Patent Application: US-201615332330-A

Abstract:
recombinant influenza virus proteins , including influenza capsomers , subviral particles , virus - like particles , vlp complexes , and / or any portions of thereof , are provided as a vaccine for influenza viruses . the invention is based on the combination of two vaccine technologies : intrinsically safe recombinant vaccine technology , and highly immunogenic , self - assembled protein macromolecules embedded in plasma membranes and comprised of multiple copies of influenza virus structural proteins exhibiting neutralizing epitopes in native conformations . more specifically , this invention relates to the design and production of functional homotypic and heterotypic recombinant influenza virus - like particles comprised of recombinant structural proteins of human influenza virus type a / sydney / 5 / 94 and / or avian influenza virus type a / hong kong / 1073 / 99 in baculovirus - infected insect cells and their application as a vaccine in the prevention of influenza infections and as a laboratory reagent for virus structural studies and clinical diagnostics .

Description:
as used herein , the term “ baculovirus ,” also known as baculoviridae , refers to a family of enveloped dna viruses of arthropods , members of which may be used as expression vectors for producing recombinant proteins in insert cell cultures . the virion contains one or more rod - shaped nucleocapsids containing a molecule of circular supercoiled double - stranded dna ( m r 54 × 10 6 - 154 × 10 6 ). the virus used as a vector is generally autographa californica nuclear polyhedrosis virus ( nvp ). expression of introduced genes is under the control of the strong promoter that normally regulates expression of the polyhedron protein component of the large nuclear inclusion in which the viruses are embedded in the infected cells . as used herein , the term “ derived from ” refers to the origin or source , and may include naturally occurring , recombinant , unpurified , or purified molecules . the proteins and molecules of the present invention may be derived from influenza or non - influenza molecules . as used herein the term “ first ” influenza virus protein , i . e ., a first influenza virus m1 protein , refers to a protein , such as m1 , ha , na , and m2 , that is derived from a particular strain of influenza virus . the strain or type of the first influenza virus differs from the strain or type of the second influenza virus protein . thus , “ second ” influenza virus protein , i . e ., the second influenza virus m1 protein , refers to a protein , such as m1 , ha , na , and m2 , that is derived from a second strain of influenza virus , which is a different strain or type than the first influenza virus protein . as used herein , the term “ hemagglutinin activity ” refers to the ability of ha - containing proteins , vlps , or portions thereof to bind and agglutinate red blood cells ( erythrocytes ). as used herein , the term “ neuraminidase activity ” refers to the enzymatic activity of na - containing proteins , vlps , or portions thereof to cleave sialic acid residues from substrates including proteins such as fetuin . as used herein , the term “ heterotypic ” refers to one or more different types or strains of virus . as used herein , the term “ homotypic ” refers to one type or strain of virus . as used herein , the term “ macromolecular protein structure ” refers to the construction or arrangement of one or more proteins . as used herein , the term “ multivalent ” vaccine refers to a vaccine against multiple types or strains of influenza virus . as used herein , the term “ non - influenza ” refers to a protein or molecule that is not derived from influenza virus . as used herein , the term “ vaccine ” refers to a preparation of dead or weakened pathogens , or of derived antigenic determinants , that is used to induce formation of antibodies or immunity against the pathogen . a vaccine given to provide immunity to the disease , for example , influenza , which is caused by influenza viruses . the present invention provides vaccine compositions that are immunogenic and provide protection . influenza remains a pervasive public health concern despite the availability of specific inactivated virus vaccines that are 60 - 80 % effective under optimal conditions . when these vaccines are effective , illness is usually averted by preventing viral infection . vaccine failure can occur as a result of accumulated antigenic differences ( antigenic shift and antigenic drift ). for example , avian influenza virus type a h9n2 co - circulated with human influenza virus type a sydney / 97 h3n2 in pigs and led to genetic reassortment and emergence of new strains of human influenza virus with pandemic potential ( peiris et al ., 2001 ). in the event of such antigenic shift , it is unlikely that current vaccines would provide adequate protection . another reason for the paucity of influenza vaccine programs is the relatively short persistence of immunity elicited by the current vaccines . further inadequacy of influenza control measures reflects restricted use of current vaccines because of vaccine reactogenicity and side effects in young children , elderly , and people with allergies to components of eggs , which are used in manufacturing of commercially licensed inactivated virus influenza vaccines . additionally , inactivated influenza virus vaccines often lack or contain altered ha and na conformational epitopes , which elicit neutralizing antibodies and play a major role in protection against disease . thus , inactivated viral vaccines , as well as some recombinant monomeric influenza subunit protein vaccines , deliver inadequate protection . on the other hand , macromolecular protein structures , such as capsomers , subviral particles , and / or vlps , include multiple copies of native proteins exhibiting conformational epitopes , which are advantageous for optimal vaccine immunogenicity . the present invention describes the cloning of avian influenza a / hong kong / 1073 / 99 ( h9n2 ) virus ha . na , and m1 genes into a single baculovirus expression vector alone or in tandem and production of influenza vaccine candidates or reagents comprised of recombinant influenza structural proteins that self - assemble into functional and immunogenic homotypic macromolecular protein structures , including subviral influenza particles and influenza vip , in baculovirus - infected insect cells . the present invention further features the cloning of human influenza a / sydney / 5 / 94 ( h3n2 ) virus ha , na , m1 , m2 , and np genes into baculovirus expression vectors and production influenza vaccine candidates or reagents comprised of influenza structural proteins that self - assemble into functional and immunogenic homotypic macromolecular protein structures , including subviral influenza particles and influenza vlp , in baculovirus - infected insect cells . in addition , the instant invention describes the cloning of the ha gene of human influenza a / sydney / s / 94 ( h3n2 ) virus and the ha , na , and m1 genes of avian influenza a / hong kong / 1073 / 99 ( h9n2 ) into a single baculovirus expression vector in tandem and production influenza vaccine candidates or reagents comprised of influenza structural proteins that self - assemble into functional and immunogenic heterotypic macromolecular protein structures , including subviral influenza particles and influenza vlp , in baculovirus - infected insect cells . this invention is further illustrated by the following examples which should not be construed as limiting . the contents of all references , patents and published patent applications cited throughout this application , as well as the figures and the sequence listing , are incorporated herein by reference . avian influenza a / hong kong / 1073 / 99 ( h9n2 ) virus ha , na , and m1 genes were expressed in spodoptera frugiperda cells ( sf - 9s cell line ; atcc pta - 4047 ) using the baculovirus bacmid expression system . the ha , na , and m1 genes were synthesized by the reverse transcription and polymerase chain reaction ( pcr ) using rna isolated from avian influenza a / hong kong / 1073 / 99 ( h9n2 ) virus ( fig1 , and 3 ). for reverse transcription and pcr , oligonucleotide primers specific for avian influenza a / hong kong / 1073 / 99 ( h9n2 ) virus ha , na , and m1 genes were used ( table 1 ). the cdna copies of these genes were cloned initially into the bacterial subcloning vector , pcr2 . 1topo . from the resulting three pcr2 . 1topo - based plasmids , the ha , na , and m1 genes were inserted downstream of the acmnpv polyhedrin promoters in the baculovirus transfer vector , pfastbac1 ( invitrogen ), resulting in three pfastbac1 - based plasmids : pha , pna , and pm1 expressing these influenza virus genes , respectively . then , a single pfastbac1 - based plasmid pham was constructed encoding both the ha and m1 genes , each downstream from a separate polyhedrin promoter ( fig4 ). the nucleotide sequence of the na gene with the adjacent 5 ′- and 3 ′- regions within the pna plasmid was determined ( seq id no : 1 ) ( fig1 ). at the same time , the nucleotide sequences of the ha and m1 genes with the adjacent regions were also determined using the pham plasmid ( seq id nos : 2 and 3 ) ( fig2 and 3 ). finally , a restriction dna fragment from the pham plasmid that encoded both the ha and m1 expression cassettes was cloned into the pna plasmid . this resulted in the plasmid pnaham encoding avian influenza a / hong kong / 1073 / 99 ( h9n2 ) virus ha , na , and m1 genes ( fig4 ). plasmid pnaham was used to construct a recombinant baculovirus containing influenza virus na , ha , and m1 genes integrated into the genome , each downstream from a separate baculovirus polyhedrin promoter , infection of permissive sf - 9s insect cells with the resulting recombinant baculovirus resulted in co - expression of these three influenza genes in each sf - 9s cell infected with such recombinant baculovirus . the expression products in infected sf - 9s cells were characterized at 72 hr postinfection ( p . i .) by sds - page analysis , coomassie blue protein staining , and western immunoblot analysis using ha - and m1 - specific antibodies ( fig5 ). western immunoblot analysis was carried out using rabbit antibody raised against influenza virus type a / hong kong / 1073 / 99 ( h9n2 ) ( cdc , atlanta , ga ., usa ), or mouse monoclonal antibody to influenza m1 protein ( serotec , uk ). the ha , na , and m1 proteins of the expected molecular weights ( 64 kd , 60 kd , and 31 kd , respectively ) were detected by western immunoblot analysis . compared to the amount of ha protein detected in this assay , the na protein showed lower reactivity with rabbit serum to influenza a / hong kong / 1073 / 99 ( h9n2 ) virus . explanations for the amount of detectable na protein included lower expression levels of the na protein from sf - 9s cells infected with recombinant baculovirus as compared to the ha protein , lower reactivity of the na with this serum under denaturing conditions in the western immunoblot assay ( due to the elimination of important na epitopes during gel electrophoresis of membrane binding ), lower na - antibody avidity as compared to ha - antibody , or a lower abundance of na - antibodies in the serum . the culture medium from the sf - 9s cells infected with recombinant baculovirus expressing a / hong kong / 1073 / 99 ( h9n2 ) ha , na , and m1 proteins was also probed for influenza proteins . the clarified culture supernatants were subjected to ultracentrifugation at 27 , 000 rpm in order to concentrate high - molecular protein complexes of influenza virus , such as subviral particles , vlp , complexes of vlp , and possibly , other self - assembled particulates comprised of influenza ha , na , and m1 proteins . pelleted protein products were resuspended in phosphate - buffered saline ( pbs , ph 7 . 2 ) and further purified by ultracentrifugation on discontinuous 20 - 60 % sucrose step gradients . fractions from the sucrose gradients were collected and analyzed by sds - page analysis , western immunoblot analysis , and electron microscopy . influenza ha and m1 proteins of the expected molecular weights were detected in multiple sucrose density gradient fractions by coomassie blue staining and western immunoblot analysis ( fig6 ). this suggested that influenza viral proteins from infected sf - 9s cells are aggregated in complexes of high - molecular weight , such as capsomers , subviral particles , vlp , and / or vlp complexes . the na proteins , although inconsistently detected by coomassie blue staining and western immunoblot analysis , which was likely due to the inability of the rabbit anti - influenza serum to recognize denatured na protein in the western immunoblot assay , were consistently detected in neuraminidase enzyme activity assay ( fig1 ). the presence of high - molecular vlps was confirmed by gel filtration chromatography . an aliquot from sucrose density gradient fractions containing influenza viral proteins was loaded onto a sepharose cl - 4b column for fractionation based on mass . the column was calibrated with dextran blue 2000 , dextran yellow , and vitamin b12 ( amersham pharmacia ) with apparent molecular weights of 2 , 000 , 000 ; 20 . 000 ; and 1 , 357 daltons , respectively , and the void volume of the column was determined . as expected , high - molecular influenza viral proteins migrated in the void volume of the column , which was characteristic of macromolecular proteins , such as virus particles . fractions were analyzed by western immunoblot analysis to detect influenza and baculovirus proteins . for example , m1 proteins were detected in the void volume fractions , which also contained baculovirus proteins ( fig7 ). the morphology of influenza vlps and proteins in sucrose gradient fractions was elucidated by electron microscopy . for negative - staining electron microscopy , influenza proteins from two sucrose density gradient fractions were fixed with 2 % glutaraldehyde in pbs , ph 7 . 2 . electron microscopic examination of negatively - stained samples revealed the presence of macromolecular protein complexes or vlps in both fractions . these vlps displayed different sizes including diameters of approximately 60 and 80 nm and morphologies ( spheres ). larger complexes of both types of particles were also detected , as well as rod - shaped particles ( fig8 ). all observed macromolecular structures had spikes ( peplomers ) on their surfaces , which is characteristic of influenza viruses . since the size and appearance of 80 nm particles was similar to particles of wild type influenza virus , these structures likely represented vlps , which have distinct similarities to wild type influenza virions , including similar particle geometry , architecture , triangulation number , symmetry , and other characteristics . the smaller particles of approximately 60 nm may represent subviral particles that differ from vlps both morphologically and structurally . similar phenomenon of recombinant macromolecular proteins of different sizes and morphologies was also reported for other viruses . for example , recombinant core antigen ( hbcag ) of hepatitis b virus forms particles of different sizes , which have different architecture and triangulation number t = 4 and t = 3 , respectively ( crowther et al ., 1994 ). to characterize the functional properties of the purified influenza a / hong kong / 1073 / 99 ( h9n2 ) vlps , samples were tested in a hemagglutination assay ( fig9 ) and a neuraminidase enzyme assay ( fig1 ). for the hemagglutination assay , 2 - fold dilutions of purified influenza vlps were mixed with 0 . 6 % guinea pig red blood cells and incubated at 4 ° c . for 1 hr or 16 hr . the extent of hemagglutination was inspected visually and the highest dilution of recombinant influenza proteins capable of agglutinating red blood cells was determined and recorded ( fig9 ). again , many fractions from the sucrose density gradient exhibited hemagglutination activity , suggesting that multiple macromolecular and monomeric forms of influenza proteins were present . the highest titer detected was 1 : 4000 . in a control experiment , wild - type influenza a / shangdong virus demonstrated a titer of 1 : 2000 . the hemagglutination assay revealed that the recombinant vlps consisting of influenza a / hong kong / 1073 / 99 ( h9n2 ) virus ha , na , and m1 proteins were functionally active . this suggested that the assembly , conformation , and folding of the ha subunit proteins within the vlps were similar or identical to that of the wild type influenza virus . additionally , a neuraminidase enzyme assay was performed on samples of purified h9n2 vlps . the amount of neuraminidase activity in sucrose density gradient fractions was determined using fetuin as a substrate . in the neuraminidase assay , the neuraminidase cleaved sialic acid from substrate molecules to release sialic acid for measurement . arsenite reagent : was added to stop enzyme activity . the amount of sialic acid liberated was determined chemically with thiobarbituric acid that produces a pink color that was proportional to the amount of free sialic acid . the amount of color ( chromophor ) was measured spectrophotometrically at wavelength 549 nm . using this method , neuraminidase activity was demonstrated in sucrose gradient fractions containing influenza vlps ( fig1 ). as expected , the activity was observed in several fractions , with two peak fractions . as a positive control , wild type influenza virus was used . the wild type influenza virus exhibited neuraminidase enzyme activity comparable to that of purified influenza vlps . these findings corroborated the ha results with regard to protein conformation and suggested that purified vlps of influenza a / hong kong / 1073 / 99 ( h9n2 ) virus were functionally similar to wild type influenza virus . the results from the above analyses and assays indicated that expression of influenza a / hong kong / 1073 / 99 ( h9n2 ) ha , na , and m1 proteins was sufficient for the self - assembly and transport of functional vlps from baculovirus - infected insect cells . since these influenza vlps represented self - assembled influenza structural proteins and demonstrated functional and biochemical properties similar to those of wild type influenza virus , these influenza vlps conserved important structural conformations including surface epitopes necessary for effective influenza vaccines . example 2 : rt - pcr cloning of avian influenza a / hong kong / 1073 / 99 viral genes it is an object of the present invention to provide synthetic nucleic acid sequences capable of directing production of recombinant influenza virus proteins . such synthetic nucleic acid sequences were obtained by reverse transcription and polymerase chain reaction ( pcr ) methods using influenza virus natural genomic rna isolated from the virus . for the purpose of this application , nucleic acid sequence refers to rna , dna , cdna or any synthetic variant thereof which encodes the protein . avian influenza a / hong kong / 1073 / 99 ( h9n2 ) virus was provided by dr . k . subbarao ( centers for disease control , atlanta , ga ., usa ). viral genomic rna was isolated by the acid phenol rna extraction method under biosafety level 3 ( bsl3 ) containment conditions at cdc using trizol ls reagent ( invitrogen , carlsbad , calif . usa ). cdna molecules of the viral rnas were obtained by reverse transcription using mulv reverse transcriptase ( invitrogen ) and pcr using oligonucleotide primers specific for ha , na , and m1 proteins and taq i dna polymerase ( invitrogen ) ( table 1 ). the pcr fragments were cloned into the bacterial subcloning vector , pcr2 . 1topo ( invitrogen ), between eco ri sites that resulted in three recombinant plasmids , containing the ha . na , and m1 cdna clones . example 3 : rt - pcr cloning of human influenza a / sydney / 5 / 94 ( h3n2 ) viral genes influenza a / sydney / 5 / 94 ( h3n2 ) virus was obtained from dr . m . massare ( novavax , inc ., rockville , md .). viral genomic rna was isolated by the rna acid phenol extraction method under bsl2 containment conditions at novavax , inc . using trizol ls reagent ( invitrogen ). cdna molecules of the viral rnas were obtained by reverse transcription and pcr using oligonucleotide primers specific for ha , na , m1 , m2 , and np proteins ( table 2 ). the pcr fragments were cloned into the bacterial subcloning vector , pcr2 . 1topo , between eco ri sites that resulted in five recombinant plasmids , containing the ha , na , m1 , m2 , and np cdna clones . example 4 : cloning of avian influenza a / hong kong / 1073 / 99 viral cdnas into baculovirus transfer vectors from the pcr2 . 1topo - based plasmids , the ha , na , or m1 genes were subcloned into pfastbac1 baculovirus transfer vector ( invitrogen ) within the polyhedron locus and tn7 art sites and downstream of the baculovirus polyhedrin promoter and upstream of the polyadenylation signal sequence . the viral genes were ligated with t4 dna ligase . for the ia gene , a bam hi - kpn i dna fragment from pcr2 . 1topo - ha was inserted into bam hi - kpn i digested pfastbac1 plasmid dna . for the na gene , an eco ri dna fragment from pcr2 . 1topo - na was inserted into eco ri digested pfastbac1 plasmid dna . for the m1 gene , an eco ri dna fragment from pcr2 . 1topo - m1 was inserted into eco ri digested pfastbac1 plasmid dna . competent e . coli dh5α bacteria ( invitrogen ) were transformed with these dna ligation reactions , transformed colonies resulted , and bacterial clones isolated . the resulting pfastbac1 - based plasmids , pfastbac1 - ha , pfastbac1 - na , and pfastbac1 - m1 were characterized by restriction enzyme mapping on agarose gels ( fig4 a ). the nucleotide sequences as shown on fig1 - 3 of the cloned genes were determined by automated dna sequencing . dna sequence analysis showed that the cloned influenza ha , na , and m1 genes were identical to the nucleotide sequences for these genes as published previously [ na , ha ., and m1 genes of influenza a / hong kong / 1073 / 99 ( h9n2 ) ( genbank accession numbers aj404629 , aj404626 , and aj278646 , respectively )]. example 5 : cloning of human influenza a / sydney / 5 / 94 viral cdnas into baculovirus transfer vectors from the pcr2 . 1topo - based plasmids , the ha , na , m1 , m2 , and np genes were subcloned into pfastbac1 baculovirus transfer vector within the polyhedron locus and tn7 art sites and downstream of the baculovirus polyhedrin promoter and upstream of the polyadenylation signal sequence . the viral genes were ligated with t4 dna ligase . for the ha gene , a bam hi - kpn i dna fragment from pcr2 . 1topo - hha3 was inserted into bam hi - kpn i digested pfastbac1 plasmid dna . for the na gene , an eco ri dna fragment from pcr2 . 1topo - hna was inserted into eco ri digested pfastbac1 plasmid dna . for the m1 gene , an eco ri dna fragment from pcr2 . 1topo - hm1 was inserted into eco ri digested pfastbac1 plasmid dna . for the m2 gene , an eco ri dna fragment from pcr2 . 1topo - hm2 was inserted into eco ri digested pfastbac1 plasmid dna . for the np gene , an eco ri dna fragment from pcr2 . 1topo - hnp was inserted into eco ri digested pfastbac1 plasmid dna . competent e . coli dh5α bacteria were transformed with these dna ligation reactions , transformed colonies resulted , and bacterial clones isolated . the resulting pfastbac1 - based plasmids , pfastbac1 - hha3 , pfastbac1 - hna2 , pfastbac1 - hm1 , pfastbac1 - hm2 , and pfastbac1 - hnp were characterized by restriction enzyme mapping on agarose gels . the nucleotide sequences of the cloned genes were determined by automated dna sequencing . dna sequence analysis showed that the cloned influenza ha , na , m1 , m2 , and np genes were identical to the nucleotide sequences for these genes as published previously . example 6 : construction of multigenic baculovirus transfer vectors encoding multiple avian influenza a / hong kong / 1073 / 99 viral genes in order to construct pfastbac1 - based bacmid transfer vectors expressing multiple influenza a / hong kong / 1073 / 99 ( h9n2 ) virus genes , initially a sna bi - hpa i dna fragment from pfastbac1 - m1 plasmid containing the m1 gene was cloned into hpa i site of pfastbac1 - ha . this resulted in pfastbac1 - ham plasmid encoding both ha and m1 genes within independent expression cassettes and expressed under the control of separate polyhedrin promoters . finally , a sna bi - avr ii dna fragment from pfastbac1 - ham containing the ha and m1 expression cassettes , was transferred into hpa 1 - avr ii digested pfastbac1 - na plasmid dna . this resulted in the plasmid pfastbac1 - naham encoding three independent expression cassettes for expression of influenza ha , na , and m1 genes and expressed under the control of separate polyhedrin promoters ( fig4 b ). in another example , the h3 gene from pfastbac1 - hha3 ( see example 5 ) was cloned into pfastbac1 - naham as a fourth influenza viral gene for the expression and production of heterotypic influenza vlps . example 7 : generation of multigenic recombinant baculovirus encoding na , ha , and m1 genes of avian influenza a / hong kong / 1073 / 99 virus in insect cells the resulting multigenic bacmid transfer vector pfastbac1 - naham was used to generate a multigenic recombinant baculovirus encoding avian influenza a / hong kong / 1073 / 99 ( h9n2 ) ha , na , and m1 genes for expression in insect cells . recombinant bacmid dnas were produced by site - specific recombination at polyhedrin and tn7 art dna sequences between pfastbac1 - naham dna and the acmnpc baculovirus genome harbored in competent e . coli dh10bac cells ( invitrogen ) ( fig4 b ). recombinant bacmid dna was isolated by the mini - prep plasmid dna method and transfected into sf - 9s cells using the cationic lipid c ellfectin ( invitrogen ). following transfection , recombinant baculoviruses were isolated , plaque purified , and amplified in sf - 9s insect cells . virus stocks were prepared in sf - 9s insect cells and characterized for expression of avian influenza viral ha , na , and m1 gene products . the resulting recombinant baculovirus was designated bnaham - h9n2 . example 8 : expression of recombinant avian influenza a / hong kong / 1073 / 99 proteins in insect cells sf - 9s insect cells maintained as suspension cultures in shaker flasks at 28 ° c . in serum - free medium ( hyq sfm , hyclone , ogden , utah ) were infected at a cell density of 2 × 10 6 cells / mil with the recombinant baculovirus , bnaham - h9n2 , at a multiplicity of infection ( moi ) of 3 pfu / cell . the virus infection proceeded for 72 hrs . to allow expression of influenza proteins . expression of avian influenza a / hong kong / 1073 / 99 ( h9n2 ) ha and m1 proteins in infected insect cells was confirmed by sds - page and western immunoblot analyses . sds - page analysis was performed on 4 - 12 % linear gradient nupage gels ( invitrogen ) under reduced and denaturing conditions . primary antibodies in western immunoblot analysis were polyclonal rabbit antiserum raised against avian influenza a / hong kong / 1073 / 99 ( h9n2 ) obtained from cdc and monoclonal murine antiserum to influenza m1 protein ( serotec , uk ). secondary antibodies for western immunoblot analysis were alkaline phosphatase conjugated goat igg antisera raised against rabbit or mouse igg ( h + l ) ( kirkegaard and perry laboratories , gaithersburg , md ., usa ). results of these analyses ( fig5 ) indicated that the ha and m1 proteins were expressed in the baculovirus - infected insect cells . example 9 : purification of recombinant avian influenza h9n2 virus - like particles and macromolecular protein complexes culture supernatants ( 200 ml ) from sf - 9s insect cells infected with the recombinant baculovirus bnaham - h9n2 that expressed avian influenza a / hong kong / 1073 / 99 ( h9n2 ) ha , na , and m1 gene products were harvested by low speed centrifugation . culture supernatants were clarified by centrifugation in a sorval rc - 5b superspeed centrifuge for 1 hr at 10 , 000 × g and 4 ° c . using a gs - 3 rotor . virus and vlps were isolated from clarified culture supernatants by centrifugation in a sorval otd - 65 ultracentrifuge for 3 hr at 27 , 000 rpm and 4 ° c . using a sorval th - 641 swinging bucket rotor . the virus pellet was resuspended in 1 ml of pbs ( ph 7 . 2 ), loaded onto a 20 - 60 % ( w / v ) discontinuous sucrose step gradient , and resolved by centrifugation in a sorval otd - 65 ultracentrifuge for 16 hr at 27 , 000 rpm and 4 ° c . using a sorval th - 641 rotor . fractions ( 0 . 5 ml ) were collected from the top of the sucrose gradient . influenza proteins in the sucrose gradient fractions were analyzed by sds - page and western immunoblot analyses as described above in example 6 . the ha and m1 proteins were found in the same sucrose gradient fractions ( fig6 ) as shown by western blot analysis and suggested that the ha and m1 proteins were associated as macromolecular protein complexes . also the ha and m1 proteins were found in fractions throughout the sucrose gradient suggesting that these recombinant viral proteins were associated with macromolecular protein complexes of different densities and compositions . example 10 : analysis of recombinant avian influenza h9n2 vlps and proteins by gel filtration chromatography protein macromolecules such as vlps and monomeric proteins migrate differently on gel filtration or size exclusion chromatographic columns based on their mass size and shape . to determine whether the recombinant influenza proteins from sucrose gradient fractions were monomeric proteins or macromolecular protein complexes such as vlps , a chromatography column ( 7 mm × 140 mm ) with a resin bed volume of 14 ml of sepharose cl - 4b ( amersham ) was prepared . the size exclusion column was equilibrated with pbs and calibrated with dextran blue 2000 , dextran yellow , and vitamin b12 ( amersham pharmacia ) with apparent molecular weights of 2 , 000 , 000 ; 20 , 000 ; and 1 , 357 , respectively , to ascertain the column void volume . dextran blue 2000 eluted from the column in the void volume ( 6 ml fraction ). as expected , the recombinant influenza protein complexes eluted from the column in the void volume ( 6 ml fraction ) also . this result was characteristic of a high molecular weight macromolecular protein complex such as vlps . viral proteins in the column fractions were detected by western immunoblot analysis as described above in example 6 . the m1 proteins were detected in the void volume fractions ( fig7 ). as expected baculovirus proteins were also in the void volume . to determine whether the macromolecular protein complexes isolated on sucrose gradients and containing recombinant avian influenza proteins had morphologies similar to influenza virions , electron microscopic examination of negatively stained samples was performed . recombinant avian influenza a / hong kong / 1073 / 99 ( h9n2 ) protein complexes were concentrated and purified from culture supernatants by ultracentrifugation on discontinuous sucrose gradients as described in example 7 . aliquots of the sucrose gradient fractions were treated with a 2 % glutaraldehyde in pbs , ph7 . 2 , absorbed onto fresh discharged plastic / carbon - coated grids , and washed with distilled water . the samples were stained with 2 % sodium phosphotungstate , ph 6 . 5 , and observed using a transmission electron microscope ( philips ). electron micrographs of negatively - stained samples of recombinant avian influenza h9n2 protein complexes from two sucrose gradient fractions showed spherical and rod - shaped particles ( fig8 ) from two sucrose gradient fractions . the particles had different sizes ( 60 and 80 nm ) and morphologies . larger complexes of both types of particles were also detected , as well as rod - shaped particles ( fig8 ). all observed protein complex structures exhibited spike like surface projections resembling influenza virus ha and na peplomers . since the size and appearance of the 80 nm particles was similar to that of wild type influenza virus particles , these structures likely represented enveloped influenza vlps . the smaller particles of approximately 60 nm probably represented subviral particles that differed from the above vlps both morphologically and structurally . example 12 : analysis of functional characteristics of influenza proteins by hemagglutination assay to determine whether the purified influenza vlps and proteins possessed functional activities , such as hemagglutination and neuraminidase activity , which were characteristic for influenza virus , the purified influenza vlps and proteins were tested in hemagglutination and neuraminidase assays . for the hemagglutination assay , a series of 2 - fold dilutions of sucrose gradient fractions containing influenza vlps or positive control wild type influenza virus type a were prepared . then they were mixed with 0 . 6 % guinea pig red blood cells in pbs ( ph 7 . 2 ) and incubated at 4 ° c . for 1 to 16 hr . as a negative control , pbs was used . the extent of hemagglutination was determined visually , and the highest dilution of fraction capable of agglutinating guinea pig red blood cells was determined ( fig9 ). the highest hemagglutination titer observed for the purified influenza vlps and proteins was 1 : 4000 , which was higher than the titer shown by the wild type influenza control , which was 1 : 2000 . example 13 : analysis of functional characteristics of influenza proteins by neuraminidase assay the amount of neuraminidase activity in influenza vlp - containing sucrose gradient fractions was determined by the neuraminidase assay . in this assay the na ( an enzyme ) acted on the substrate ( fetuin ) and released sialic acid . arsenite reagent was added to stop enzyme activity . the amount of sialic acid liberated was determined chemically with the thiobarbituric acid that produced a pink color in proportion to free sialic acid . the amount of color ( chromophor ) was measured in a spectrophotometer at wavelength 594 nm . the data , as depicted in fig8 , showed that a significant amount of sialic acid was produced by vlp - containing fractions of the sucrose gradients and that these fractions corresponded to those fractions exhibiting hemagglutination activity . example 13 : immunization of balb / c mice with functional homotypic recombinant influenza h9n2 vlps the immunogenicity of the recombinant influenza vlps was ascertained by immunization of mice followed by western blot analysis of immune sera . recombinant vlps ( 1 μg / injection ) comprised of viral ha , na , and m1 proteins from avian influenza virus type a / honk kong / 1073 / 99 and purified on sucrose gradients were inoculated subcutaneously into the deltoid region of ten ( 10 ) female balb / c mice at day 0 and day 28 ( fig1 ). pbs ( ph 7 . 2 ) was administered similarly as a negative control into five ( 5 ) mice . the mice were bled from the supraorbital cavity at day - 1 ( pre - bleed ), day 27 ( primary bleed ), and day 54 ( secondary bleed ). sera were collected from blood samples following overnight clotting and centrifugation . for western blot analysis , 200 ng of inactivated avian influenza virus type a h9n2 or cold - adapted avian influenza virus type a h9n2 , as well as see blue plus 2 pre - stained protein standards ( invitrogen ), was denatured ( 95 ° c ., 5 minutes ) and subjected to electrophoresis under reduced conditions ( 10 mm β - mercaptoethanol ) on 4 - 12 % polyacrylamide gradient nupage gels ( invitrogen ) in mes buffer at 172 volts until the bromophenol blue tracking dye disappeared . for protein gels , the electrophoresced proteins were visualized by staining with colloidal coomassie blue reagent ( invitrogen ). proteins were transferred from the gel to nitrocellulose membranes in methanol by the standard western blot procedure . sera from vlp - immunized mice and rabbits immunized with inactivated avian influenza virus h9n2 ( positive control sera ) were diluted 1 : 25 and 1 : 100 , respectively , in pbs solution ( ph 7 . 2 ) and used as primary antibody . protein bound membranes , which were blocked with 5 % casein , were reacted with primary antisera for 60 minutes at room temperature with constant shaking . following washing of primary antibody membranes with phosphate buffered saline solution containing tween 20 , secondary antisera [ goat anti - murine igg — alkaline phosphatase conjugate ( 1 : 10 , 000 ) or goat anti - rabbit igg — alkaline phosphatase conjugate ( 1 : 10 , 000 )] were reacted 60 minutes with the membrane . following washing of secondary antibody membranes with phosphate buffered saline solution containing tween 20 , antibody - binding proteins on the membranes were visualized by development with the chromogenic substrate such as nbt / bcip ( invitrogen ). the results of western blot analysis ( fig1 ) were that proteins with molecular weights similar to viral ha and m1 proteins ( 75 and 30 kd , respectively ) bound to positive control sera ( fig1 b ) and sera from mice immunized with the recombinant influenza h9n2 vlps ( fig1 a ). these results indicated that the recombinant influenza h9n2 vlps alone were immunogenic in mice by this route of administration . berglund , p ., fleeton , m . n ., smerdou , c ., and liljestrom , p . 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( 1995 ). generation of cytotoxic and humoral immune responses by non - replicative recombinant semliki forest virus . proc . natl . acad . sci . usa 92 , 3009 - 3013 . those skilled in the art will recognize , or be able to ascertain using no more than routine experimentation , many equivalents to the specific embodiments of the invention described herein . such equivalents are intended to be encompassed by the following claims :