal's body, is subjected to the interaction with intended for diagnostic reference virus, which is derived from the influenza virus, but contains the hemagglutinin molecule OF influenza virus, which has an amino acid substitution in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitutions in its receptor binding site, resulting in increased reactivity against antisera. In some embodiments of the invention, the animal is a human. In other embodiments of the invention, the animal is a ferret.

The following objects of the invention are other ways to determine if an animal is infected with influenza virus. In some embodiments of the invention the method is that anticigarette obtained from an animal's body, is subjected to the interaction with the diagnostic reference virus, which is derived from the influenza virus, but contains a modified molecule ON the influenza virus, which has the amino acid asparagine at the position corresponding to position 223 in the And H5, while the inclusion of asparagine leads to increased reactivity against antisera derived from an animal's body, infected with influenza virus with a molecule of the wild type, resulting in higher reactivity against antisera. In some embodiments of the invention a method of determining if an animal is infected with influenza virus, is that anticigarette obtained from an animal's body, is subjected to the interaction with the diagnostic reference virus, which is derived from the influenza virus, but contains a modified molecule ON the influenza virus, which has the amino acid asparagine at the position corresponding to position 223 in H5, where the molecule is not derived from isolate human H5 And a/HK/213/03 and the inclusion of asparagine at position 223 leads to increased reactivity against antisera derived from an animal's body, infected with influenza virus. In some embodiments of the invention, the animal is a human. In other embodiments of the invention, the animal is a ferret.

Under the scope of the invention are also intended to receive the vaccine influenza viruses containing the hemagglutinin molecule (ON), having amino acid substitution in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific for a molecule that lacks amino acid substitutions in its receptor binding site, and where the modification increases the immunogenicity of the vaccine virus.

One of the objects of the present invention are selected nucleic acid molecule encoding hemagglutinin () influenza virus containing amino acid substitution in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific against a molecule that lacks amino acid substitution in its receptor binding site. In some embodiments of the invention, the molecule of influenza virus encoded by the selected nucleic acid that contains the amino acid asparagine at the position corresponding to position 223 in H5, where the molecule is not derived from an isolate of human H5 And a/HK/213/03 and the inclusion of asparagine at position 223 leads to increased reactivity against antisera derived from an animal's body, infected with influenza virus.

In addition, the invention relates to methods of producing nucleic acids encoding the hemagglutinin molecule OF influenza virus, which contains amino acid substitution in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific in respect to the research Institute of the molecule, in which there is no amino acid substitution in its receptor binding site. In some embodiments of the invention the method is that being introduced nucleotide sequence in a nucleic acid encoding a molecule, in which there is no amino acid substitution in the receptor binding site, resulting in amino acid substitution in the sequence of the molecule, giving the molecule the greater antigenicity against antibodies specific against a molecule that lacks amino acid replacement.

Under the scope of the present invention are subject to these and other objects of the invention, as it is in more detail described in "Detailed description of the invention" and "Examples".

Description of the drawings

In the drawings shown:

Figure 1 is a graph illustrating the HI antibody titers in ferrets, which were inoculable H5N1 influenza viruses, isolated in 2003 and 2004 (A); HI-titers and neutralizing antibody titers in ferrets immunized with viruses Δ51/03 and Δ51/04 (B). Presented at Figo data obtained using serum samples taken at day 28 after inoculation 106EID50(median infective dose for chicken embryo) virus H5N1, which was titrated against 4 hemagglutinin units (HAU) of homologous virus. Data represent repr the representative value, received 2 or 4 serum samples. Presented at figb data obtained using serum samples taken from the body of ferrets vaccinated twice with each time 7 µg viruses ΔH5N1/03 and ΔH5N1/04, which was titrated against 4 HAU and 100 TCID50(average cytopathology dose) homologous virus, respectively.

Figure 2 is a graph illustrating the viral titers in nasal swabs obtained from vaccinated and control ferrets after controlling virus A/Vietnam/I 203/04 (H5N1). The ferrets vaccinated with recombinant viruses ΔH5N1/04 or Δ5/04, intranasal were inoculable 106EID50virus A/Vietnam/1203/04. Titers are the average (log10EID50/0.1 ml) ± SKO defined for nasal swabs have 3 ferrets.

It should be noted that differences in the values of titles for the vaccinated and control groups are statistically significant and are characterized by values of R that make up 0,0028 - 0,0173 according to unpaired t-test.

Figure 3 - model of a molecule of the polypeptide ON the N5, which shows the position of the amino acids (position 154 and 223) in three-dimensional (3D) structure of the influenza A/duck/Singapore/3/97 (H5N3). On figa specified receptor binding site amino acids in the 3D structure. On figb circle denotes the boundary surface between presents MES the mayor and two other monomers (not shown) in a three-dimensional molecule. The amino acid at position 223 is located in the 220-loop of the receptor binding domain between glutamine residue, which is normally present at position 222, and a residue of glycine, which is normally present at position 224.

Detailed description of the invention

In the present invention proposed changes in the amino acid sequence of the hemagglutinin molecule OF influenza virus, which give the molecule the greater antigenicity against antibodies specific against molecules in which there are no changes. Such sequence changes include substitutions and deletions. Formed TO represent as with high antigenicity".

Molecule with high antigenicity can be used to test the effectiveness of the vaccine, because the change increases the sensitivity of diagnostic methods for antibodies to influenza virus in serum. In a specific embodiment of the invention, the virus is an influenza virus A. In another embodiment of the invention, the virus can be a virus of influenza, and in another variant of the invention, it may be a virus influenza C. In the embodiment of the invention, in which the virus is an influenza virus, he can be the H5 subtype of influenza virus A. more specifically, the embodiment of the invention the molecule ON the H5-subtype modify to include the amino acid asparagine at position 223 (N 223), which leads to increased reactivity against antisera derived from an animal infected with H5 subtype influenza virus. In a specific embodiment of the invention, the molecule ON the proposed invention does not represent a molecule IN A/NC/213/03, which refers to naturally occurring H5-subtype containing asparagine at position 223. The influenza virus may be a virus influenza a man H5-subtype, including virus A/Vietnam/1203/04 (H5N1).

Amino acid changes, which give the molecule the greater antigenicity, can be done in the receptor binding domain, for example, as shown in figa, especially in the field 220-loop of the receptor binding domain. This change can reduce binding TO sialic acid receptors on the red blood cells, thereby increasing the ability of antibodies to inhibit hemagglutination, which leads to increased ability to bind with the antibody in the analysis of response inhibition of haemagglutination. Alternatively, amino acid change, which makes the molecule AT greater antigenicity, can be done to change or be removed by deletion of the site of glycosylation on protein, primarily the site of glycosylation, which masks the epitope on, readable the e specific in relation TO antibodies. In another embodiment of the invention, the amino acid changes can be made to the amino acid residue corresponding to residue 223 IN H5-subtype. In all variants of the invention, the amino acid modifications, which give the molecule the greater antigenicity, can be easily identified by immunoassays using antibodies specific against a particular subtype. The modification, which makes the molecule AT greater antigenicity, should lead to significantly higher binding activity against titrated antibody compared to the binding activity of a molecule to which antibodies. Such analyses include analyses using response inhibition of haemagglutination (HI).

In a specific embodiment of the invention, the substitution in the molecule ON the H5-subtype of serine residue (which may be a glycosyl residue) to asparagine (which may be either deglycosylation, or have a different scheme glycosylation) leads to increased antigenicity. However, other substitutions can also lead to a noticeable increase of antigenicity. Such replacement can represent conservative substitutions, such as replacing a threonine to serine, or glutamine for asparagine, but not necessarily conservati is by replacement. They can keep the relative polarity, for example, substitution of asparagine for serine or lysine to aspartic acid, which preserves the polarity that existed prior to these changes. You can also consider replacing these residues as glycine and alanine, which leads to the elimination of reactive side chain, without exerting significant influence on the structure of the polypeptide. And finally, you can implement a fully non-conservative changes. And in this case using a simple immunoassays can be installed, does this particular change is to increase antigenicity.

Some H5N1-avian viruses, isolated in Central and South America, are the basic amino acid arginine at position 223. Neutral amino acid asparagine is found at position 223 IN the human isolate And/HK/213/03. This amino acid residue is located in the 220-loop of the receptor binding domain between the glutamine residue normally present at position 222, and a glycine residue normally present at position 224 (Fig 3). Experimental data suggest that higher HI titers reflect changes in the specificity of the receptor. In fact, glutamine, normally present at position 222, and glycine, as a rule, present in position 224, bind directly to the receptor sielow the th acid. Amino acids present in the 220-loop or related areas, play an important role in the conformation of the receptor binding pocket (and others, Proc. Natl. Acad. Sci. USA, 98, 2001, x). Although the present invention is not approved by any particular explanation of the observed effect, it may be possible that the substitution of asparagine for serine at position 223 N5 leads to conformational changes and alters the specificity of the receptor.

According to the next object of the invention molecules, modified in accordance with the invention in order to achieve greater antigenicity, also have greater immunogenicity. Such molecules as a component of the influenza vaccine can cause more powerful or effective immune response that, in turn, leads to more effective protection against influenza infection.

The influenza viruses, the source of which birds are carriers of infectious agents, have special significance for public health. It is believed that these viruses most likely to cause outbreaks of pandemic influenza in the human population. Therefore, the molecule ON from avian subtype with high antigenicity and/or immunogenicity may be most suitable for use in immunoassays conducted during the development of vaccines. The strategy is illustrated in the crust is present for example H5, you can apply to increase the titles in HI-analysis (inhibition of haemagglutination) against other subtypes, which is especially important for evaluation of immunity against avian influenza. In some embodiments of the invention the molecule, having amino acid substitutions in the receptor binding site, can be obtained from the virus of avian influenza subtypes H1 to H16, inclusive.

One of the objects of the present invention is the use of recombinant influenza virus containing a molecule with high antigenicity, as the reference virus in the immunoassay, first of all HI-analysis, and also set to perform such analysis. For example, replacement of the amino acid asparagine at position 223 in the molecule AT N5 increases binding to sialic acid receptors of erythrocytes (RBC)with alpha-2,6-linkage, such that are present in the body of chickens, but reduces binding to receptors with N-glycosidically acid with alpha-2,3-communication, such as those present in the horse's body. Thus, one object of the invention lies in the fact that as a result achieved a reduced ability to bind with receptors on horse RBC should be required fewer antibodies for inhibition of haemagglutination. This principle, which consists in the introduction of amino acid substitutions on usausa antigenicity, consider binding antibody can be applied to all 16 subtypes, including subtype influenza a viruses of birds.

Under the scope of the invention covers methods of assessing the effectiveness of the vaccine on the basis of influenza virus to the animal. This method consists in the fact that anticigarette obtained from the body of the vaccinated animal is subjected to interaction with influenza virus containing a molecule of hemagglutinin (ON) with high antigenicity. In some embodiments, the method of evaluating the effectiveness of the vaccine on the basis of influenza virus in animals, as demonstrated by the examples, anticigarette obtained from the body of the vaccinated animal, is subjected to the interaction with the molecule ON the H5-subtype influenza virus containing the amino acid asparagine at position 223 (N223), for example, a molecule ON the isolate from human H5N1 And a/HK/213/03. The presence of asparagine at position 223 leads to increased reactivity against antisera derived from an animal's body, infected other H5 subtype influenza virus. Vaccinated animal can refer to any of the numerous species of animals, including ferrets and humans.

Under the scope of the present invention also includes methods of increasing the sensitivity of HI-analysis by using the reference virus, which contains the Molek is ON with high antigenicity. In some embodiments of the invention, such that the molecule ON derived from the isolate strain of human H5N1 And a/HK/213/03, amino acid replacement is the introduction of asparagine at position 223, which leads to increased reactivity against antisera derived from an animal's body, infected with influenza virus H5 with another amino acid residue in this position, such as A/Vietnam/1203/04. This increase in reactivity can achieve any level, including at least 2-fold or at least 4-fold increase in reactivity. 2-fold or 4-fold increase in sensitivity may be particularly important in situations where the final concentration when using conventional methods of titration are below the detection limit.

A specific embodiment of the invention is also the use as a diagnostic reference virus recombinant influenza virus, which contains a modified molecule ON the H5-subtype having the amino acid asparagine at position 223, which leads to increased reactivity against antisera derived from an animal's body, infected with influenza virus H5. In a specific embodiment of the invention, the molecule ON the N5 in the diagnostic reference virus derived from isolate the Tamm human H5N1 And a/HK/213/03. In another embodiment of the invention, the residue of aspartic acid (or glutamate) substituted at position 223 molecule of H5 from a different strain of influenza virus. Obviously, the same approach can be applied to any molecule from any strain of influenza virus. In various embodiments of the invention, the animal may be any of numerous species of animals, including ferrets and humans.

In addition to the application as having increased sensitivity of the reference viruses in clinical studies of vaccines for humans, these viruses can be used in seroepidemiological studies. Obtaining data showing how many people are infected with H5N1 viruses using quick and simple detection methods, such as HI-analyses, allows to obtain important information on the prevalence of H5N1 viruses among humans. These data can be used to assess the probability of transmission of H5N1 viruses from person to person or from different species of birds to man.

Other objects of the invention are methods of determining if an animal is infected with influenza virus, namely, that anticigarette obtained from an animal's body, is subjected to the interaction with the diagnostic reference virus. Diagnostic reference virus derived from the same strain of influenza virus that is picirywi virus but the reference virus contains a molecule with high antigenicity. In one of the embodiments of the invention the reference virus contains a molecule, having an asparagine residue at the position corresponding to position 223 in H5, where the inclusion of the residue asparagine leads to increased reactivity against antisera derived from an animal's body, infected with influenza virus containing a molecule of the wild type, resulting in higher reactivity against antisera. In another embodiment of the invention the reference virus contains the hemagglutinin molecule OF influenza virus having the amino acid asparagine at the position corresponding to position 223 in H5, and H5 molecule is not derived from isolate human H5 And a/HK/213/03 and the inclusion of the residue asparagine at position 223 leads to increased reactivity against antisera derived from an animal's body, infected with influenza virus. In a specific embodiment of the invention the infecting virus is a virus H5N1 and the reference virus is an isolate of human H5N1 And a/HK/213/03, which contains the residue asparagine at position 223 amino acid sequence that leads to increased reactivity against H5N1-specific antisera. In other embodiments, domestic the invention, the animal may be any of numerous species of animals, including ferrets and humans.

Under the scope of the invention apply also to the influenza vaccine containing molecule with high antigenicity. In a specific embodiment of the invention, the virus is an isolate of human H5N1. In a more specific embodiment, the invention is obtained from the isolate And/HK/213/03. In another embodiment, the invention is a H5, modified by the inclusion of the amino acid asparagine at position 223.

The introduction of modifications in the molecule ON requires manipulation at the genetic level, as is well known in this field and are described in more detail below. After creating a modified gene ON you can use a number of approaches, such as reverse genetic methods for the introduction of modified molecules ON the influenza virus, resulting in it can be used as a reference virus for testing the efficacy of vaccines or diagnostic reference virus for monitoring epidemics of influenza, including the transmission of the virus from animal to man and the transmission of the virus from person to person.

Some of the objects of the invention relate to recombinant influenza viruses containing the hemagglutinin molecule OF influenza virus having at least one change in the amino acid placentas the work, which makes the molecule AT greater antigenicity against antibodies specific against a molecule in which there are no changes. Changes in amino acid sequences may include the replacement or deletion of one or more amino acids. The modified molecule ON can be obtained from the H5N1 influenza virus in the genetic environment of influenza virus A. In some embodiments of the invention, the influenza virus And is a source of vaccine strain virus. Recombinant viruses containing the modified molecule with high antigenicity against antibodies specific against molecules in which there is no change in amino acid sequence, can be used as a diagnostic reference virus in the analysis of response inhibition of haemagglutination (HI). The recombinant virus can also be included in the kit for analysis of response inhibition of haemagglutination (HI).

Other objects of the invention relate to methods for creating computer viruses containing the hemagglutinin molecule (). For example, one of the objects of the invention is a method of creating computer viruses containing the hemagglutinin molecule OF influenza virus, which has at least one change in the amino acid sequence is, which makes the molecule AT greater antigenicity against antibodies specific against molecules in which there are no changes. The method can consist in the introduction of a recombinant vector expressing modified FOR reverse genetic system. Way to create an influenza virus containing a molecule of hemagglutinin (ON)can be created by using a reverse genetic method, a reverse genetic system in which DNA encoding, encodes amino acid substitution in the receptor binding site that makes the molecule AT greater antigenicity against antibodies specific against a molecule that lacks amino acid substitution in the receptor binding site.

Under the scope of the invention also includes methods of obtaining nucleic acid molecule encoding hemagglutinin (ON). The molecule may have at least one change in the amino acid sequence, which gives the molecule the greater antigenicity against antibodies specific against molecules in which there are no changes. In some embodiments of the invention, the method consists in the fact that the nucleotide sequence being introduced to a nucleic acid encoding a molecule ON which lacks amino acid for the s in the receptor binding site, that leads to amino acid substitution in the sequence of the molecule, giving the molecule the greater antigenicity against antibodies specific against a molecule that lacks amino acid replacement.

Definition

In the context of the present description the term "influenza" refers to the types of the virus, pathogenic strains which cause the disease known as influenza or the flu.

The concept of "original vaccine strain of the virus" means a virus strain, which is used to create used in the vaccine strains with the capacity for rapid growth, or weakened strains. Such original vaccine strains usually contain six gene segments used in the vaccine virus (RV, RV, PA, NP, NA, M and NS). The original vaccine strain of the virus can be a strain, which is also used as a component of vaccines, in particular the virus strain A/PR/8/34.

The term "polypeptide" refers to a polymer of amino acids and is not associated with a specific length of the product; thus, under the definition of "polypeptide" fall peptides, oligopeptides and proteins. This concept does not apply or does not include post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, etc.

In the context of the present description, the term "infection"refers to the ability of the virus to multiply in the cell to produce viral particles. Infectivity can be estimated either by detection of the virus, i.e. viral load, or by identifying the development of the disease in the animal.

The terms "individual" or "subject" or "animal" in the context of the present description refers to a vertebrate, which are susceptible to infection caused by a virus with the antisense strand of RNA, particularly infections caused by influenza virus, including (but not limited to birds such as waterfowl and chickens) and representatives of various mammalian species, such as canids, felines, wolves, ferrets, rodents (rats, mice etc), horses, cows, sheep, goats, pigs and primates, include the last man. In a specific embodiment, the subject invention represents a ferret, which is a suitable animal model for studying influenza. In another embodiment of the invention the subject is a human.

In the context of the present description, the term "immunogenic" means that the virus or the polypeptide has the ability to induce a humoral or cell-mediated immune response, and preferably both. Immunogenic substance is also antigenic. Immunogenic composition is a composition that induces a humoral or cell-mediated immune response, and preferably both answers in the introduction the Institute of animal.

A molecule is "antigenic"if she has the ability to interact specifically with recognizing the antigen molecule of the immune system, such as immunoglobulin (antibody) or T-cell antigen receptor. Antigenic polypeptide contains an "epitope", consisting of at least about five and preferably at least about 10 amino acids. Antigenic fragment of the polypeptide, which in the present description denotes as "epitope"may be a fragment that is immunodeterminants for recognition by the antibody or T-cell receptor, or it may be a fragment that is used to create antibodies to the molecule by conjugation of antigenic fragment of the polypeptide carrier for the purpose of immunization. It is not necessary to antigenic molecule was itself immunogenic, i.e. have the ability to induce an immune response in the absence of the media.

In the context of the present description, the term "amino acid substitution" refers to the incorporation of amino acids at a particular position in the amino acid sequence of the considered molecules. Amino acid substitution is a substitution of a particular amino acid with any other amino acid, which may take a considered position. The polypeptide produced as a result of changes in amino acid sequence, may also contain changes that represent post-translational modifications such as glycosylation, acetylation, phosphorylation, or any other amino acid modifications, as well as amino acid replacement.

In the context of the present description the term "reverse genetic system" refers to methods of creating particles of influenza virus polypeptides, virions or nucleic acids using methods of genetic engineering. Such methods include, but are not limited to) "plasmid system", described by Hoffmann (Hoffmann and others, Vaccine, 20, 2002, s; patent publication US 2002/A, November 7, 2002, which is incorporated into this description by reference in its entirety). In General, a reverse genetic system allow you to create viral particles, polypeptides and/or nucleic acid containing a specific sequence, using genetic engineering techniques known to experts in this field. Such systems are also described in more detail below.

In the context of the present description, the term "receptor binding site" refers to a fragment of a molecule, which binds interest receptors, such as receptor sialic acid on the erythrocyte. The structure of a molecule of H5 A/duck/Singapore and the position of the receptor binding site of the hemagglutinin of the virus H5-subtype and is known and described in literature (and other, Proc. Natl. Acad. Sci. USA, 98, 2001, x). Molecule model specified ON the N5, including the receptor binding site, are presented in figure 3.

The concept of diagnostic reference virus" refers to a virus that contains AT with high antigenicity. Such diagnostic reference virus can be used in immunoassay, for example, in the analysis of the hemagglutinin inhibition.

The term "infectious virus" refers to a virus that infects a particular animal. This infection can be carried out in the course of daily activities, such as contact with an infected subject, for example, leading to human infection infectious influenza virus. This disease may be the result of special clinical control of infection, for example, when conducting laboratory experiments, for example, when a laboratory animal, such as a ferret, specifically infect with a virus. This infection can be made by immunization with the use of influenza vaccine.

The term "pharmaceutically acceptable" refers to molecules and compositions that are physiologically tolerable and, as a rule, with the introduction of one does not cause allergic or similar adverse reaction, such as gastric upset, dizziness and the like, In the context of the present description the term "farmacevtichesky acceptable" preferably means "approved" by the regulatory authority of the Federal government or the state government or "permitted" by the U.S. Pharmacopoeia or other generally recognized Pharmacopoeia for use in animals and more specifically for a person.

The term "carrier" refers to a diluent, adjuvant, excipient or filler which is injected compound. Such pharmaceutical carriers can be a sterile liquid such as water or oil, including oil derived from petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. as carriers, particularly for injectable solutions, preferably using water or aqueous saline solutions and aqueous dextrose and glycerol. Suitable pharmaceutical carriers are described in "Remington''s Pharmaceutical Sciences" Ed. by E.W. Martin, 18th ed.

In the context of the present description, the term "adjuvant" refers to a compound or mixture that enhances the immune response to the antigen. Adjuvant can serve as a depot in the tissue, which slowly releases the antigen and also as an activator of the lymphatic system, nonspecific immune response (Hood and others, Immunology, 2nd ed., published by Benjamin/Cummings, Memo Park, CA, 1984, s). Often the primary control infection using only the antigen in the absence of adjuvant cannot cause a humoral or cell-mediated immune response. Adjuvants include (n is not limited to) full beta-blockers, incomplete adjuvant's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomies polyols, polyanion, peptides, oil or hydrocarbon emulsions, hemocyanine lymph snails and potentially suitable for human adjuvants such as N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)ethylamine, BCG (Bacillus of Calmet-Guerin) and Corynebacterium parvum. Preferably the adjuvant is pharmaceutically acceptable.

In the context of the present description, the term "isolated" means that the material removed from its native environment, for example, from a cell or virus. Thus, the selected biological material may be free of some or all of the cellular components, i.e. components of the cells in which the native material found in natural conditions (for example, cytoplasmic or membrane component). It should be assumed that the material selected, if it is present in the cell extract or supernatant. In the case of nucleic acid molecules to a selected nucleic acids refers PCR product, isolated mRNA, cDNA or obtained by restriction fragment. In another embodiment, implementation is tvline of the invention the selected nucleic acid is preferably cut from a chromosome in which it may be present, and more preferably not associated with non-coding regions, and is not in the immediate vicinity (but it may be associated with its native regulatory regions or fragments), or with other genes located against the course or in the middle of transcription relative to the gene contained in the selected nucleic acid molecule, when it is present in the chromosome. In another embodiment of the invention in a selected nucleic acid is missing one or more introns. To the selected nucleic acid molecules include sequences that are built into the plasmid, Comedy, artificial chromosomes and the like, i.e. when they are part of a chimeric recombinant structure of nucleic acids. Thus, in a specific embodiment of the invention the recombinant nucleic acid is a selected nucleic acid. Isolated protein may be associated with other proteins or nucleic acids with which it is associated in the cell, or both, or with cellular membranes, if it is a associated with a membrane protein. The selected organelle, cell or tissue is removed from the anatomical region in which it is present in the body. The separated material can be cleaned, butthis is not required.

In the context of the present description, the term "purified" refers to a material that has been isolated under conditions that reduce or eliminate the presence of irrelevant documentation, i.e. pollutants, including native materials, which received the material. For example, the purified virion preferably practically free from components of the host cell or culture, including tissue culture or egg whites, non-specific pathogens, etc. In the context of the present description the term "almost free" is used in the specific context of the analytical determination of the purity of the material. Preferably the purified material, practically free from pollutants, has at least 50%purity, more preferably at least 90%purity, even more preferably at least 99%purity. The degree of purity can be assessed by using chromatography, gel electrophoresis, immunoassay, composition analysis, biological analysis, and other methods known in this field.

Purification methods are well known in this field. Viral particles can be purified by ultrafiltration or ultracentrifugation, preferably continuous centrifugation (see Furminger, above). You can apply other Metodicheskie, described in this description. The purified material may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90% of cellular components, environments, proteins or other unwanted components or impurities (depending on context), with whom he was associated in the initial state. The notion of "almost pure" means the highest degree of purity that can be achieved using conventional cleaning methods known in this field.

In a specific embodiment of the invention, the term "about" or "approximately" means that the value is statistically determined range of values. This range may be in the order of magnitude, preferably 50%, more preferably 20%, more preferably 10%, and even more preferably 5% from the specified value or range. Permissible variations that fall under the term "about" or "approximately"would depend on the system in question and the ordinary person skilled in the art can easily appreciate them.

Hemagglutinin

Hemagglutinin (ON) represents the main envelope glycoprotein of influenza viruses a and B. the Hemagglutinin-esterase (NOT) influenza viruses With is a homologue of these viruses. New subtypes of molecules, the source of the ICOM which, as a rule, are aquatic birds, which, as you know, are natural carriers of influenza viruses that cause influenza pandemics (see Suzuki and Nei, Mol. Biol. Evol., 19(4), 2002, s-509).

ON has two polypeptide chains, H1 and H2, encoded by a single gene and resulting from the proteolysis of one molecule predecessor, including the loss of the signal peptide (Suzuki and Nei, above; Air, Proc. Natl. Acad. Sci. USA, 78(12), 1981, SS-7643). HA1 contains approximately 320 amino acids and is a protein that links the receptor, which is the main target of the immune response. H2 contains approximately 220 amino acids and provides zakalivanie" viral membrane (Suzuki and Nei, above).

Genes of the influenza virus And is divided into 16 subtypes according to their antigenic properties. Genes ON (NOT) of influenza virus b and C are not divided into subtypes. Sequences of subtypes H1-H15 influenza virus and ON (NOT) of influenza virus b and C presented in the article by Suzuki and Nei, above, in figure 1, which is incorporated into this description by reference. Comparison of the sequences of subtypes H1, H2, H3, H4, H5, h6, H7, H8, H9, H10, H11 and H12 of the influenza a virus, including conservative amino acid residues of all the twelve subtypes presented in figure 1 in the article Air above, which is incorporated into this description by reference. In both references described the strains, of which were obtained from these sequences.

New molecules ON the proposed invention, is generated by the introduction of such changes in the amino acid sequence of a molecule, which lead to increased antigenicity. Nucleic acid encoding such a molecule is a standard procedure (see Air, above), as well as modification of nucleic acids with the aim of introduction of changes in amino acid sequence, which is carried, for example, using siteprovides mutagenesis.

In an alternative embodiment of the invention molecules ON one specific strains of subtype ON already contain the amino acid sequence that enhances antigenicity compared with molecules of the same subtype of the other strains that are illustrated in the present description on the example of the strain a/HK/213/03, related, for example, strain A/Vietnam/1203/04. In this case, the strain of influenza virus containing a molecule with greater antigenicity, can serve as a diagnostic reference strain. In another embodiment of the invention amino acid residues from AT more antigenicity can be introductionat or to carry out their use of substitution in the sequence of the molecules ON the lower antigenicity, thereby giving the molecule ON a substantial the antigenicity.

To increase the antigenicity can cause various changes in the sequence of the molecule. As indicated above, the circuit in which it is possible to implement such changes is a circuit HA1, which is a receptor binding domain and serves as the primary target of the immune response. Changes can increase the affinity of the antibody molecule ON, for example, by deleting the glycosylation site, which forms the epitope, or by the formation of a more favorable conformation with high affinity to bind with the antibody. Alternatively, the change may lead to a reduction in the receptor binding TO, for example, receptor sialic acid on red blood cells, resulting in antibody specific against the molecules TO become more effective competitors in the reaction of haemagglutination. Various immunoassays allow the detection of such changes, including the analysis of response inhibition of haemagglutination (HI), well known in this field.

In HI-analysis using whole particles of influenza virus that are currently used in vaccines. Thus, the invention proposed by the influenza virus containing a molecule with high antigenicity. Such viruses are most conveniently be created by using reverse genetic method is, although you can also use the classic method of rearrangement.

Reverse genetic methods

Currently developed reverse genetic methods allow manipulation of the genome of influenza virus (Palese and others, Proc. Natl. Acad. Sci. USA, 93, 1996, s; Neumann and Kawaoka, Adv. Influenza Res., 53, 1999, s; Neumann and others, Proc. Natl. Acad. Sci. USA, 96, 1999, s; Fodor and others, J. Virol., 73, 1999, s; application for U.S. patent 20040029251). For example, it was demonstrated that provided by plasmid expression of eight wrnc under the control of the promoter and a polI and all mRNA under the control of the promoter and a polII leads to the formation of infectious influenza A (Hoffmann and others, Proc. Natl. Acad. Sci. USA, 97, 2000, s; publication of U.S. patent No.20020164770, which is incorporated into this description by reference in part, related to the description of the minimal plasmid reverse genetic system and describe the methods of genetic engineering). Such recombination methods allow for specific building types of the influenza virus with specific changes in the amino acid sequence of the polypeptide. The molecule, containing the desired replacement can be a part of a recombinant influenza virus. Recombinant influenza virus you can create any methods known to experts in this field, including such method of genetic engineering, as the method based on the use of"only plasmid system (Hoffmann and others Vaccine, 20, 2002, s). Recombinant influenza virus can be obtained from the H5N1 virus. The recombinant virus can be genetically surrounded by H1N1 virus used to create vaccines, such as influenza A/PR/8/34 or any influenza a viruses, including adapted for protection against cold strains A/Leningrad/134/17/57, A/Leningrad/134/47/57 and A/Ann Arbor/6/60. Nucleic acid corresponding to a sequence of a molecule can be distinguished from virus and sequenced.

Methods for selection and modification of specific nucleic acids and proteins is well known to specialists in this field. According to the present invention can be applied generally accepted methods of molecular biology, Microbiology and recombinant DNA, known to specialists in this field. Such methods are described in detail in the literature, see, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., published by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 (in the present description is cited as "Sambrook and others, 1989"); DNA Cloning: A Practical Approach, volumes I and II, Ed. by D.N. Glover, 1985; Oligonucleotide Synthesis, Ed. by M.J. Gait, 1984; Nucleic Acid Hybridization, edited by B.D. Hames and S.J. Higgins, 1985; reduced And Translation, edited by B.D. Hames and S.J. Higgins, 1984; Animal Cell Culture, Ed. by R.I. Freshney, 1986; Immobilized Cells And Enzymes, published by IRL Press, 1986; B. Perbal, A Practical Guide To Molecular Cloning, 1984; Current Protocols in Molecular Biology, edited by Ausubel, F.M., published by John Wiley & Sons, Inc., 1994. These methods include siteprovides mutagenesis with ISOE what Itanium oligonucleotides with modified nucleotides to obtain PCR products containing mutations (for example, using a set of "Quikchange"manufactured by Stratagene).

The immunoassays

To detect binding and increased antigenicity, created according to the present invention, it is possible to apply a variety of detection methods immunospecific binding of an antibody to an antigen, known in this area. One of the first methods for the detection of interaction between antigen and antibody was based on the detection and analysis of complex precipitation in gels. Another method of detection associated pair of the analyzed substance-identifying antibody based on the use of labeled radioactive iodine identifying antibodies or labeled with radioactive iodine protein with reactivity against IgG, such as protein A. These first methods well known to specialists in this field, and review of these methods is given in Methods in Enzymology, 70, 1980, SS-198.

Later methods of determining the presence of the analyzed substance in the sample with only one of the antibodies is based on the use of analysis of competitive binding. In this method, the antibody, which often must be immobilized on a solid substrate, is brought into contact with the sample, which is assumed to contain the analyzed substance, in combination with known quantities of the m labeled the analyzed substance. After that, two of the analyzed substance, namely, labeled the analyzed substance and analyzed substance contained in the sample, must compete for the binding sites of antibodies. Determine quantitatively or free labeled the analyzed substance, or associated labeled analyzed the substance and the results of this measurement to determine the number of competing analyzed substance in the sample. More details on this method are described in "Basic Principles of Antigen-Antibody Reaction" (Labat, Methods in Enzymology, 70, 1980, SS-70). In this case, labeled the analyzed substance can bear or radioactive, or enzymatic tag.

In more modern immunoassays used the double antibody to detect the presence of the analyzed substance. These methods are also described in the cited volume of Methods in Enzymology. So according to one of embodiments of the present invention, the presence of individual markers was determined by using a pair of antibodies for each used for detecting markers. In the context of the present description one of the specified pair of antibodies known as "identifying the antibody, and the second from the specified pairs of antibodies referred to as "immobilized antibody". So, in one of the embodiments of the present invention use the sandwich method is used in which of the two antibodies to detect the analyzed substance in the sample of biological fluid. In this method, the analyzed substance is placed between identifying antibody and the immobilized antibody and the immobilized antibody irreversibly immobilized on a solid substrate. Identifying the antibody should be detectable label to identify the presence of antibody sandwich-being analyzed substance and thereby the presence of the analyzed substance.

The first conventional solid substrate was in the form of polystyrene plates, tubes or pellets, which are all well known in the field of radioimmunoassay analysis and immunoassay. In recent years, as the solid substrates used many porous materials, such as nylon, nitrocellulose, cellulose acetate, glass fiber and other porous polymers.

You can apply a variety of methods and appropriate touch devices. Automated device for analysis include devices for the analysis of continuous/random access. Examples of such systems are OPUS™ firm PB Diagnostic System, Inc. and the IMX analyzer™, created by company Abbott Laboratories of North Chicago, Ill. Devices for automatic analysis of company PB Diagnostic Systems, Inc. described in U.S. 5051237; 5138868; 5141871 and 5147609.

Other classes of systems for immunochemical analysis, which can be used in the implementation in practice this is part II of the invention, are optical immunosensoric system. In General, optical immunosensor is a device that uses the principles of optics for the quantitative conversion of interest chemical or biochemical concentrations or activities into electrical signals. These systems can be grouped into four main categories: devices for measuring reflection; devices for measuring surface plasmon resonance; optical fiber (light guide) devices and integrated optical devices. Device for measuring reflection include ellipsometric device for multiple integral reflective spectroscopy and capillary device with fluorescent filling. Fiber optical devices include a device for measuring fluorescence in a rapidly changing field, the optical fiber capillary tube and fiber optic fluorescence sensors. Integrated optical devices include a device for measuring fluorescence in a flat rapidly changing field, immunosensor with gradebook input device, an interferometer of the Mach-Zehnder interferometers, the interferometer Hartman and differential (differential) interferometric sensors. Holographic detection of the coupling reaction is carried out by the detect the presence of a holographic image, which is created in the assigned position of the image when one reagent binding pair binds to the immobilized second reagent binding pair (see U.S. No.5352582, issued October 4, 1994 Board name Lichtenwalter and others). Examples of optical immunosensors described in General in a review article by G. A. Robins, Advances in Biosensors, 1, 1991, SS-256. A more specific description of these devices can be found, for example, in U.S. 4810658; 4978503 and 5186897; R. A. Brady and others in the Phil. Trans. R. Soc. Land. Century, 316, 1987, SS-160 and G. A. Robinson and others in Sensors and Actuators, published by Elsevier, 1992).

For the diagnosis of influenza, as well as search engine studies on epidemiology and antigenicity of viral strains that are widely used serological tests. In particular, the widely used analysis using the response inhibition of haemagglutination (HI) due to the minimal requirements for laboratory equipment and ease of use. It is assumed that the present invention should increase the applicability of HI-analysis by increasing its sensitivity. HI-analysis can also be used to determine the antigenicity of the modified molecules and to facilitate characterization of the modified molecule as a molecule having a higher or lower antigenicity than the unmodified molecule.

HI-analysis allows to determine the ability of antibodies from the sample with the adjustable tap wrenches contact with the standardised benchmark. In HI-analysis of serial dilution (titer) serum sample is mixed with a standard amount of erythrocytes and visually identify their Association in the complexes. The result of the analysis is to establish the lower limit of the titer of the serum, which can visually detect the complex.

As indicated above, the present invention proposed an improved method of obtaining and validation of vaccines to treat or prevent infections caused by influenza virus. In particular, the present invention is applicable to the vaccines that are produced using reverse genetic techniques. It is assumed that the invention should find application for the validation and verification of the immune response after vaccination. In particular, due to the increased antigenicity of the modified molecule TO the invention allows for (but not limited to) enhanced detection of antibodies after an individual has been infected with the influenza virus. This increased antigenicity provides increased sensitivity analysis is used to detect the immune response, such as HI-analysis.

The creation of a vaccine

As shown in the following examples, the modified virus containing a molecule with high antigenicity, by itself, has a greater immunogenicity, which in turn leads to a stronger immune the WMD response and higher potential vaccines.

Strategies to improve the effectiveness of vaccines include the use of adjuvants (Wood and Williams, above), co-administration of immunostimulatory molecules (Salgaller, and Lodge, J. Surg. Oncol., 68, 1998, s) and vaccination strategies through the mucous membrane. Strategies of immunization through the mucous membrane include encapsulation of the virus in microcapsules (U.S. 5075109, 5820883 and 5853763) and use immunopotentiating membrane carrier (WO 98/0558). In addition, the immunogenicity of oral input by immunogens can be improved by the use of red blood cells (RBC) or hemolysed RBC (U.S. 5643577), or by using antigen "blue language" (blue tongue antigen) (U.S. 5690938). Although these approaches are promising for the development of future strategies for vaccination, their application in specific situations requires validation and monitoring to ensure the effectiveness of the vaccine. It is assumed that presented in the present description the invention is to improve these strategies, including by improving the ability to detect their therapeutic action.

Examples

Below the various objects of the invention are illustrated in the examples, not limiting its scope.

Example 1: the Titers of antibodies in the serum of inoculated ferrets

With high pathogenicity of H5N1 viruses were received from those in Asia laboratories, collaborating with the world Body is izala Health (who). All work with these viruses was carried out on the equipment BL3+ Children's research hospital St. Judas (St. Jude Children's Research Hospital). When creating inventions to compare the immunogenicity of influenza viruses Z-genotype, dedicated in 2003, which became predominant in 2004, with the viruses isolated in 2004, was carried out by inoculation of ferrets with the H5N1 virus isolated from the body of the deceased person (A/HK/213/03) (Guan and others, Proc. Natl. Acad. Sci. USA, 101, 2004, s), and four H5N1 viruses isolated from humans, chickens and birds in 2004 (figa). Males and females bred by outbreeding ferrets received as part of a special breeding programmes of the animal resource Center at St. Jude Children's Research Hospital. Animals were age 3-5 months and when conducting HI-analyses they gave negative serological reactions were seronegative) in relation to contamination of the currently circulating viruses of influenza A H1N1, H3N2 and H5N1) and influenza virus C. Viruses were propagated in allantoin cavities of 10-day-old fertilized chicken eggs at 35°C for 48 hours Allantoin liquid collected after one passage of the fertilized chicken eggs were frozen at -80°C and used in experiments.

Serum antibodies when conducting HI-analysis was titrated and exercised control virus infection through 28 days after inoculation. The virus is/HK/213/03 induced high titers of antibodies (from 1:640 to 1:1280), while four strains 2004 induced very low HI titres from 1:20 to 1:40).

Relatively low Hi titres of virus H5N1 2004 could be the result of General immunosuppression induced by the virus. However, the results of vaccination using vaccines ΔH5N1-A/PR/8/34 (6+2), which contained ON and neuraminidase of a/HK/213/03 and A/Vietnam/1203/04, showed that the main contribution to this effect may be due to differences in H5 (figb). Vaccination with two doses of vaccine ΔH5N1/03 7 µg induced high levels of antibodies in the serum, which can be detected with the help of HI-analysis, and test to neutralize the virus (figb). After the same vaccination using ΔH5N1/04 when HI-analysis were found very low (about 1:20) titles, while neutralizing titers were much higher (they accounted for about half of the titles, induced Δ51/03). In earlier studies it was found that inactivated vaccine derived from A/duck/Singapore/3/97 (H5N3), induced low or undetectable levels of antibodies in the serum (Nicholson and others, Lancet, 357, 2001, s 7; Stephenson and others, Vaccine, 21, 2003, s). Taken together, these results suggest that some isolates H5 may have unusual immunogenic and/or antigenic properties. A comparative analysis of primary structuralintegrity sequences H5 has allowed to establish, which of viruses And/HK/213/03 and A/Vietnam/1203/04 influenza differ 10 amino acids in 1 region (table 1B). Virus A/Vietnam/1203/04 has the potential site of glycosylation at the asparagine residue of the N154(N*154-S155-T156N-X-S/T, X≠P). Comparison of the sequences identified three amino acids (S120, K189and S223), which was present in all viruses, 2004, but was not present in the a/HK/213/03. For virus A/Vietnam/1203/04 distinctive feature was the presence of a212.

Example 2: Create recombinant viruses Δ5-A/PR/8/34 and the determination of their antigenic characteristics

To assess the impact of the identified amino acids on the immunogenicity and protection from the control virus (introduction provocative tests) when creating inventions used 8-plasmid reverse genetic system to generate recombinant viruses carrying seven gene segments of A/PR/8/34 and a gene segment AT A/Vietnam/1203/04, containing a single point mutation (Hoffmann and others, Vaccine, 20, 2002, s). Recombinant viruses lacking pathogenicity as a result of modifications TO H5 in the cleavage site was created using DNA transfection (Hoffmann and others, Vaccine, 20, 2002, s). Point mutations have been built ON by PCR using a set for siteprovides mutagenesis QuikChange® (the company Stratagene, B & Creek, Texas, USA) and the set of SPE is specific for H5 primers. Rearranged viruses contain a gene or genes ON and neuraminidase (NA) of H5N1 viruses in genetic environment of the influenza A/PR/8/34 (H1N1) (see table 1A, which includes viruses, designed with the creation of the present invention and their abbreviations). Allantoin liquid collected after one passage of the fertilized chicken eggs were frozen at -80°C and used in the experiments. Genes ON recombinant viruses amplified using RT-PCR and sequenced to verify that there are only these mutations. The change in amino acid sequence was verified by sequencing of a segment of recombinant viruses (table 1A).

To assess antigenic properties and diversity recombinant in the claimed invention was performed HI-analyses using a panel of six monoclonal antibodies (table 2). Monoclonal antibody (MAB) SR, SR, SR and 406/7 to TO virus A/chicken/Pennsylvania/1370/83 (H5N3) was created in the Department of infectious diseases St. Jude Children's Research Hospital. Mat VN04-6 to ON virus A/Vietnam/I 203/04 and Mat NK-3 ON virus And/HK/213/03 was created using a modified method described by Kohler and Milstein (Kaverin and others, Journal of Virology, 78, 2004, s; Koher and others, European Journal of Immunology, 6, 1976, s). Five Mat reacts at relatively high titers of virus Δ5/03, but tol is to 3 reacts with Δ5/04. Schema reactivity viruses ΔH5S155→N,T156→A/04, ΔH5S120→N/04 and ΔH5R212→K/04 were generally similar to the scheme reactivity virus Δ5/04. The reaction of the virus ΔH5S120→N, S155→N,T→A/04 were similar to the reactions of the virus ΔH5N1/03. Four Mat recognized Δ5/04 carrying the mutation S223→N(ΔH5S223→N/04). Reverse mutation N223→S ON virus 2003 (ΔH5N223→S/03) led to a significant decrease in HI-titles or loss of ability to recognize the Mat.

Example 3: HI-analyses using chicken and horse red blood cells

Another interesting result was obtained in the HI-analysis using chicken and horse red blood cells (RBC). Interestingly, the recombinant virus ΔH5S223→N/04 had a weak ability to cause agglutination of 1%local horse RBC, but resulted in agglutination of chicken RBC at high titers (1:1024). None of the other recombinant viruses reactions with chicken and horse RBC did not differ in the extent.

Example 4: Vaccination of ferrets mutant recombinant viruses of H5-subtype

In the claimed invention was evaluated the immunogenicity and protective efficacy of inactivated vaccines by vaccination groups, including 3 of the ferret, which was carried out by intramuscular injection of drugs viruses ΔH5N1/04, Δ5/04, ΔH5S15→N ,T→A/04, ΔH5S120→N/04 and ΔH5S223→N/04, standardized in terms of content of them. For standardization ΔH5N1/03 used the method of a simple radial immunodiffusion (Webby and others, Lancet, 363, 2004, s). Other recombinant viruses were separated by electrophoresis in 12%of the LTO-polyacrylamide gel, stained gels were analyzed using densitometry on the analyzer fluorescent image type LAS-1000plus company FUJIFILM and quantitatively evaluated by comparison with the product of the reference protein. After making two injections of 7 mg FOR each animal was inoculable A/Vietnam/1203/04 (H5N1). Group 3 ferrets were vaccinated by intramuscular injection of 250 μl of sterile SFR containing 7 µg of inactivated purified virus. Vaccine viruses iactiveaware, concentrated, and purified according to the literature method (Liu and others, Virology, 314, 2003, s; Webby and others, Lancet, 363, 2004, s). Three control animals were injected by injection of 250 μl of only one sterile SFR. At day 21 after vaccination took serum samples and carried out the second intramuscular injection of 7 mg. Two weeks later again took serum samples and carried out the control of infection by inoculation of the virus.

Vaccinated and control animals were inoculable, as described above, route 106medium to inficere the proposed doses for the chicken embryo (EID 50) virus A/Vietnam/1203/04 (Govorkova and others, Journal of Virology, 79, 2005, s). Within two weeks of daily monitored for clinical signs of infection, measurement of body weight and temperature. Ferrets that have been identified signs of serious illness, were killed. To assess postinfective immune response additional groups of ferrets were inoculable 106EID50isolates And/HK/213/03, A/Vietnam/3046/04, A/Vietnam/3062/04, A/chicken/Vietnam/39/04 and A/falcon/HK/D0028/04 human and avian H5N1. At day 28 after inoculation in animals took serum samples. To determine the titers of virus in the upper respiratory tract in days 3, 5 and 7 samples were taken using a nasal lavage (Govorkova and others, Journal of Virology, 79, 2005, s). Contained in samples of the virus was titrated in 10-day-old fertilized chicken eggs and were expressed as log10EID500.1 ml of virus Titres in nasal swabs from all vaccinated animals ranged from 2.5 to 4.5 log10EID50on day 3, from 0.5 to 2.5 log10EID50in day 5 and 0.25 log10EID50or less at day 7 (figure 2). In unvaccinated ferrets average titre was equal to 4.0 log10EID50one week after infection. Two of the three control ferrets were identified signs of serious illness (large weight loss and paralysis) and they were slain, and one died from the infection. Among vaccinated Ho is ikov only one has developed a serious disease. This ferret vaccinated with virus ΔH5S120→N/04, revealed severe neurological signs and he was put to death at day 7 after inoculation. This ferret in day 4 was identified serious viral conjunctivitis with subsequent spread of the virus in the brain. Apparently, the virus was inserted into the eye in the process of implementing a nasal lavage on day 3 and the rapid spread of the neurons in the brain caused encephalitis. The rest of vaccinated ferrets during the first three days after the control virus was revealed reduced activity, loss of body weight and increased body temperature. These signs disappeared by the fifth day, and all the animals quickly returned to normal. Thus, all tested vaccine viruses protected ferrets from lethal infection with A/Vietnam/1203/04. Vaccination resulted in lower titers of virus in the upper respiratory tract and reduced the duration of excretion of the virus in the environment.

Example 5: Evaluation using HI-tests for neutralizing immunogenicity of recombinant viruses ΔH5-A/PR/8/34

Obtained from vaccinated ferrets serum was tested against the presence of recombinant viruses with HI-analysis and analysis of neutralization of the virus (table 3 and 4 respectively). Serum samples taken from ferrets were treated in those who tell the night enzyme destroying the receptor for the cholera Vibrio (Vibrio cholerae) (firm Denka-Seiken, Tokyo, Japan), iactiveaware heat treatment at 56°C for 30 min and to absorb a 0.5%suspension of chicken red blood cells (CRBC). Standard HI-analysis and neutralization of virus in MDCK cells was performed as described in the literature method (Palmer and others, Immunology series no. 6. Atlanta: Centers for Disease Control and Prevention, published by US Department of Health, Education and Welfare, 1975; Kida, etc., Virology, 122, 1982,ñ.38).

Each HI-analysis used four hemagglutinins units (HAU) of virus in each assay neutralization of virus used for 100 secondary cytopathogenic doses (TCID50). For serum obtained from an organism ferrets vaccinated pseudovirions wild type by a single gene (DN/04, reference virus) HI-titers were 1:20. Design, in which you deleted the site of glycosylation (ΔH5S155→N;Tl56→A/04), induced HI titers comprising from 1:10 to 1:20. For mutant Δ5 HAS120→N/04 HI-titers ranged from 1:20 to 1:80. In contrast, vaccination with the use of ΔH5S223→N/04 led to HI-titer of 1:640, and the other tested immune serum reacts with the virus ΔH5S223→N/04 high HI-titer (1:160 to 1:320). Thus, although the vaccine induced protective immunity, levels of detectable antibodies were different.

Example 6: Reactivity of recombinant viruses

When creating inventions to further assess the reactivity of recombinant viruses used HI-analysis to test hyperimmune mouse serum and chickens, obtained after vaccination with the use of viruses ΔH5N1/03 and a/HK/213/03, against the recombinant viruses with altered (table 5). Average HI-titers against the homologous virus ΔH5N1/03 was 1:2560. HI-titers against Δ5/04 was 1:160. HI-titers against the recombinant virus ΔH5S223→N/04 exceeded at least twice titers against other mutants.

For more information about the contribution of the amino acid present at position 223 in serological reactivity in the claimed invention was constructed recombinant virus in which N5 was obtained from a/HK/213/03, having only point mutations of the N223→S (table 1A). This recombinant virus ΔH5N223→S/03 was characterized by lower HI titers in chicken and horse RBC than the virus ΔH5N1/03. To further assess the influence of the amino acid at position 223 to recognize is avanie antigen antibody in the claimed invention was constructed recombinant virus, which contained the wild type and had ON of the A/duck/Singapore/3/97 mutation S223→N (see table 1A). These viruses were tested using HI-analysis using a panel of antisera to H5 and Mat (table 6). Replacement of S223→N has led to a sharp increase in HI titers (4 times or more). However, this mutation did not lead to a significant change in the scheme of reactivity TO A/duck/Singapore/3/97, especially in reactions with Mat: neither the source nor the mutant TO enter into reaction with the Mat, representing NC-3 and SR, and both reacts with CR with low titers (table 6). These results indicate that replacement of S223→N ON increases sensitivity HI-analysis.

Example 7: the Second generation of diagnostic reference viruses, which should improve the sensitivity of the HI-analysis

Above in example 3 has been demonstrated that the replacement amino acid at position 223 IN H5 at asparagine increased sensitivity HI-analysis using chicken erythrocytes. While the invention has been proven that the basis of this effect at the molecular level is altered receptor specificity. The mutant virus together with a heightened sensitivity does not cause agglutination of horse erythrocytes, which contain only alpha-2,3-linked. Therefore, amino acid substitutions that lead to the ore ability to cause agglutination of horse erythrocytes, are candidates for increasing capacity sensitivity HI-analysis using chicken erythrocytes. This principle can be applied to all 16 subtypes ON, first of all influenza a viruses of birds, which have specificity against 2,3-connection.

Reverse genetic methods allow you to create recombinant viruses that have minimal changes in their antigenic structures, but are "optimized" from the point of view of the recognition of different cell substrates. Preferably subjected to mutation of amino acids (91, 130-134, 149, 151, 179, 186, 190-191, 220-225 in H5-subtype), which are located near the binding site of the receptor, or a part of it. You can create a plasmid containing constructed by genetic and by cotransfection to create viruses. Recombinant viruses can be tested by analysis using parallel chicken erythrocytes and horse erythrocytes. The viruses that cause agglutination of chicken red blood cells and do not cause agglutination of horse erythrocytes, are candidates for testing with HI-analyses. With additional experiments create viruses that cause agglutination of horse red blood cells and do not cause agglutination of chicken erythrocytes. Using a combination of candidates with Odie the full amino acid substitutions, you may also improve sensitivity. We should expect that the identification of candidates may lead to the identification of diagnostic reference viruses with specific reactivity to the receptor with specificity against 2,3-connection.

Table 2
HI-analysis of recombinant viruses Δ5 using monoclonal antibodies to H5
VirusMonoclonal antibodies to H5 (HI titers)
VN04-6*NC-3+SR++SR++SR++406/7++
ΔH5N1/0351200640016001001600800
Δ5/0412800<100800<1001600<10
ΔH5S155→N,T→A/043200<100800<100800100
ΔH5S120→N/0412800200800<1001600<100
ΔH5R212→K/04128001001600<1006400<100
ΔH5S223→N/0451200320012800<10025600<100
ΔH5S120→N,S155→N,T156→A/041280016003200<1001600200
ΔH5N223→S/0312800800≤100 <100200≤100

HI assays were performed in titration microplate containing 0.5%chicken RBC. Titers are the reciprocal of the smallest dilutions Mat, which inhibit the hemagglutinin 4 hemagglutinin units (HAU) of virus.

* The Mat to AT virus A/Vietnam/1203/04;

+ Mat to AT virus a/HK/213/03;

++Mat to AT virus A/chicken/Pennsylvania/1370/83.

11-week seronegative against influenza virus ferrets were vaccinated twice with a 3-week intervals by intramuscular injection of inactivated purified and concentrated product containing 7 μg IN 250 μl SFR. Data represent HI-titers presented individually for 3 ferrets. In HI-analyses were applied to 0.5%chicken RBC.

Analysis of neutralization was performed in MDCK cells. Titers are the reciprocal of the smallest dilution that completely inhibited 100 TCID50of the virus. Homologous titles are underlined. Values represent the neutralizing titers specified individually for each of the 3 ferrets.

Table 5
HI-analyses antisera to the H5N1 2003 against mutant viruses
The polyclonal anticavity to:
Virusvaccine ΔH5N 1/03A/HK/213/03 (H5N1)
ΔH5N1/0325602560
Δ5/04160160
ΔH5S155→N; T156→A80320
ΔH5S120→N/04320320
ΔH5S223→N/041280640

HI-analysis was performed in titration microplate containing 0.5%chicken RBC (see Palmer and others in: Immunology series no. 6. Atlanta: Centers for Disease Control and Prevention, published by US Department of Health, Education and Welfare, 1975). Titers are the reciprocal of the smallest dilution of serum that inhibited the hemagglutinin 4 HAU of virus.

<100
Table 6
Analysis of the antigenicity of the virus H5/97 and H5S223→N/97 using polyclonal and monoclonal antibodies
VirusHI-titer polyclonal antisera:HI-titer Mat:
A/ck/Hi dalgo/94A/HK/1 56/97a/gs/h K/497-4/97A/ck/HK /YU22/02And/NK/2
13/03
Δ5/03VN04-6*HK03-3+SR++SR++SR++SR/7++
Δ5/03203205120128001605120256006400160012800<1001600
H5/9740640640320<1010320032001600<1003200
H5S223→N/973201024010240256080128025600<100256005120020025600
* The Mat to AT virus A/Vietnam/1203/04;
+Mat to AT virus a/HK/213/03;
++Mat to AT virus A/chicken/Pennsylvania/13 70/83.
HI-analysis was performed in titration microplate containing 0.5%chicken RBC (14). Titers are the reciprocal of the smallest dilution of serum that inhibited the hemagglutinin 4 HAU of virus.

Scope of the present invention is not limited in this description of a particular variant embodiment of the invention. In fact, experts in this field after reading the above description and accompanying drawings is be apparent various modifications of the invention other than those presented in the present description. It should be considered that such modifications fall under the scope of the attached claims.

In addition, it should be borne in mind that all values are approximate and are given for illustrative purposes.

All patents and patent applications cited in the present description, are included in the description by reference in its entirety.

1. Recombinant hemagglutinin molecule OF influenza a subtype H5 (H5)with reactivity against antisera derived from an animal's body that are infected with influenza virus or influenza vaccine and comprising the amino acid asparagine at the position corresponding to position 223 in H5, where the molecule is not derived from isolate human H5 And a/HK/213/03.

2. Recombinant molecule ON according to claim 1, characterized in that it contains one or more substitutions compared to a corresponding molecule IN wild-type, where this recombinant molecule includes AT amino acid asparagine at the position corresponding to position 223 in H5; where the indicated wild-type molecule includes an amino acid other than asparagine at the position corresponding to position 223.

3. Recombinant molecule of influenza virus according to claim 1, characterized in that the amino acid substitution leads to a change of site gli is atilirovanie.

4. Recombinant molecule of influenza virus according to claim 1, wherein the influenza virus is an influenza virus And human.

5. Recombinant molecule of influenza virus according to claim 4, wherein the influenza virus a person is a representative of H5-subtype.

6. Recombinant molecule of influenza virus according to claim 4, wherein the influenza virus a person is a virus A/Vietnam/1203/04 (H5N1).

7. Recombinant influenza a subtype H5, designed to detect antibodies to influenza in animals exposed to influenza or influenza vaccine, recombinant hemagglutinin molecule OF influenza virus And type N5 according to claim 1.

8. The vaccine virus influenza a subtype H5, having immunogenic activity and containing recombinant hemagglutinin molecule OF influenza virus And type N5, where this recombinant molecule TO contain one or more substitutions compared to a corresponding molecule IN wild-type, where this recombinant molecule includes AT amino acid asparagine at the position corresponding to position 223 in H5; where the indicated wild-type molecule includes an amino acid other than asparagine at the position corresponding to position 223.

9. Influenza vaccine, recombinant hemagglutinin molecule (ON) VIR is sa influenza a type N5 according to claim 1.



 

Same patents:

FIELD: agriculture.

SUBSTANCE: specified plant is a plant of Cucumis sativus type and includes at least one area at one chromosome, which gives resistance to closterovirus, and at least one area, which gives resistance to powdery mildew. The area of the chromosome, which gives resistance to closterovirus, is linked to at least one marker selected from the group, made of markers E16/M50-244, E16/M50-188, and E11/M48-251. The area of the chromosome, which gives resistance to powdery mildew, is linked to at least one marker selected from the group made of the following components: a marker of single-nucleotide polymorphism 39TG in SEQ ID NO:1, a marker of single-nucleotide polymorphism 29GA in SEQ ID NO:2, a marker of single-nucleotide polymorphism 193CT in SEQ ID NO:3, mutation of an insert 5'-AATTT-3' in position 221 in SEQ ID NO:4, and markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001, E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200.

EFFECT: production of the plant resistant to closterovirus and to powdery mildew of cucumbers.

25 cl, 8 dwg, 4 tbl

FIELD: medicine.

SUBSTANCE: elaborated is method of obtaining factor, which takes part in process of control of appetite and/or body weight. Also described are genes, obtained by said method, polypeptides, coded by said genes, intended for treatment, control or diagnostics of diseases, associated with eating disorders and/or control of body weight. Invention also relates to substances, which inhibit activity of said genes or said polypeptides, intended for treatment, control or diagnostics of diseases, associated with process of appetite and/or body weight control.

EFFECT: using tiasolidindions, possessing PPARγ-agonistic activity, it is possible to obtain genes and polypeptides, involved into regulation of appetite and/or body weight reduction.

27 cl, 41 dwg, 35 ex

FIELD: medicine.

SUBSTANCE: RNA is recovered from peripheral blood or synovial liquid. Further, cytokine balance is evaluated by quantitative analysis of interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10) or interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-1p (IL-1p) cytokines mRNA genes expression, as well as by quantitative analysis of interferon-gamma (IFNG) and a tumour necrosis factor (TNF) by reverse transcription and polymerase chain reaction with recording the accumulation of reaction products by direct fluorescence. The direct fluorescence is used to evaluate the cytokine balance. Further, a pair balance of expression of various cytokines is calculated on the basis a functional interrelation.

EFFECT: use of the invention reduces an evaluation error related to specific properties of the control gene expression.

4 dwg, 6 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: offered is a method and a set of primers for determining a haplotype of a DNA-target containing two heterozygous polymorphic sites. The method according to the invention provides a) taking a genome DNA sample and amplifying the area on the 3'-side of which there is a first polymorphic site, and on the 5'-side of which the second polymorphic site is found; b) conducting a PCR of said specific area with using one of two primers allele-specific relatively to the first polymorphic site and a downstream primer which is located so that to include the second polymorphic site in an amplicon; c) hybridising the prepared PCR product with two probes one of which is specific to a mutant type sequence, and another one - to a wild type sequence within the second polymorphism and d) determining a haplotype where the first polymorphism is described by a type of an extended upstream primer, and the second polymorphism - by a type of the probe hybridised with the PCR product.

EFFECT: use of the invention provides relatively simple and high precise diagnostic system.

4 cl, 5 dwg, 5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to synthetic oligonucleotide primers, complementary to high conservative VP60 gene region of a genome of rabbit viral hemorrhagic disease virus, to a method for identifying rabbit viral hemorrhagic disease virus and to a test system for identifying RNA of rabbit viral hemorrhagic disease virus. The offered invention can be used in veterinary virology. The method for identifying rabbit viral hemorrhagic disease virus involves sample preparation, RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers 5'-caa cgt get cca gtt ttg gta cg-3', 5'-att ctg tct ggt tgg ggc gtg t-3'. Further, viral RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 398 base pairs.

EFFECT: invention allows higher sensitivity of the method, as well as reduced time of diagnostic manipulations with organ and blood samples of the infected animals.

3 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to synthetic oligonucleotide primers complementary to a conservative S-segment region of a genome of sheep Nairobi disease virus, to a method for identifying sheep Nairobi disease virus and to a test system for identifying DNA of sheep Nairobi disease virus. The offered invention can be used in veterinary virology. The method for identifying sheep Nairobi disease virus involves RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers NSD F1 5'TATGCTTCTGCCTTGGTTG-3' NSD R1 5'-ATCCGATTGGC AGTGAAG-3', NSD F2 5'-AGAGCACATTGACTGGGC-3', NSD R2 5'-GCCTTCCAAAGCCAGTAG-3' Thereafter, virus RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 360 base pairs.

EFFECT: offered invention allows identifying genome RNA of sheep Nairobi disease virus by means of a nidicolous version of RT-PCR with using two synthesized pairs of oligonucleotide primers complementary to conservative gene region of core protein N.

3 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: sequence typing is used to differentiate Yersinia pestis strains. The technique provides recovery of chromosomal DNA of the investigated strain, polymerase chain reaction (PCR) with amplification of rhaS, araC, metB, asp A and thiH gene fragments to be analysed for nucleotide sequences. A genotype of the investigated strain is stated by nucleotides being in the positions 482, 494, 671 reHarhaS, in the position 773 of the gene rhaS, in the positions 988 and 989 of the gene metB, in the positions 1087-1089 of the gene aspA and in the position 552 of the gene thiH. A sequence type (ST) of the Y. pestis strain is specified by rhaS, araC, metB, aspA and thiH gene alleles, while the subspecies differentiation is enabled by comparing to the sequence types of the major and minor subspecies. The sequence type (ST) is specified for each subspecies.

EFFECT: use of the method provides fast, reliable and effective differentiation of Yersinia pestis subspecies.

2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to a synthetic oligonucleotide kit for identifying DNA of human monocytic ehrlichiosis (HME) agent - pathogenic representatives of Ehrlichia genus by a polymerase chain reaction. The offered invention can be used for the diagnostic purposes for detecting monocytic ehrlichiosis Ehrlichia spp. by the real-time polymerase chain reaction. Said kit includes primers as follows: 5'- GGG GAA AGA TTT ATC GCT ATT AG -3', 5'- CGG CAT AGC TGG ATC AGG CT -3' and a sample: (BHQl)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G - 3'P, where BHQ1 means a dark fluorescence killer attached to 5'-terminal nucleotide, while FdT is a fluorescent dye FAM attached to nucleotide T.

EFFECT: invention allows reliable identification of Ehrlichia representatives in the biological material.

1 ex

FIELD: medicine.

SUBSTANCE: live attenuated Pasteurella multocida bacterium is modified by introduing a mutation into the gene Orf-15. The mutation represents insertion and/or deletion in the gene Orf-15. As a result of modification, the bacterium is not able to express the functional protein Orf-15 that leads to its attenuation. Also, disclosed is the use of such bacterium for preparing a related vaccine for human or animal protection against Pasteurella multocida bacteria infection or pathogenic effects of the infection.

EFFECT: increased efficacy of applying the composition.

16 cl, 1 dwg, 3 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: genetic predisposition is determined by analysing polymorphism of genes ACTN3 (R577X), CNB (5I/5D), AMPD1 (C34T) through a multiplex polymerase chain reaction. For this purpose, a set of primers is used, wherein for the gene ACTN3 (R577X) the primes used are ACTGCTGCCCTTTCTGTTGCCT-3' and S'-CTGCAGGTGGCACTGACCATA3', for the gene CNB (5I/5D) the primers used are 5'-GGAGTTTAAAAGCCAGCCAGTCATACTA-3' and 5'-TGGAAGATCACACCATTTGATTAGCAGT-3', for the geneAMPD1 (C34T) the primers used are S'-GCAATCTACATGTGTCTACCCCAAAG-3' and 5'-CACTGCTGAAAAATAGCCATGTTTCTG-3'. Further, 3 pairs of primers with average melting point of 58-59°C are merged in a single test tube at standard conditions. The reaction product is analysed through analysis of polymorphism of the length of restriction fragments.

EFFECT: invention provides a simple, cheap, specific method which enables to identify functionally significant polymorphous gene loci, grow the number of analysed genes for further introduction into any standard clinical laboratory.

2 cl, 2 dwg, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: disclosed is a plasmid for producing a viral vector transporting multiple expression cassettes to a target. The plasmid contains genome nucleotide sequences packed into a polyvalent capsid and a number of cassettes to be transported. A method for producing a viral vector, a viral vector and an immunogenic composition are described besides.

EFFECT: group of inventions can be used for wide-ranging expression of target antigens.

23 cl, 3 dwg

FIELD: medicine.

SUBSTANCE: modified virus-like particle (VLP) includes hybrid protein which consists of AP205 bacteriophage protein and any antigen. Also, a composition including VLP, being a derivative of RNA-containing AP205 bacteriophage is described. Besides, the invention describes a method for producing said VLP. Modified VLP of the present invention is applicable for producing compositions for inducing immune response for prevention or treatment of diseases, disorders, including infectious diseases, allergies, cancer and drug addiction.

EFFECT: offered group of inventions can be used in medicine, immunology, virology and molecular biology.

35 cl, 3 dwg, 1 tbl, 26 ex

FIELD: medicine.

SUBSTANCE: recombinant plasmid pFastBac-B17R DNA carries a cowpox virus genome fragment. BvB 17RG recombinant baculovirus strain is produced with the use of recombinant plasmid pFastBac-B 17R DNA and deposited in the Microorganism Cultures Collection of the Federal State Research Institution 'State Research Centre for Virology and Biotechnology 'Vector' of Federal Service for Supervision of Consumer Rights Protection and Human Welfare' (FGUN GNC VB 'Vector' of Rospotrebnadzor) under No. V-388, characterised as a producer of a soluble analogue protein of cowpox interferons type 1 cell receptor.

EFFECT: extended spectrum of preparations of new generation.

2 cl, 5 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: viral particle consists of non-alphavirus structural elements and contains an alphavirus vector replication-defective by deletion or substitution by at least one transgene. Besides said particle contains structural elements not coded by an alphavirus vector genome. A method for preparing said particle according to the invention implies the expression in trans of the genes coding the non-alphavirus structural elements, and the alphavirus vector, in a cell line and then recovery of the viral particles found in the cell culture supernatant.

EFFECT: invention allows preparing the viral particles with lower recombination risk.

24 cl, 5 dwg, 3 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology. Recombinant adenovirus expresses a protein selected from a group comprising E1B and E4 proteins and their combination, and then a second protein which is E1A. Recombinant adenovirus consists of an expression cassette which codes E1B and E4 proteins under the control of a promoter which is different from the promoter which regulates expression in the wild type adenovirus. The recombinant adenovirus optionally contains nucleic acid which codes YB-1, expression cassettes which contain promoters and nucleic acids which code different genes. Disclosed also is a nucleic acid which codes such a recombinant adenovirus and use thereof.

EFFECT: recombinant adenovirus can be used in medicine for treating tumoural diseases.

30 cl, 26 dwg, 19 ex

FIELD: medicine.

SUBSTANCE: recombinant virus containing human gene p53, its application, manufacture method and pharmaceutical composition are offered.

EFFECT: invention can be used for gene therapy in human malignant neoplasm treatment and prevention.

8 cl, 14 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention is related to preparation of protein, binding tumour necrosis factor (TNF), and may be used in medicine. Strain-producer of baculovirus BvG2RIgG is created with the help of recombinant plasmid DNA pFastBac-G2R-IgG with size of 6444 p.n. and molecular mass 4.18 mDa, which bears fragment of smallpox virus genome of strain India-1967, which codes protein that binds TNF, and fragment of human genome, which codes fragment of heavy chain of human antibody G. Produced strain produces soluble chimeric protein, which consists of smallpoz virus protein, which binds TNF, and fragment of heavy chain of human antibody G.

EFFECT: wider spectrum of new generation preparations intended for treatment of human diseases related to hyperproduction of tumour necrosis factor.

2 cl, 3 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and biotechnology. There is disclosed application of coding DNA for making virus-like particles of hepatitis C virus, as well as the method for making the specified particles and a pharmaceutical composition using the same. The virus-like particles cause induction of interferon system in vivo.

EFFECT: invention can be used for making a preparation for prevention and treatment of the HCV-related conditions, as well as diagnostics thereof.

5 cl, 4 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention claims synthetic DNS molecule encoding L1 HPV58 protein, where codons are optimised for high expression level in yeast cell. Also invention claims expression vector, yeast host cell, HPV58 virus-like particle and method of its obtainment, and pharmaceutical composition including such VLP.

EFFECT: invention can be applied in medicine for efficient immune prevention of papillomavirus infection by neutralising antibodies and cell-mediated immunity, and for treatment of developed HPV infections.

10 cl, 10 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention concerns virology and medicine area. The synthetic DNA-molecule coding protein L1 HPV45 is presented. Thus DNA-molecule was Codonum-optimised for high-level protein expression in a yeast cell. The given synthetic molecules can be used for reception of virus-like particles (VLP) HPV45 and for reception of vaccines and the pharmaceutical compositions containing VLP-particles HPV45.

EFFECT: effective immunologic prophylaxis of infections with papilloma virus due to neutralised antibodies and cellular immunity.

9 cl, 6 dwg, 8 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to molecular biology, virology and genetic engineering. There are disclosed method of producing recombinant influenza virus with mutant gene NB of membrane protein, recombinant influenza virus produced by this method, and vector composition based on parts of recombinant influenza virus.

EFFECT: developed method of recombinant influenza virus with mutant gene NB of membrane protein.

24 cl, 8 dwg

Up!