according to who data , 250 000 to 500 000 people ( most of them older than 65 years ) die each year from of all variants of the virus during seasonal epidemics in the world ; in some years the number of deaths could reach a million . however , existing vaccines only have effectiveness against certain strains of influenza virus . a common focus for creating a universal vaccine is the development of substances that affect the virus structure , which is conservative and highly homologous among the different types of viruses . at the moment , the flu vaccine should be created every year because the virus is constantly mutating , forming new antigenic determinants , and every time the disease is caused by a new type of virus . influenza viruses belong to the ortomyxoviridae family , which includes the genera influenza a , b , and c , which are defined depending on presence of 2 internal virion proteins ( m1 and np ) . further division is carried out in accordance with sub - types ( serotypes ) of the surface proteins hemagglutinin ( ha ) and neuraminidase ( na ) . it is of epidemiological importance that human have viruses containing 3 subtypes of hemagglutinin ( h1 , h2 , and h3 ) and 2 subtypes of neuraminidase ( n1 , n2 ) . the major virion antigenic components of influenza a and b is the na and ha ; the c virus does not contain neuraminidase . antibodies produced in response to the hemagglutinin constitute the basis of immunity against a specific subtype of influenza virus [ 79 ] . the immune system usually does not respond to the  stem  part of hemagglutinin , which is difficult to reach , however , their high variability allows the virus to remain unrecognized . even in case of administration of specially isolated conserved regions of hemagglutinin , the immune system will respond with the formation of a wide spectrum of antibodies , most of which will not be able to recognize a live virus because this area is deeply hidden . our study was conducted in an attempt to create 2 recombinant short conserved regions of viral surface proteins  hemagglutinin and neuraminidase integrated epitopes  against which both t- and na- and ha - coding genes were obtained and expressed in e. coli during the decade of the 1980s to study their immunological properties [ 1113 ] . the first hemagglutinin subunit ( ha1 ) amino acids ( aa ) 193199 forming qnpttyi epitope remained conservative among influenza h5n1 virus strains recently in circulation and could be recognized by monoclonal antibodies to ha1 of influenza virus a / vietnam/1203/04 ( h5n1 ) . n - terminus ) in h5n1 viruses , aa 262295 in h1n1 viruses , aa 5790 in h3n2 viruses , and aa 2861 in h7n7 viruses were shown to be highly conserved . ha conservative epitope sequences of influenza h3n2 virus ( aa 173181 , 227239 ) and antibodies to these epitopes neutralizing strains of this subtype were demonstrated . highly homologious ha areas among h1 , h3 , and h5 subtypes of influenza a virus were detected . nine ha1 sites have similar secondary structure ( aa : 3137 , 8996 , 118130 , 200208 , 238246 , 272277 , 304312 , 317334 , 341348 ) among h1 , h2 , h3 , and h5 subtypes . similar structure regions were identified among influenza h1n1 and h5n2 virus subtypes , probably being the main epitope inducing formation of neutralizing antibodies to a vast variety of influenza virus strains . these regions located behind the receptor - binding ha sites are conservative among h3n2 subtype of influenza virus strains and highly conservative in h1n1 and h5n1 . nucleotide sequences coding rha and rna of influenza a and b virus and antigenic determinants with codon optimization for expression in e. coli are unknown . the aim of the present work was to study immunogenicity of obtained rha and rna . fragments aa 83469 of na and aa 61287 of ha were chosen because they include the main b - cell epitopes and are the minimal structures for correct folding of target proteins . fragments were cut from the vector and amplified using the polymerase chain reaction ( pcr ) method . nucleotide sequences coding ha and na were synthesized by overlapping of elongation of oligonucleotides   and cloned into plasmid pet151/d - topo . for gene engineering work , induction of rha and rna expression was performed by 0.2% lactose in induction medium ( 1% peptone , 0.5% yeast extract , 50 mm na2hpo4 , 50 mm k2hpo4 , 25 mm ( nh4)2so4 , 2 mm mgso4 , 0.5% glycerol , 0.05% glucose , 0.2% lactose ) . fermentation was performed in induction medium containing ampicillin ( 100 g / ml ) in thermostatic rotor shaker ( 37c , 250 rpm , 1620 hours ) . disc - electrophoresis in paag of cell lysate of bl21(de3)ppvna and bl21(de3)ppvha e. coli strains was performed for densitometric measurement of rha and rna nucleotide sequences expression level in e. coli . proteins were purified according to the ni - nta purification system protocol ( invitrogen , catalog number k950 - 01 ) . balb / c mice ( 16 per each group , 1618 g ) were immunized by 20 g rha and rna in the ratio 1 : 1 with 0.5 mg of alhydrogel . blood samples ( from 56 mice ) were collected from the ventral vein 2 weeks after the second immunization . influenza viruses a / california/04/2009 ( h1n1 ) , a / pr/8/34 ( h1n1 ) , a / perth/16/2009 ( h3n2 ) , a / chicken / kurgan/05/2005 r.g . immunized mice were challenged intranasally with 1ld/50 of a / california/04/2009 ( h1n1 ) and a / pr/8/34 ( h1n1 ) or 5ld/50 of a / california/04/2009 strain ( 50 l per mouse)s . tween-20 was added to the sample  the final concentration 1% ( w / v ) , followed by water bath incubation ( 1 h , 37c ) with further centrifugation ( 1 h , 20c , 2000 g ) . supernatant including viral envelope proteins of influenza viruses a / pr/8/34 and a / chicken / kurgan/05/2005 r.g . was taken . detergent was removed using the detergent - outmicro kit ( millipore ) and the sample was concentrated on speedvac by achieving initial volume . the preparation of additional elaboration was held with 8 m carbamide in presence of ddt ( 0.02 m ) with further night dialysis versus carbonate buffer ( ph 8.5 ) . specific igg levels were determined by elisa in 96-well elisa plates (  greiner  , germany ) coated overnight at 4c with 100 l / well of rha , rna ( 3 g / ml ) and influenza strains a / california/04/2009 , a / pr/8/34 , a / perth/16/2006 , a / chicken / kurgan/05/2005 r.g . , b / florida/04/2006 ( 2 g / ml ) resuspended in carbonate coating buffer . plates were blocked with blocking buffer ( pbs with 5% fcs )  300 l / well , 1 h at room temperature . hrp - labeled rabbit anti - mouse igg antibodies ( abcam , uk ) and goat anti - rabbit igg antibodies ( sigma - aldrich ) diluted in blocking buffer were added ( 100 l / well ) ( incubation at room temperature for 1 h ) . plates were developed with tmb substrate ( bd bioscience ) according to the manufacturer s instructions . elisa endpoint titers were defined as inverse value of the highest dilution that yielded an od 450 nm value above 2 times the mean of negative control wells . significant differences in survival in mouse groups were analyzed by mantel - cox ( log - rank ) and gehan - breslow - wilcoxon tests . fragments were cut from the vector and amplified using the polymerase chain reaction ( pcr ) method . nucleotide sequences coding ha and na were synthesized by overlapping of elongation of oligonucleotides   and cloned into plasmid pet151/d - topo . for gene engineering work , induction of rha and rna expression was performed by 0.2% lactose in induction medium ( 1% peptone , 0.5% yeast extract , 50 mm na2hpo4 , 50 mm k2hpo4 , 25 mm ( nh4)2so4 , 2 mm mgso4 , 0.5% glycerol , 0.05% glucose , 0.2% lactose ) . fermentation was performed in induction medium containing ampicillin ( 100 g / ml ) in thermostatic rotor shaker ( 37c , 250 rpm , 1620 hours ) . disc - electrophoresis in paag of cell lysate of bl21(de3)ppvna and bl21(de3)ppvha e. coli strains was performed for densitometric measurement of rha and rna nucleotide sequences expression level in e. coli . proteins were purified according to the ni - nta purification system protocol ( invitrogen , catalog number k950 - 01 ) . balb / c mice ( 16 per each group , 1618 g ) were immunized by 20 g rha and rna in the ratio 1 : 1 with 0.5 mg of alhydrogel . blood samples ( from 56 mice ) were collected from the ventral vein 2 weeks after the second immunization . influenza viruses a / california/04/2009 ( h1n1 ) , a / pr/8/34 ( h1n1 ) , a / perth/16/2009 ( h3n2 ) , a / chicken / kurgan/05/2005 r.g . immunized mice were challenged intranasally with 1ld/50 of a / california/04/2009 ( h1n1 ) and a / pr/8/34 ( h1n1 ) or 5ld/50 of a / california/04/2009 strain ( 50 l per mouse)s . tween-20 was added to the sample  the final concentration 1% ( w / v ) , followed by water bath incubation ( 1 h , 37c ) with further centrifugation ( 1 h , 20c , 2000 g ) . supernatant including viral envelope proteins of influenza viruses a / pr/8/34 and a / chicken / kurgan/05/2005 r.g . was taken . detergent was removed using the detergent - outmicro kit ( millipore ) and the sample was concentrated on speedvac by achieving initial volume . the preparation of additional elaboration was held with 8 m carbamide in presence of ddt ( 0.02 m ) with further night dialysis versus carbonate buffer ( ph 8.5 ) . specific igg levels were determined by elisa in 96-well elisa plates (  greiner  , germany ) coated overnight at 4c with 100 l / well of rha , rna ( 3 g / ml ) and influenza strains a / california/04/2009 , a / pr/8/34 , a / perth/16/2006 , a / chicken / kurgan/05/2005 r.g . , b / florida/04/2006 ( 2 g / ml ) resuspended in carbonate coating buffer . plates were blocked with blocking buffer ( pbs with 5% fcs )  300 l / well , 1 h at room temperature . hrp - labeled rabbit anti - mouse igg antibodies ( abcam , uk ) and goat anti - rabbit igg antibodies ( sigma - aldrich ) diluted in blocking buffer were added ( 100 l / well ) ( incubation at room temperature for 1 h ) . plates were developed with tmb substrate ( bd bioscience ) according to the manufacturer s instructions . elisa endpoint titers were defined as inverse value of the highest dilution that yielded an od 450 nm value above 2 times the mean of negative control wells . significant differences in survival in mouse groups were analyzed by mantel - cox ( log - rank ) and gehan - breslow - wilcoxon tests . alignment of 837 aa rna and rha sequences of influenza a and b virus strains ( ncbi , 2009 ) resulted in aa sequence creation representing na and ha consensus aa variants . fragments including the main a ( h1n1 , h3n2 , h5n1 ) and a influenza virus b - cell epitopes were chosen . substitutions in an active center of the na molecule and those typical for russian virus isolates were made . the obtained expression of ppvna and ppvha plasmids provide rna and rha synthesis in e. coli under specific promoter induction ( figures 1 and 2 ) . na accumulation assessed with densitometry was 23% of total cell protein amounts in bl21(de3)ppvna e. coli cells and 43% in bl21(de3)ppvha e. coli cells . after purification , rha was obtained with 96% purity and rna with 95% purity . the specific serum igg antibodies to rha , rha+rna , a / california/04/2009 , a / pr/8/34 , a / perth/1620/09 , and a / chicken / kurgan/05/2005 r.g were determined by elisa . immunization induced high titers of antibodies to rha and rna and titers were 2 times lower to influenza viruses surface glycoproteins ( table 1 ) . after denaturation of influenza virus surface glycoproteins , the antibody titers increased by 4 times to a / pr/8/34 and by 8 times to a / chicken / kurgan/05/2005 r.g . anti - ha and anti - na serum igg induced by immunization with inactivated a / california/04/2009 were shown to be capable of binding to rha and rna of this virus . two months following immunization of mice , igg titers to influenza virus a / california/04/2009 were 102 400 , titers to rha were 12 800 , and titers to rna were 3200 . serum of rabbits immunized with influenza b virus was shown to be capable of binding to both rha and influenza virus b / florida/04/2006 . we evaluated the ability of mice to resist a lethal dose after immunization with rha+rna containing virus . immunized mice challenged with 1ld/50 of influenza virus a / california/04/2009 showed 100% survival compared to 43% ( p<0.05 ) survival in the control group ( figure 3 ) . immunized mice lost less body weight and started rapidly gaining weight compared to the control group ( p<0.05 ) . administration of 5ld/50 of influenza virus a / california/04/2009 resulted in 57% survival level among immunized mice compared to 14% among control animals ( figure 3 ) and also resulted in less body weight loss than in unvaccinated controls . inoculation with 1ld / p50 of a / pr/8/34 showed 100% survival in the group of immunized mice ( figure 4 ) and 53% in the control group ( p<0.05 ) . animals of the control group had more pronounced body weight loss compared with immunized mice ( p<0.05 ) . alignment of 837 aa rna and rha sequences of influenza a and b virus strains ( ncbi , 2009 ) resulted in aa sequence creation representing na and ha consensus aa variants . fragments including the main a ( h1n1 , h3n2 , h5n1 ) and a influenza virus b - cell epitopes were chosen . substitutions in an active center of the na molecule and those typical for russian virus isolates were made . the obtained expression of ppvna and ppvha plasmids provide rna and rha synthesis in e. coli under specific promoter induction ( figures 1 and 2 ) . na accumulation assessed with densitometry was 23% of total cell protein amounts in bl21(de3)ppvna e. coli cells and 43% in bl21(de3)ppvha e. coli cells . the specific serum igg antibodies to rha , rha+rna , a / california/04/2009 , a / pr/8/34 , a / perth/1620/09 , and a / chicken / kurgan/05/2005 r.g were determined by elisa . immunization induced high titers of antibodies to rha and rna and titers were 2 times lower to influenza viruses surface glycoproteins ( table 1 ) . after denaturation of influenza virus surface glycoproteins , the antibody titers increased by 4 times to a / pr/8/34 and by 8 times to a / chicken / kurgan/05/2005 r.g . anti - ha and anti - na serum igg induced by immunization with inactivated a / california/04/2009 were shown to be capable of binding to rha and rna of this virus . two months following immunization of mice , igg titers to influenza virus a / california/04/2009 were 102 400 , titers to rha were 12 800 , and titers to rna were 3200 . serum of rabbits immunized with influenza b virus was shown to be capable of binding to both rha and influenza virus b / florida/04/2006 . we evaluated the ability of mice to resist a lethal dose after immunization with rha+rna containing virus . immunized mice challenged with 1ld/50 of influenza virus a / california/04/2009 showed 100% survival compared to 43% ( p<0.05 ) survival in the control group ( figure 3 ) . immunized mice lost less body weight and started rapidly gaining weight compared to the control group ( p<0.05 ) . administration of 5ld/50 of influenza virus a / california/04/2009 resulted in 57% survival level among immunized mice compared to 14% among control animals ( figure 3 ) and also resulted in less body weight loss than in unvaccinated controls . inoculation with 1ld / p50 of a / pr/8/34 showed 100% survival in the group of immunized mice ( figure 4 ) and 53% in the control group ( p<0.05 ) . animals of the control group had more pronounced body weight loss compared with immunized mice ( p<0.05 ) . nowadays recombinant proteins based on the conservative epitopes of influenza virus ha , m2 , and np genes obtained in e. coli are used for the development of influenza vaccines , including the so - called  universal  vaccines requiring little seasonal modifications and designed with gene engineering methods representing minimal outlay [ 4,2022 ] . antibodies to conservative ha regions can induce a broad cross - reactive immune response to new influenza a virus strains , hence ha1-conservative regions could become potential targets for universal influenza vaccines . strain - specific antibodies to ha are considered to have a weak cross - activity with other ha types . furthermore , h1n1-induced infection stimulates the production of protective cross - reacting antibodies to h5n1 , which are supposed to serve as the basis for development of universal vaccines against flu and theurapeutic immunbiological medications . descriptive studies have been conducted of antibodies interacting with different influenza virus subtypes , as well as corresponding antigen determinants [ 2528 ] . synthetic immunogens based on such epitopes inducing the formation of antibodies displaying high cross - reactivity with h1n1 , h3n2 , h2n2 , h5n1 , h7n2 , h7n3 , h7n7 , and h9n2 virus antigens were described and their protective immune response were shown . recombinant protein consisting of 6 ha epitopes highly protective against influenza virus a strains h1n1 , h2n2 , and h3n2 was characterized . according to the obtained data , the synthesis of neutralizing antibodies to influenza h1n1 virus strains that emerged during the last 73 years was induced by h1 ha - based immunogens . the originality of the suggested approach is that ha and na ( inducing immunogenic reactivity spectrum in vaccine composition ) antigen determinants of 2 influenza virus types ( a and b ) were used for the first time and sequences presenting combination of b- and t - cell conservative epitopes ( ha  61287 aa , na 83469 aa ) are codon - optimized for increase of rha and rna nucleotide sequences expression level . the obtained rha and rna are influenza a ( h1n1 ) virus aa sequences consensus variants also containing immunogenic epitopes typical for other influenza virus strains ( h3n1 and h5n1 ) . intraperitoneal immunization efficiently stimulated specific serum igg production in equal concentrations to both rha and rna . antibodies to influenza virus a / california/04/2009 a / pr/8/34 and a / perth/16/2006 surface glycoproteins were revealed in lesser titers . nearly 12% of total antibodies to rha+rna bind with influenza virus a / california/04/2009 , likely because synthesized recombinant proteins are not structurally identical to influenza virus ha and can not bind with antibodies specific to ha tetrameric molecule conformational epitopes . to make some linear ha and na epitopes accessible for post - immunization antibodies , surface proteins denaturation was performed ; igg titers to them immunization with rha+rna was shown to protect mice , fully or partially , from lethal challenge with a / california/04/2009 and a / pr/8/34 viruses ( depending on injected virus dose ) and to significantly reduce weight loss . most current efforts to create universal vaccines hinge on the idea of generating antibodies against a portion of the virus that is relatively unchanged year - to - year . a vaccine , based on using antibodies simultaneously to two conservative and highly homologous virus structures serves to increase the degree of defence from different types of viruses . the results obtained for rha and rna immunogenicity and protective efficacy demonstrate the possibility and necessity of this approach for development of a universal pandemic influenza vaccine .
backgroundrecombinant hemagglutinin ( rha ) and neurominidase ( rna ) developed in our investigation are amino acid sequence consensus variants of h1n1 2009 subtype influenza virus strain , also including immunogenic epitopes typical for other influenza virus subtypes ( h3n1 and h5n1 ) . 
 substitutions were made : typical for russian virus isolates ( in ha 
 s220 t , na 
 d248n ) and in active centers of molecules 
 r118l , r293l , r368l ; c92s , c417s to increase recombinant proteins stability in e. coli . 
 the aim of the present work was to study immunogenicity of the obtained rha and rna.material/methodsfragments aa 83469 of na and aa 61287 of ha were chosen because they include the main b - cell epitopes and are the minimal structures for correct folding of target proteins . 
 the designed nucleotide sequences were synthesized and purified and the expression of rna and rna were analyzed . for immunization and virus challenge we used influenza viruses a / california/04/2009 ( h1n1 ) , a / pr/8/34 ( h1n1 ) , a / perth/16/2009 ( h3n2 ) , a / chicken / kurgan/05/2005 r.g . 
 ( h5n1 ) , and b / florida/04/2006 . 
 specific igg levels were determined by elisa in 96-well elisa plates . 
 significant differences of survival in mouse groups were analyzed by mantel - cox ( log - rank ) and gehan - breslow - wilcoxon tests.resultsthe obtained results demonstrate the high immunogenicity and ability of indicated proteins mixture to provide similar cross - protection against influenza viruses of the h1n1 subtype.conclusionsthe data obtained suggest efficient pluripotent vaccine creation based on ha and na conservative regions .