effective influenza vaccines need to satisfy three major criteria : safety , immunogenicity , and efficacy . according to the regulatory documentation in russia , the hemagglutination inhibition ( hai ) however , the goal of vaccination is not only the induction of serum antibodies , but also the generation of effective immunological memory for longterm protection . in recent years , substantial research efforts have been devoted toward vaccine assessment methods that measure memory cell responses to a wide range of pathogens and cancers . 
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  in the case of vaccines delivered to mucosal surfaces , development of fast , easy , and costeffective assays for the measurement of immunological memory continues to be a challenge because of the different nature of the immunity elicited by such vaccines . in this report , an analysis of the laiv ability to stimulate b and tcell immunological memory was performed not only to demonstrate the benefit of this vaccine but also to refine the appropriate immunological assays for comprehensive measurement of live vaccine immunogenicity . there are several approaches for memory bcell assessment : ( i ) by igm to igg switching of antibodyproducing plasma cells ; 
  ( ii ) bcell phenotype changing to cd27  cd38  igd  bcl2 subpopulation ; 
  and ( iii ) increases in serum antibody avidity . 
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  here , we report the modification of the latter approach for the measurement of local iga avidity in human nasal swabs . effective antiinfluenza immunity depends on the induction of tcell immunological memory as well as bcell immune responses . 
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  previously , the ability of inactivated influenza vaccine to induce cd4  cd45ro and cd8  cd45ro cells in volunteers was demonstrated without determination of virusspecific cells . later , the production of influenzaspecific ifnproducing cd4 and cd8 t cells was studied upon vaccination with inactivated or live influenza vaccines . 
  in our study , we modified the [ tcell recognition of antigenpresenting cells ( trap ) by protein capture ] assay that measures antigenactivated cells involved in trogocytosis , i.e. , membrane fragments interchange between t cells and apcs during antigen presentation . the trap method has been previously used to study trogocytosis in vitro 

  and to determine in vivo t cells specific for herpes virus , 
  lymphocytic choriomeningitis virus , 
  and ovalbumin . after desirable sizes of groups were chosen , persons were randomly ( by chance ) assigned to receive laiv or placebo . avidity of local iga antibodies was observed in 56 volunteers vaccinated with attenuated reassortant strain for laiv a/17/solomon islands/06/9 ( h1n1 ) at a dose of 10  eid50/02  ml and 21 volunteers inoculated with placebo ( sterile physiological solution ) . levels of cd4 
 cd45ro 
 cells were measured in 32 volunteers vaccinated with trivalent laiv [ a/17/new  caledonia/99/145 ( h1n1 ) at a dose of 10  eid50/02  ml , a/17/california/04/6  ( h3n2 ) at a dose of 10  eid50/02  ml , and b/60/jilin/01/1 at a dose of 10  eid50/02  ml ] and 25 volunteers inoculated with placebo . virusspecific memory t cells were studied in 20 volunteers vaccinated with attenuated reassortant strain for laiv a/17/solomon islands/06/9 ( h1n1 ) and 10 volunteers inoculated with placebo . vaccines or placebo were intranasally administered once , 025  ml per nostril . physical examination and venous blood collection nasal swab samples were collected before and 21  days after vaccination ( day 0 and day 21 , respectively ) . dry cotton buds were inserted in the volunteer s nostrils along the lateral nasal walls 23  cm inside , to inferior nasal conches ( volunteer should sit in a comfortable position with the head slightly tilted backward ) . the bud tips were put into a plastic vial containing 05  ml of sterile pbs . after 2hour incubation at 4c , cotton buds were pulled out of vial and placed into standard 1ml pipette tip that was further inserted back into the vial and centrifuged for 10  minutes , 200  g , + 4c . vial containing ready nasal swab sample was stored at 70c until analysis ( but no longer than 3  months ) . separation of human peripheral blood mononuclear cells ( pbmc ) was carried out by the standard methods 
  using histopaque1077 ( sigmaaldrich co. , st . immunogenicity of laiv was assessed in standard hai assay using 4 hau of appropriate viruses . before the analysis , serum samples were pretreated with receptordestroying enzyme ( denka seiken , tokyo , japan ) overnight at 37c and subsequently heated at 56c for 45  minute . local iga in nasal swabs was measured by elisa 
 , 
  using 16 hau per 005  ml of whole purified viruses for absorption and peroxidaselabeled antibodies to human iga ( bd biosciences , franklin lakes , nj , usa ) . the endpoint elisa titers was expressed as the inversed highest dilution that gave an optical density ( od ) equal or greater than twice the mean od of the control ( blank ) wells . increases in antibody titers after vaccination by four times or more were considered as antibody conversions . local antibody avidity was estimated by elisa ( urea test ) as described by de souza . the method is based on the ability of urea ( chaotropic agent ) to dissociate antigen highavidity antibodies were detected by elisa , measuring the difference between the od values because of antibody binding in the absence and in the presence of 5  m urea . then , 5  m urea solution was added into three wells , and three extra wells were used as controls ( pbs instead of urea ) . then , antibodies to human iga were added to the wells ( bd biosciences ) . after 1  hour of incubation , opd substrate was added to the wells for 20  minutes , and finally stop solution ( 2n h2so4 ) was added . avidity index ( ai ) was calculated as the ratio between mean od of sample treated with urea and the mean od of controls 100 . cells were stained with respective monoclonal antibodies ( bd biosciences ) using standard manufacturer s protocols . analysis was performed using coulter epics xl ( beckman coulter , inc . , brea , ca , usa ) . virusspecific t cells were detected by the trap method based on previous protocols 
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  human pbmcs were divided into half the targets were loaded with virus by exposure to 15 moi of purified a/17/solomon islands/06/9 ( h1n1 ) influenza virus for 1  hour in serumfree rpmi at 37c in 5% co2 . the other half of the target cells were stimulated with pbs instead of virus and used as a control . loaded targets were washed in rpmi10%fbs and incubated overnight at 37c in 5% co2 followed by labeling of the target cells . target cells were washed twice with pbs and surface biotinylated using a biotinylation reagentezlink sulfonhslcbiotin ( merck & co. , inc . , whitehouse station , nj , usa ) at a final concentration of 1  mg / ml . cells were incubated for 10  minute at 25c , 10  minute with equal volume of fbs ( fetal bovine serum ) at 4c , and washed three times with rpmi10%fbs . effector cells were labeled with 005  m cfse ( sigmaaldrich co. ) , then incubated for 5  minute at room temperature in the dark , and washed three times in pbs10%fbs . the effector cells were then cocultured with loaded or control targets in a 1:1 ratio for 1  hour at 37c in 5% co2 to allow the trogocytosis to occur . edta , washed in pbs , and stained with the phycoerythrinconjugated monoclonal antibodies to human cd4 ( cd4pe ; bd biosciences ) and phycoerythrin  texas red streptavidin ( secondstep reagent for the indirect immunofluorescent staining of biotinylated cells the samples were analyzed by flow cytometry using a coulter epics altra ( beckman coulter , inc . ) . pbmc , peripheral blood mononuclear cells ; e , effector cells ; t , target cells ( antigen presenting cells ) ; cfse , carboxyfluorescein succinimidyl ester ; stav , fluorescently labeled streptavidin . after desirable sizes of groups were chosen , persons were randomly ( by chance ) assigned to receive laiv or placebo . avidity of local iga antibodies was observed in 56 volunteers vaccinated with attenuated reassortant strain for laiv a/17/solomon islands/06/9 ( h1n1 ) at a dose of 10  eid50/02  ml and 21 volunteers inoculated with placebo ( sterile physiological solution ) . levels of cd4 
 cd45ro 
 cells were measured in 32 volunteers vaccinated with trivalent laiv [ a/17/new  caledonia/99/145 ( h1n1 ) at a dose of 10  eid50/02  ml , a/17/california/04/6  ( h3n2 ) at a dose of 10  eid50/02  ml , and b/60/jilin/01/1 at a dose of 10  eid50/02  ml ] and 25 volunteers inoculated with placebo . virusspecific memory t cells were studied in 20 volunteers vaccinated with attenuated reassortant strain for laiv a/17/solomon islands/06/9 ( h1n1 ) and 10 volunteers inoculated with placebo . vaccines or placebo were intranasally administered once , 025  ml per nostril . physical examination and venous blood collection nasal swab samples were collected before and 21  days after vaccination ( day 0 and day 21 , respectively ) . dry cotton buds were inserted in the volunteer s nostrils along the lateral nasal walls 23  cm inside , to inferior nasal conches ( volunteer should sit in a comfortable position with the head slightly tilted backward ) . the bud tips were put into a plastic vial containing 05  ml of sterile pbs . after 2hour incubation at 4c , cotton buds were pulled out of vial and placed into standard 1ml pipette tip that was further inserted back into the vial and centrifuged for 10  minutes , 200  g , + 4c . vial containing ready nasal swab sample was stored at 70c until analysis ( but no longer than 3  months ) . separation of human peripheral blood mononuclear cells ( pbmc ) was carried out by the standard methods 
  using histopaque1077 ( sigmaaldrich co. , st . immunogenicity of laiv was assessed in standard hai assay using 4 hau of appropriate viruses . before the analysis , serum samples were pretreated with receptordestroying enzyme ( denka seiken , tokyo , japan ) overnight at 37c and subsequently heated at 56c for 45  minute . local iga in nasal swabs was measured by elisa 
 , 
  using 16 hau per 005  ml of whole purified viruses for absorption and peroxidaselabeled antibodies to human iga ( bd biosciences , franklin lakes , nj , usa ) . the endpoint elisa titers was expressed as the inversed highest dilution that gave an optical density ( od ) equal or greater than twice the mean od of the control ( blank ) wells . increases in antibody titers after vaccination by four times or more were considered as antibody conversions . local antibody avidity was estimated by elisa ( urea test ) as described by de souza . the method is based on the ability of urea ( chaotropic agent ) to dissociate antigen highavidity antibodies were detected by elisa , measuring the difference between the od values because of antibody binding in the absence and in the presence of 5  m urea . then , 5  m urea solution was added into three wells , and three extra wells were used as controls ( pbs instead of urea ) . then , antibodies to human iga were added to the wells ( bd biosciences ) . after 1  hour of incubation , opd substrate was added to the wells for 20  minutes , and finally stop solution ( 2n h2so4 ) was added . avidity index ( ai ) was calculated as the ratio between mean od of sample treated with urea and the mean od of controls 100 . cells were stained with respective monoclonal antibodies ( bd biosciences ) using standard manufacturer s protocols . analysis was performed using coulter epics xl ( beckman coulter , inc . , brea , ca , usa ) . virusspecific t cells were detected by the trap method based on previous protocols 
 , 
  human pbmcs were divided into half the targets were loaded with virus by exposure to 15 moi of purified a/17/solomon islands/06/9 ( h1n1 ) influenza virus for 1  hour in serumfree rpmi at 37c in 5% co2 . the other half of the target cells were stimulated with pbs instead of virus and used as a control . loaded targets were washed in rpmi10%fbs and incubated overnight at 37c in 5% co2 followed by labeling of the target cells . target cells were washed twice with pbs and surface biotinylated using a biotinylation reagentezlink sulfonhslcbiotin ( merck & co. , inc . , whitehouse station , nj , usa ) at a final concentration of 1  mg / ml . cells were incubated for 10  minute at 25c , 10  minute with equal volume of fbs ( fetal bovine serum ) at 4c , and washed three times with rpmi10%fbs . effector cells were labeled with 005  m cfse ( sigmaaldrich co. ) , then incubated for 5  minute at room temperature in the dark , and washed three times in pbs10%fbs . the effector cells were then cocultured with loaded or control targets in a 1:1 ratio for 1  hour at 37c in 5% co2 to allow the trogocytosis to occur . edta , washed in pbs , and stained with the phycoerythrinconjugated monoclonal antibodies to human cd4 ( cd4pe ; bd biosciences ) and phycoerythrin  texas red streptavidin ( secondstep reagent for the indirect immunofluorescent staining of biotinylated cells the samples were analyzed by flow cytometry using a coulter epics altra ( beckman coulter , inc . ) . pbmc , peripheral blood mononuclear cells ; e , effector cells ; t , target cells ( antigen presenting cells ) ; cfse , carboxyfluorescein succinimidyl ester ; stav , fluorescently labeled streptavidin . geometric mean titers ( gmts ) of local iga in 56 volunteers immunized with a ( h1n1 ) laiv strain increased from 689 to 1640 by 21  days after vaccination ( p  <  001 ) . conversion rate ( percentage of volunteers showing a fourfold or higher hi antibody titer increase 21  days after vaccination ) was 50% in vaccinated persons . subject received placebo had no conversions with gmt of 465 ( figure  2 ) . thus , a ( h1n1 ) laiv strain was able to induce marked local antibody response . ab titers were measured before vaccination and 21  days after vaccination in volunteers vaccinated with attenuated reassortant strain for laiv a/17/solomon islands/06/9 ( h1n1 ) . analysis of the local iga avidity was carried out in the same volunteers divided into three groups : ( i ) vaccinated people with significant increases in local iga antibody titers in elisa ; ( ii ) vaccinated people without such increases ; and ( iii ) placebo group ( table  1 ) . wide range of avidity indexes ( ais ) of local iga was observed in volunteers before vaccination ( day 0 ) : from 33 to 96  units . we divided all volunteers into three subgroups : low ais ( 3360 ) , medium ais ( 6180 ) , and high ais ( 8196 ) . the percentage of volunteers with high baseline ais among elisa(+ ) persons ( with fourfold or higher conversions in local iga at day 21 ) was 79 times lower than among elisa( ) persons ( without local iga conversions ) : 36% compared to 286% ( p the opposite distribution was observed in volunteers with low baseline ais , 500% of elisa(+ ) subjects , and 214% of elisa( ) subjects . after vaccination with laiv , the percentage of subjects with high ais among the elisa(+ ) volunteers increased 89 times : from 36% to 321% . in elisa( ) group , the vaccineinduced increase was less : from 286% to 357% . during the period between nasal swab sample collections ( from day 0 to day 21 ) some people demonstrated decreased ai results . the percentage of persons with ai decreases was minimal ( 143% ) among the elisa(+ ) volunteers , medium ( 321% ) among the elisa( ) volunteers , and largest ( 571% ) in the control group . we established the cutoff point based on placebo data ( increases by 15 and more ) to consider the changes in vaccinated subjects to be reliable . it is important to note that such increases were observed not only in elisa(+ ) , but also in elisa( ) volunteers ( 286% and 214% of persons , respectively ) . thus , enhancement of local iga levels after vaccination was accompanied by increase in their avidity . there is a strong and significant negative correlation between ais before vaccination and fold changes in ais after vaccination ( r  =  063 , p  < 005 ) , meaning that the higher the baseline avidity , the lower the intensity of vaccinecaused changes in ab avidity . a weak but significant positive correlation was also found between iga antibody titers , and ai was 031 ( p  <  005 ) . avidity indexes ( ais ) of local iga antibodies to a ( h1n1 ) laiv prior and after vaccination of volunteers * increases exceeding maximal value observed in the placebo group were considered significant ( ai increase by 15 and more ) laiv , live attenuated influenza vaccines . the cd4 memory tcell responses were measured by surface cd45ro staining showing memory phenotype  total memory t cells ( in volunteers vaccinated with trivalent laiv )  and by new trap assay adapted to study influenzaspecific memory tcell responses in human pbmc [ in persons vaccinated with a ( h1n1 ) reassortant strain ] ( figure  4 ) . examples of lymphocyte gating and quantification of cfse+ cd4 + t cells are presented in figure 3 . changes in cd45roexpressing total memory cd4 cells and trogocytosisreactive virusspecific cd4 t cells were analyzed in three groups of volunteers : ( i ) hai(+ ) persons  with seroconversions in hai test , ( ii ) hai( ) persons  without seroconversions , and ( iii ) placebo group . increases in both total cd4  cd45ro cells and virusspecific cd4 were more marked in hai(+ ) volunteers than in hai (  ) . the average cd4 tcell levels before vaccination in hai(+ ) group were much lower than in hai( ) group : 0014% and 0170% , respectively ( p  <  001 ) . as has been shown in the mucosal iga avidity results , here we observed a similar decrease in the total and virusspecific cd4 t cells in some volunteers within 1month period after vaccination . proportion of such persons among hai(+ ) persons was 286% ( total ) and 200% ( virus specific ) , among hai( ) was 433% and 400% , and in control group 720% and 700% .  tcell immunological memory in volunteers after live attenuated influenza vaccines ( laiv ) vaccination . levels of cd4cd45ro cells were measured in volunteers vaccinated with trivalent laiv [ a/17/new caledonia/99/145 ( h1n1 ) , a/17/california/04/6 ( h3n2 ) and b/60/jilin/01/1 ] . virusspecific memory t cells were studied in persons vaccinated with attenuated reassortant strain for laiv a/17/solomon islands/06/9 ( h1n1 ) . [ hemagglutination inhibition hai ( + ) ] : volunteers with serum antibody conversions in hai test ; hai (  ) : volunteers without seroconversions . increases were more marked in hai(+ ) volunteers , than in hai ( p  <  0.01 ) . wilcoxon matched pair test was used for comparison of vaccine and placebo groups , mann whitney utest was used for estimation of changes within groups before and after vaccination . ( c ) quantification of cfse  cd4 t cells involved in trogocytosis ( stavpetr+ ) . white histogram : virusstimulated sample ; grey histogram : control sample ( rpmi medium only ) . percent of virusspecific cells was calculated as a difference between percents of cells involved in trogocytosis in virusstimulated and control samples . the gate of trogocytosispositive cells ( c ) was stated using negative control ( non biotinylated targets stained with anti cd4 antibodies and streptavidin ) . analysis of the individual data using nfold changes in total cd4  cd45ro cells and virusspecific cd4 cells in vaccinated volunteers and placebo group ( figure  5 ) allowed us to establish the following cutoff points : more than 15fold increase for total and more than 100fold increase for virusspecific cd4 memory t cells . considering these criteria , the percentages of persons who provided reliable increases in total and influenzaspecific cd4 t cells were 429% and 700% in hai(+ ) group and 222% and 400% in hai( ) group . thus , vaccination with laiv enhanced the levels of total cd4  cd45ro and virusspecific cd4 memory cells not only in volunteers with seroconversions , but in persons with no seroconversions as well . furthermore , we found a strong and significant negative correlation between cell levels before vaccination and fold changes in cell levels after vaccination ( r  =  048 with p  <  005 for total cd4 t cells ; r  =  052 with p  <  005 for influenzaspecific ones ) .  tcell immunological memory in volunteers after levels of cd4cd45ro cells were measured in volunteers vaccinated with trivalent laiv [ a/17/new caledonia/99/145 ( h1n1 ) , a/17/california/04/6 ( h3n2 ) and b/60/jilin/01/1 ] . virusspecific memory t cells were studied in persons vaccinated with attenuated reassortant strain for laiv a/17/solomon islands/06/9 ( h1n1 ) . triangles designate individual foldchanges ( % of cells before vaccination/% of cells 1  month after vaccination ) . the following increases were accepted as significant : for total cd4  cd45romemory cells , results exceeding maximal value in placebo group in 1.5 times and more ; for virusspecific cd4 cells , results exceeding maximal value in placebo group in 10.0 times and more . mann the ability of longliving plasma cells to produce highavidity antibodies ( effector antibodies ) is one of the most important components of bcell memory ; 
  therefore , virusspecific bcell memory can be estimated by the measurement of antibody avidity . concerning antiinfluenza vaccination , local ( mucosal ) memory b cells are of the greatest interest as mucosal antibodies provide the first immunological barrier against influenza infection . 
  in our study of local iga antibody avidity , we applied an assay that has been previously used only for serum antibody avidity evaluation . , 
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  it has been observed that clearance of laiv influenza strains was completed at day 7day 10 . 
  following antigen elimination , more than 90% of lymphocytes die by apoptosis with the remaining cells transforming into memory subpopulation . we chose cd4 t cells for memory tcell assessment as they have been shown to participate in the regulation of both cytotoxic and antibody immunity . total memory cd4 t cells were determined by the expression of cd45ro as a general marker of memory phenotype . influenzaspecific memory cd4 t cells were measured by functional test using the new trap assay based on recently described phenomenon of trogocytosis . the seasonal laiv vaccine was able to stimulate bcell memory responses by increasing local iga avidity in the upper airways , i.e. , in the region of the first contact of influenza virus with a host ( table  1 ) . interestingly , some persons provided reliable increases in iga avidity without simultaneous reliable increases in mucosal antibody titers . the same phenomenon was previously described for serum antibody avidity in influenzainfected patients and volunteers vaccinated with influenza vaccines . these data show that production of antibodies with high avidity can occur without significant increases in their titers . intranasal vaccination with laiv increased the levels of both total and virusspecific cd4 memory t cells in human peripheral blood ( 4 , 5 ) . it has been previously shown that vaccination of young subjects with inactivated influenza vaccine slightly enhanced the total cd4  cd45ro memory t cells , but the proportion of people with such increases was < 20% . stimulation of total memory t cells seems to be associated with the phenomenon of polyclonal activation of heterotypic lymphocyte subpopulations specific to other antigens ( bystander activation ) . 
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  the laiv flumist ( medimmune , gaithersburg , md , usa ) was shown to be able to stimulate influenzaspecific ifnproducing cd4 t cells . 
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  activation of total and virusspecific memory t cells is an important tool for homo and heterosubtype protection against influenza viruses . however , the data of vaccineinduced polyclonal activation of memory t cell specific to allergens or autoantigens are very limited . we consider this question requires further investigation concerning yearly influenza vaccination particularly in immunocompromised populations , although our recent studies showed that laiv did not stimulate the production of serum and local antibodies to ovalbumin both in mice sensitized to this antigen 
  and in patients with high total ige levels . the analysis of antibody avidity , total , and influenzaspecific memory cd4 t cells indicated a strong negative correlation between baseline levels before vaccination and the rate of vaccineinduced immune responses ( fold changes in ais or cell levels ) . thus , immunogenicity of influenza vaccines depends on immune status before vaccination , which greatly varies in population . unfortunately , this factor is not taken into account for the massive immunization , which means that significant proportion of vaccine recipients , particularly those with long vaccination history , may not be able to respond appropriately to the actual vaccine . therefore , we consider the measurement of b and tcell memory before vaccination is necessary for selecting volunteers in newly developed vaccine trials or for evaluating the epidemiological potential of influenza a viruses and pandemic laiv candidates to make a clear assessment of the actual vaccine benefits . significant increases in total and virusspecific memory cd4 tcell levels were observed not only in vaccinated persons with seroconversions in hai assay , but apparently also in seronegative individuals ( 2240% of volunteers , 4 , 5 ) . these results indicate that the hai assay ( currently standard method for laiv immunogenicity evaluation ) does not properly reflect the frequency and quality of the immune responses elicited by laiv vaccination and should be combined with other assays related to cellular and mucosal immune responses . seasonal laiv is highly effective in stimulating mucosal bcell memory proved by significant increases in iga avidity even in persons without conversions of mucosal ab titers after vaccination . iga avidity and memory cd4 tcell responses do not correspond to immunogenicity rate measured by standard hai assay . mucosal iga avidity assessment and trap assay can be used for studies of antiinfluenza immunity after infection and vaccination in both volunteers and animal studies . the main advantage of the avidity assay is its ability to estimate memory bcell function via antibodies secreted by longlived plasma cells localized in the mucosa without having to harvest lymphoid tissue from the upper airways . trap assay is easier to perform than standard cytokine tests 
  and can reveal lymphocyte further comparative studies will be necessary to determine whether the trap assay is superior to the intracellular cytokine staining test for analyzing influenza vaccine immunity .
please cite this paper as : petukhova et  al . 
 ( 2011 ) b and tcell memory elicited by a seasonal live attenuated reassortant influenza vaccine : assessment of local antibody avidity and virusspecific memory tcells using trogocytosisbased method . 
 influenza and other respiratory viruses doi : 10.1111/j.17502659.2011.00279.x . 
 purpose   the main purpose of vaccination is to generate immunological memory providing enhanced immune responses against infectious pathogens . the standard and most commonly used assay for influenza vaccine immunogenicity evaluation is a hemagglutination inhibition assay ( hai ) . 
 it is clear now that hai assay is unable to properly assess the proven protective immunity elicited by live attenuated influenza vaccines ( laiv ) . 
 new methods need to be developed for more accurate laiv immunogenicity assessment and prediction of vaccine efficacy among target populations . 
 objective   randomized placebocontrolled study of memory b and tcell responses to intranasal laiv in young adults . 
 methods   a total of 56 healthy young adults 1820  years old received seasonal monovalent laiv . 
 mucosal memory bcell responses were measured by iga avidity assessment in nasal swabs . 
 cd4 memory t cells in peripheral blood were examined by the expression of cd45ro marker and in functional test by the ability of virusspecific t cells to maintain the trogocytosis with antigenloaded target cells . 
 results   intranasal laiv immunization enhances mucosal iga avidity even without reliable increases in antibody titers . at the day 21 after vaccination , 
 up to 40% of subjects demonstrated significant increases in both total and virusspecific cd4 memory t cells that were observed regardless of seroconversion rate measured by hai assay . 
 conclusion   the data suggest that immunogenicity of laiv vaccines should be evaluated on the mucosal and cellular immunity basis . 
 the assays applied could be used to support influenza clinical trials through preliminary screening of volunteers and subsequent measurement of antiinfluenza in immunity .