characterization of cross protection of swine-origin influenza virus

Lin et al Journal of Biomedical Science 2013 2019httpwwwjbiomedscicomcontent20119

RESEARCH Open Access

Characterization of cross protection ofSwine-Origin Influenza Virus (S-OIV) H1N1 andreassortant H5N1 influenza vaccine in BALBcmice given a single-dose vaccinationHui-Tsu Lin1 Chuan-Chang Chuang1 Hsieh-Ling Wu1 Der-Ming Chu2 and Yeau-Ching Wang1

Abstract

Background Influenza virus has antigen drift and antigen shift effect vaccination with some influenza vaccinemight not induce sufficient immunity for host to the threat of other influenza virus strains S-OIV H1N1 and H5N1influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge ofACalifornia72009 H1N1 or NIBRG-14 H5N1 virus

Results Both H1N1 and H5N1 induced significant homologous IgG HAI and microneutralization antibodyresponses in the mice while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibodyresponses Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicitprotective HAI antibody titers from 005 μg to 0001 μg Vaccines alone did not protect mice from challenge withheterogeneous influenza virus while H5N1 vaccine plus alum and MPLA adjuvants did Mouse body weight losswas also less significant in the presence of adjuvant than in the vaccine without adjuvant Furthermore both H1N1and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses

Conclusion Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viraltiters post H1N1 virus challenge and not vice versa MPLA adjuvant induced cross protection with a single dosevaccination to the challenge of heterogeneous influenza virus in mice Lung viral titer seemed to be a betterindicator compared to IgG neutralization antibody and HAI titer to predict survival of mice infected with influenzavirus

Keywords S-OIV H1N1 H5N1 Adjuvant Alum MPLA

BackgroundThe swine-original influenza virus A (S-OIV) H1N1 wasdetermined to be a novel strain of AInfluenza H1N1serotype which had been derived by reassortment ofswine avian and human influenza viruses The WHOdeclared that the infections caused by the new strainhad reached pandemic proportions on June 2009 andhas reported approx 14700 deaths in more than 209countries resulting from pandemic influenza H1N1 [1]

Correspondence yeauchingmailndmctsghedutw1Institute of Preventive Medicine National Defense Medical Center PO Box90048ndash700 San-Hsia TaiwanFull list of author information is available at the end of the article

copy 2013 Lin et al licensee BioMed Central LtdCommons Attribution License (httpcreativecreproduction in any medium provided the or

Immunization provides the best preventive strategyagainst influenza virus illness The current trivalent vac-cine is unlikely to provide significant protection againstthe novel pandemic H1N1 strain especially for childrenand young adults because of absence or low immunity tothe novel 2009 H1N1 strains [23] It has been reportedthat previous vaccination of children with trivalent vaccineof the last four seasons did not elicit a cross-reactive anti-body response to the pandemic H1N1 strain [4] Thus amonovalent vaccine based on the novel H1N1 strain willbe required to induce protective immunityCurrent influenza virus vaccines aim to induce strong

antibody (Ab) responses to the ectodomains of hemag-glutinin (HA) and neuraminidase (NA) molecules since

This is an Open Access article distributed under the terms of the Creativeommonsorglicensesby20) which permits unrestricted use distribution andiginal work is properly cited

Lin et al Journal of Biomedical Science 2013 2019 Page 2 of 13httpwwwjbiomedscicomcontent20119

these antibodies (Abs) can provide potent protectionagainst infection andor disease The main deficiency ofthis protection is that it targets highly variable viral de-terminants Failure to anticipate the emergence of anepidemic strain with significant antigenic changes com-pared to the vaccine strain will greatly reduce vaccine-induced protection Several studies have suggested thatproper adjuvant might improve the immunity of influ-enza vaccine and reduce the dose of vaccine [5-10]Aluminum hydroxide (alum) is currently the onlyhuman vaccine adjuvant approved for use in the UnitedStates and although it is effective at boosting antibodyresponses these responses require repeated administra-tion and tend to generate antiparasitic T helper 2 (TH2)rather than antiviral and antibacterial TH1 T cell im-munity [11] As a consequence there is much effort de-voted to develop prospective adjuvant that can establishprotective immunity with fewer vaccinations and lessinjected material through durable antibody and TH1-dependent cytotoxic T cell activity Other potentialimmune adjuvant might be considered and developedAs demonstrated previously Monophosphoryl lipid A(MPLA) is a low-toxicity derivative of LPS with usefulimmunostimulatory properties which is nearing regula-tory approval for use as a human vaccine adjuvant Mostrecently it has been demonstrated that the use of Al(OH)3 with MPLA as an emulsion induced a furtherincrease in HAI titers of AreassortantNIBRG-14VietNam 11942004 times Puerto Rico81934 H5N1 (NIBRG-14) inactivated whole virus and split virion influenzavaccines [12] These finding may have important impli-cations for the development of future vaccine adjuvantAlthough there are studies evaluating immune re-

sponse and protection of influenza H1N1 vaccine to2009 S-OIV in ferrets and mice most recently [13-16]Some studies also approached immune response andprotection of vaccinated animals against other influenzavirus in the absence or the presence of adjuvant In thesestudies some useful information has been revealed Forexample pandemic H1N1 vaccination is effective inmice [17] the adjuvant MPLA can reduce the effectiveimmunization dose of H5N1 and H3N2 influenza vac-cines in mice [18] and some other adjuvant can reducethe effective immunization dose of pandemic vaccines inmice [1920] However most of these studies focus onthe protection of BALBc mice immunized in a two-dose regimen of vaccination Since it is impossible tovaccinate people twice at the emergent time during in-fluenza virus is pandemic worldwide effective vaccinecomponent and dosage of influenza vaccine for singleimmunization is more practical and critical for the pre-vention of influenza virus epidemic In this study single-dose immunization of 2009 H1N1 vaccine based onNIBRG-121 vaccine strain and avian H5N1 vaccine

based on NIBRG-14 vaccine strain were evaluated for itseffectiveness to elicit protective immunity of mice tothe lethal challenge of ACalifornia072009 H1N1 virusMeanwhile the minimum effective dose of single immu-nization in the absence or the presence of adjuvant (eitheralum or MPLA) as well as the cross protection of hetero-geneous vaccine the changes of adjuvant-induced im-mune responses and the reduction of vaccine dose usedin the presence of adjuvant were also elucidated

MethodsVaccine strains and reagentsInfluenza A virus NIBRG-121 (NIBSC ACalifornia72009 H1N1 virus) virus (ACalifornia072009 H1N1CDC 2009712112) hemagglutinin (HA) antigen(ACalifornia72009 NIBSC 09146) and antiserum(ACalifornia72009 NIBSC 09152) were obtainedfrom the Centers for Disease Control and Prevention(CDC Taiwan) or the National Institute for BiologicalStandards and Control (NIBSC UK) NIBRG-121 wasused as vaccine strain and virus ACalifornia072009H1N1 was used as challenge virus

Vaccine productionNIBRG-121 virus strain was amplified in embryonic eggsof 10-day special pathogen free (SPF) for 48 h it wasthen harvested and inactivated with 004 formalin at4degC for overnight Harvested virus was then filteredthrough 045 μm filter and concentrated with Lab scaleTM TFF System treatment with benzonase (cat no1016560001 Merck KGaA Germany) (9 ~ 90 uml at4degC for overnight) and purified with 10-50 sucrosegradient by Alfa Wassermann PKII Pilot-Scale Ultracen-trifuge System Purified virus was dialyzed with PBSfiltered with 045 μm filter and stocked in 15-ml stocktube at minus80degC until use Hemagglutinin (HA) antigenconcentration was determined by single radial immuno-diffusion assay (SRD) [21] for the quantification of vac-cine S-OIV H1N1

Quality control assays of vaccineTo monitor the quality of produced vaccine severalitems of assays were evaluated Vaccine was quantifiedwith SRD method ovalbumin was evaluated withChicken Egg Ovalbumin Elisa Kit (Cat No 6050) fromAlpha Diagnostic International (ADI) endotoxin wasmeasured with Toxin Sensor TM Chromogenic LALEndotoxin Assay Kit (Cat No L00350) obtained fromthe GenScript Corporation Above assays were followedthe protocols of commercial kits Formalin content ofvaccine was evaluated according to the guideline of ver-sion 6th of Pharmacopoeia Chinensis For abnormal tox-icity assay male BALBc mice (body weight 17ndash22 gm)were inoculated intraperitoneal with 05 ml of produced


vaccine (at least 5 mice per dose of vaccine) and thenobserved their changes of body weight It fit the demandcriteria only if there is no death accident symptoms ofmice and mice should recovery of their body weight tothe level of pre-inoculation 7 days after inoculation Theantigenicity of vaccine was evaluated with hemagglutination(HA) assay as described with HAI assay without antibody

Determination of vaccine HA concentrationThe HA concentration of S-OIV candidate vaccine wasmeasured with single-radial immunodiffusion (SRD) ac-cording the protocol described previously [2223]

Ethics statementAll animal experiments were reviewed by the InstitutionalAnimal Care and Use Committee and approved by theregulatory authorities of Taiwan All animal experimentswere conducted in accordance with Taiwan laws on animalexperimentation and guidelines set out by the Associationfor Assessment and Accreditation of Laboratory AnimalCare International (AAALAC) and the Office of Labora-tory Animal Welfare (OLAW) The IACUC certificateNo of this study were AN-99-05 AN-100-07 andAN-101-06 Animal were housed according to OLAWand AAALAC guidelines in housing facilities accreditedby the Center of Disease Center (CDC) of Taiwan

Immunization of miceFemale BALBc mice (8-week-old) were immunized in-traperitoneal with different dosage of produced vaccine(PBS control 0001 μg 001 μg and 005 μg) in the ab-sence or presence of adjuvant alum (final 5 ofAluminum hydroxide gel cat noA8222 Sigma) orMPLA (Monophosphoryl Lipid A) (Sigma Adjuvant Sys-tem cat noS6322 Sigma) using a single-vaccinationregimen

IgG subclass determinationELISA plates were coated with 10 ng HA per well ofpurified vaccine at 4degC for overnight After blockingnonspecific binding (5 skim milk in PBST 1 h at 37degC)and subsequent washing PBST diluted mice sera (1400)of pre-immune or immunized were added to wells andplated were incubated for 1 h at 37degC washed againprior to further incubation for 1 h with IgG subclass-specific peroxidase-conjugated goat or rabbit anti-mouseIgG antibodies (IgG goat anti-mouse IgG (H + L) HRPcat no 115-035-146 Jackson ImmunoResearch USAIgG1 rabbit anti-mouse IgG1 HRP cat no 61ndash0120Invitrogen USA IgG2b rabbit anti-mouse IgG2a HRPcat no61-0220 Invitrogen USA) Bound IgG subclassantibodies were detected colorimetrically using TMBsubstrate by OD450 nm endpoint reading The cutoffvalue was set at ODn plus three standard deviations

where ODn is the mean of ODs of six preimmune serumspecimens

Cell and virusMDCK (Madin-Darby canine kidney) cells were obtainedfrom the American Type Culture Collection (ATCC) andmaintained in Dulbeccorsquos minimum essential medium(DMEM GIBCO Invitrogen USA) supplemented with100 Uml of penicillin 100 μgml of streptomycin and10 fetal bovine serum (GIBCO Invitrogen) at 37degC in ahumidified atmosphere with 5 CO2 Influenza A virusNIBRG-121 (NIBSC ACalifornia72009 H1N1 virus)obtained from the CDC of Taiwan) was amplified in10-day-old embryonic eggs at 35degC for 48 h Virus washarvested from allantoic fluid To determine the LD50 ofeach batch of virus female BALBc mice (6ndash7 weeks)(n = 10group) were anesthetized subcutaneously withZoletil 50 (Virbac Laboratories France) (0375 mgmice)and inoculated intranasally with serial dilutions of thevirus The LD50 was the dilution of the virus that pro-duced lethality in 50 of the mice and LD50 titers werecalculated by the method of Reed and Muench [24] TheLD50 was over 10

5 TCID50 for NIBRG-121

Plaque-assayTo measure virus titer MDCK cells (5times106 well) wereinoculated into 6-well microplates and were incubated at37degC in a humidified atmosphere with 5 CO2 for over-night In the second day a serial of 10-fold dilutions ofvirus were prepared in PBS MDCK cells were washedtwo times with PBS and 100 μl of viral dilutions were in-oculated into 6-well microplates for adsorption Afterone hour of adsorption virus suspensions were removedand cells were washed two times with PBS then 1Oxoid agars in DMEMBSA medium were inoculatedinto microwells microplates were incubated at 37degC in ahumidified atmosphere with 5 CO2 for three daysMDCK cells were fixed with 10 formalin for 1 h thenagars were removed and stained with crystal violet solu-tion (05) for 1 hour stained cells were washed withtap water and were observed Microplates were air driedat room temperature for several hours and plaques werecalculated for virus concentration

TCID50

To calculate virus titer MDCK cells (15 times 104 well) inDMEM medium with 10 FBS were inoculated into96-well microplates and were incubated at 37degC in ahumidified atmosphere with 5 CO2 for overnight Inthe second day a serial of 10-fold dilutions of virus wereprepared in PBS MDCK cells were washed two timeswith PBS and 100 μl of viral dilutions were inoculatedinto 96-well microplates for adsorption After one hourof adsorption virus suspensions were removed and cells


were washed two times with PBS then DMEMBSAmedium were inoculated into microwells and microplateswere incubated at 37degC in a humidified atmosphere with5 CO2 for additional five days The TCID50 titers of viruswere calculated by the method of Reed and Muench [24]

Hemagglutination inhibition (HI) assayFunctional H1N1 HA-specific antibody titers were deter-mined by HI assay using chicken erythrocytes Prior toserological analysis sera were treated with receptor-destroying enzyme (RDEII Denka Seiken Tokyo Japan)Serum (01 ml) was mixed with 03 ml receptor-destroyingenzyme incubated at 37degC for 18 h and adjusted to a final110 dilution by adding PBS and inactivated the enzymeactivity by incubation at 56degC for 30 min Sera giving anegative signal in the first dilution (110) were assigned anominal HI score of 15 HI titers are expressed as recipro-cal value of the highest serum dilution that inhibitedhemagglutination Animals with a serum HI titer of 140were considered seroprotected

Intranasal influenza challengeFour weeks after immunization with S-OIV H1N1 orNIBRG-14 H5N1 vaccine immunized mice were blood-letting for antibody assay and were then lightlyanaesthetized with Zoletil 50 (VIRBAC LaboratoriesFrance) and challenged intranasal with 1 times 106 TCID50

ACalifornia72009 H1N1 virus Over the following14 days body weights and survival rates of each groupof mice were monitored daily

Microneutralization assayNeutralization antibodies of mice post vaccinated withS-OIV H1N1 or NIBRG-14 H5N1 vaccines were evaluatedusing microneutralization assay Mice sera (pre-immune asnegative ACalifornia72009 NIBSC 09152 and anti-serum to NIBRG-14 H5N1 HA as positive control) weremixed with viruses (100 TCID50 of NIBRG-121 H1N1 orNIBRG-14 H5N1 virus) at room temperature for 1 hourand were inoculate into 96-well MDCK cells (15 times 104ml)Experiment was performed following WHO protocol ofmicroneutralization assay [2125]

Statistical analysisIn all figures vertical error bars denote the standarddeviation (SD) Significances of differences in antibodyresponses and cellular responses were evaluated byone-way analysis of variance (ANOVA) T test was usedfor the comparison of two specific groups in one-wayANOVA To test the significance of survival rates be-tween each group of immunized mice a Zrsquo (alternativecritical ratio) test was used [1626] For the comparisonof HAI antibody titer MannndashWhitney U test was usedA P value of lt 005 was considered significant

Results and discussionProduction of S-OIV H1N1 and NIBRG-14 H5N1 vaccineIt is previously reported that a single candidate seasonalH1N1 and H3N2 vaccine produced by an identical processwas highly immunogenic and protective in mammalian(Vero) cells [17] These studies were subsequently demon-strated to be highly predictive of the immunogenicitydemonstrated in human trials particularly with respect toimmunogenicity at low doses and the lack of immune en-hancement by use of an alum based adjuvant [27] Noclear cut correlate for protection has been established forpotential pandemic vaccines such as the H5N1 vaccine orthe novel H1N1 vaccine at present Data obtained fromanimal protection studies could be of value in combin-ation with data obtained from human dose finding andobservational efficacy studies following vaccine use in apandemic situation such as presently exists for the novelH1N1 virus This study was designed to assess the im-munogenicity and protective efficacy of S-OIV H1N1 andNIBRG-14 H5N1 influenza vaccines to the challenge ofACalifornia72009 H1N1 or NIBRG-14 H5N1 virus in aBALBc mice model with a single-dose immunizationregimenTo produce a novel vaccine for S-OIV H1N1 and

NIBRG-14 H5N1 NIBRG-121 and NIBRG-14 virusstrains were amplified in 10-day special pathogen free(SPF) embryonic chicken eggs for 48 h and were thenharvested Viruses were inactivated with 004 formalin(4degC overnight) and were purified using the AlfaWassermann PKII Pilot-Scale Ultracentrifuge System(Alfa Wassermann Inc (AWI) USA) Hemagglutinin(HA) antigen content was determined by single radialimmunodiffusion assay Results showed that the S-OIVH1N1 vaccine contained about 38 μgml of HA proteinwhich was estimated to be equal to 1 to 15 doses of vac-cine (15 μg HA was regarded as one dose) for each eggThe vaccine produced was quality control tested andcharacterized for antigenicity (HA titer 128ndash256) ov-albumin (le 4 μgml) formalin (0373ndash0548 μgml) andendotoxin (28ndash51 EUml) (Table 1) All tested items ofvaccine produced had qualities fitting the vaccine criteriaof the international standard Furthermore two lots ofS-OIV H1N1 vaccine produced were tested for abnor-mal toxicity in mice The results showed no abnormaltoxicity in mice (data not shown) Results demonstratedthe S-OIV H1N1 vaccines produced conformed to thequality control (QC) requirements of the World HealthOrganization (WHO) and European criteria for influ-enza vaccines Some QC items of NIBRG-14 H5N1 vac-cine did not conform to the international standard Thecandidate vaccines produced were used to evaluate theirprotective immune effects in miceSingle-dose immunization of S-OIV H1N1 or NIBRG-

14 H5N1 vaccine elicited a sufficient immune response

Table 1 Quality control of candidate S-OIV H1N1 and NIBRG-14 H5N1 vaccines

Test items Summary of results Notes

Abnormal toxicity S-OIV pass H5N1 not done Body weight of tested mice increased

Antigenicity HAI titer S-OIV 128 ~ 256 Also interacted with NIBSC sntisera

H5N1 1024

Endotoxin S-OIV Between 28 ~ 51 International criteria le 200 EUml

H5N1 314 EUml

Ovalbumin S-OIV asymp 4 μgml Europe le 2 μgml WHO le 5 μgml

H5N1 55 μgml

Formaldehyde S-OIV 0373 ~ 0548 μgml WHO le 002 ~001 (asymp1 mgml)

H5N1 not done

Note Result showed that all items evaluated for quality control of produced candidate vaccine fit the demand of international criteria

Figure 1 Immunization scheme of candidate S-OIV H1N1 andavian H5N1 vaccine BALBc mice were immunized with differentdoses of the produced vaccine S-OIV H1N1 or NIBRG-14 H5N1(A) PBS control 0001 μg 001 μg and 005 μg of S-OIV H1N1 orNIBRG-14 H5N1 vaccine (B) PBS control 001 μg and 005 μg ofS-OIV H1N1 or NIBRG-14 H5N1 with or without alum or MPLAadjuvant About 3 weeks after immunization the mice werechallenged with 106 TCID50 of ACalifornia72009 H1N1 or 1000TCID50 NIBRG-14 H5N1


to the 2009 pandemic H1N1 and avian H5N1 virusesrespectively in a dose-dependent manner in the miceIn actual practical work it seems impossible to vaccin-

ate people twice on an emergency basis during aworldwide influenza virus pandemic Effective vaccinecomponents and influenza vaccine dosage for singleimmunization are more practical and critical for the pre-vention of influenza virus epidemics In this study asingle-dose immunization of 2009 S-OIV (NIBRG-121)H1N1 and NIBRG-14 H5N1 vaccine was evaluatedfor its effectiveness to elicit protective and cross protect-ive immunity in mice to the lethal challenge ofACalifornia072009 H1N1 virus (Figure 1) Firstgroups (10 mice per group) of mice were primed withPBS or different doses (0001 μg 001 μg and 005 μg)of S-OIV H1N1 vaccine in the absence or presence ofalum or MPLA adjuvant in a single-dose immunizationregimen The mice were then challenged with 106

TCID50 of ACalifornia072009 H1N1 virus 28th dayspost vaccination (panel A) Second the cross protectionof S-OIV H1N1 and NIBRG-14 H5N1 vaccines wereevaluated As shown in Figure 1 panel B the mice wereimmunized with PBS 001 μg or 005 μg of S-OIVH1N1 and NIBRG-14 H5N1 vaccines in the absence orpresence of alum or MPLA adjuvant in a single-dosevaccination regimen Mouse spleen and lungs were col-lected 3 days post challenge with H5N1 or H1N1 virusrespectively and were used for microneutralization assayand mice lung viral assays As shown in Figure 2A and2B S-OIV H1N1 and NIBRG-14 H5N1 vaccines elicitedimmune responses in a dose-dependent manner in themice

Adjuvant promoted IgG responses to homologous S-OIVH1N1 and NIBRG-14 H5N1 influenza virusesThe candidate vaccine elicited an immune response in adose-dependent manner in the mice In the absence ofadjuvant 005 μg of S-OIV H1N1 and NIBRG-14 H5N1were required to elicit a significant antibody response

When either alum or MPLA adjuvants were included inthe vaccine only 0001 μg of S-OIV H1N1 (Figure 2A)and NIBRG-14 H5N1 vaccine (Figure 2B) were neededto significantly induce specific immune responses TheNIBRG-14 H5N1 vaccine had better antigenicity thanthe S-OIV H1N1 vaccine

Figure 2 Adjuvant promoted strong protective immune responses to S-OIV H1N1 virus To realize the least immunization dose of S-OIVand H5N1 vaccine needed for mice to raise sufficient protective immune responses to ACalifornia72009 virus mice (n = 7 to 8 per group)immunized with PBS 0001 μg 001 μg or 005 μg of vaccine in the presence or absence of adjuvant Three weeks after the single immunizationmice sera were evaluated for (A) H1N1 IgG (B) H5N1 IgG and (C) H1N1 IgG1 and IgG2a and (D) H5N1 IgG1 and IgG2a immune responses toACalifornia72009 virus The data represent the mean titers plusmn SD (error bars) of antibodies in each group of animals


Adjuvant improved S-OIV H1N1 vaccine to elicit a Th1-type antibody response (IgG2a)The effects of different adjuvants were compared for theirenhancement of immune response to influenza virus Bothalum and MPLA adjuvants improved the S-OIV H1N1vaccine IgG2a immune response an indicator of cellularimmunity IgG antibody (Figure 2C) Alum adjuvant im-proved both humoral and cellular immune responseswhile MPLA adjuvant improved the IgG2a immune re-sponse the IgG2aIgG1 ratio increased from 7926 (vaccinealone) to 17846 (vaccine plus MPLA) (Figure 2C) Contro-versially both alum and MPLA adjuvants were more ef-fective in eliciting Th2-type antibody (IgG1) responsesthan Th1 antibody (IgG2a) responses in mice immunizedwith NIBRG-14 H5N1 vaccine (Figure 2D) Thus a candi-date vaccine containing adjuvant might be able to elicitboth humoral and cellular immune responses Thereforean S-OIV H1N1 vaccine containing alum as the adjuvantmight be effective in preventing and eliminating an influ-enza virus infection by eliciting both Th1 and Th2 immuneresponses while a vaccine containing MPLA as the adju-vant would eliminate influenza virus by enhancing mainlythe Th1 immune response [6] As shown in Figure 3A

alum and MPLA adjuvants enhanced mouse survival ratespost challenge after immunization with a single-dose vac-cine These data imply that MPLA is more suitable thanalum for use as an adjuvant for influenza vaccines Anotherstudy also implied that MPLA could reduce the minimumeffective immunization dosage of H5N1 and H3N2 influ-enza vaccines in mice [18] For H5N1 vaccines bothalum and MPLA adjuvants effectively promoted Th2 anti-body immune responses they enhanced H5N1 vaccine-immunized mouse Th1 immune response less effectively

Adjuvant enhanced the vaccinersquos hemagglutinationinhibition (HAI) antibody responseSince the HAI antibody titer is considered an effectiveimmunity indicator of host defense against influenzavirus we evaluated the adjuvant-induced HAI antibodyresponse of the S-OIV H1N1 vaccine Both alum andMPLA adjuvants enhanced HAI antibody titers when in-cluded in the 005-μg S-OIV H1N1 vaccine (P lt 005)(Figure 4) Furthermore the dosage required for thecandidate S-OIV H1N1 vaccine to elicit productionof a protective HAI antibody response (HAI antibodytiter ge 40) was 005 μg With this dosage the positive


rate of HAI antibody was 5714 the average HAI titerranged from 80 to 160 The required vaccine dosage in-ducing a protective antibody response was reduced to001 μg (average HAI titer of 80 to 320) or 0001 μg(average HAI titer of 80) when the vaccines were mixedwith alum or MPLA adjuvants respectively Similar re-sults were also demonstrated in a study of H5N1(NIBRG-14) inactivated whole virus and split virioninfluenza vaccines in which the use of Al(OH)3 withMPLA as an emulsion induced a further increase ofHAI titer [12] This implies that the addition of anadjuvant to the vaccine could reduce the vaccine dos-age required to elicit a protective immune responseto the S-OIV H1N1 virus

Figure 3 Survival Rates and Body Weight Loss of immunized mice pogroup) immunized with PBS and 0001 μg 001 μg and 005 μg of vaccinewith 106 TCID50 of ACalifornia72009 virus intranasally Following 14 daysgroup are shown

Minimum dosage of the S-OIV H1N1 single-dose vaccinerequired for generating protective immunity to a lethalchallenge of aCalifornia072009 H1N1Since a single-dose vaccination can elicit an immune re-sponse to S-OIV H1N1 virus the minimum effectivedosage of S-OIV H1N1 vaccines was evaluated Our pre-vious data showed that a 05 μg to 01 μg dosage ofS-OIV H1N1 vaccine could provide mice with completeprotection against the virus (data not shown) Thereforegroups of mice (n = 5 to 7) were immunized via the in-traperitoneal (ip) route with different dosages of vac-cine ranging from 005 μg to 0001 μg in the absence orpresence of alum or MPLA adjuvant Three weeks postvaccination the mice were bled and were challenged

st challenge with ACalifornia72009 Virus Mice (n = 4 to 7 perwith or without adjuvant (alum or MPLA) followed by lethal challengeof observation survival rates (A) and weight changes (B) in each

Figure 4 Adjuvant enhanced vaccine to elicit ahemagglutination inhibition (HAI) antibody responseMice (n = 7 to 8 per group) were immunized with single differentdose of S-OIV H1N1 or NIBRG-14 H5N1 influenza vaccine with orwithout alum or MPLA as the adjuvant The titers of serum specificantibodies were evaluated using the hemagglutination inhibition(HAI) test Data are representative of two separate experiments ofH1N1 vaccine immunized mice sera (No sera from H5N1-immunizedmice contained HAI to NIBRG-14 H5N1 virus using chicken redblood cells) Comparisons of HAI antibody titer in mice immunizedwith PBS 0001 μg 01 μg or 05 μg of HA in the produced vaccinewith or without adjuvant For comparison of HAI antibody titersStudentrsquos t test was used to examine the significance of differencesbetween HAI positive rates (with HAI titer 3 40) of each vaccinatedgroup and control group (mice immunized with PBS only) A P valueof lt 005 was considered significant The star ldquordquo indicatessignificant differences


with ACalifornia072009 H1N1 virus one day later asdescribed in Materials and Methods The body weightand survival condition of the mice were recorded dailyfor 14 days post challenge The vaccine provided miceprotection in a dose-dependent manner (Figure 3A) Mice

Table 2 HAI antibody titer of mice post immunized with S-OI

Adjuvant Sample dose 1 2 3 4 5

Pre-immune ndash ndash ndash ndash ndash

Non adjuvant PBS ndash ndash ndash ndash ndash

005 μg 80 160 ndash ndash ndash

001 μg 160 ndash ndash ndash ndash

0001 μg ndash ndash ndash 40 ndash

Alum PBS ndash ndash ndash ndash ndash

005 μg 640 1280 80 640 12

001 μg 160 40 80 160 1

0001 μg 80 ndash ndash ndash 4

MPLA PBS ndash ndash ndash ndash ndash

005 μg 160 320 320 ndash 1

001 μg 320 80 160 ndash 1

0001 μg 80 ndash ndash ndash

Did not include into calculation because of failing to immunization

in the control group immunized with PBS had no protec-tion Single-dose vaccination of mice with the 005-μg can-didate vaccine S-OIV H1N1 elicited sufficient protectiveimmunity to ACalifornia072009 H1N1 challenge

Adjuvants reduced minimum protective dosages of bothS-OIV H1N1 and NIBRG-14 H5N1 influenza vaccinesBecause a large amount of influenza vaccine could be re-quired during a pandemic or epidemic of influenza virusan appropriate adjuvant should be considered as a com-ponent of an influenza vaccine to reduce the vaccinedosage for practical vaccination In this study alum andMPLA adjuvants were evaluated and were compared fortheir capabilities to reduce vaccine dosages required forprotecting mice from a lethal challenge of S-OIV H1N1or NIBRG-14 H5N1 influenza virus As shown in Table 2and Figure 3A 005 μg of S-OIV H1N1 vaccine was re-quired to yield a 65 to 857 survival rate upon chal-lenge with S-OIV H1N1 virus When alum was includedas the adjuvant in the S-OIV H1N1 vaccine the lowestdosage of vaccine for complete protection of the micewas reduced to 001 to 0001 μg Even when the micewere vaccinated with 0001 μg of S-OIV H1N1 vaccinewith alum the survival rate was 6667 (data notshown) Furthermore when the adjuvant included in thevaccine was MPLA the lowest dosage of S-OIV H1N1vaccine providing mice complete protection from a le-thal challenge of ACalifornia072009 H1N1 virus wasreduced to 0001 μg Meanwhile body weight loss wasless significant when mice were immunized with vaccineplus alum or MPLA adjuvants than with vaccine only(Figure 3B upper panel) Body weight loss exceeded 25of the total pre-challenge weight for mice immunizedwith PBS or vaccine only while for mice immunizedwith vaccine plus adjuvant body weight loss was less

V H1N1 vaccine in the absence or presence of adjuvant

6 7 HAI ge40 HAI titer Mean of HAI titer

ndash ndash 07 0 0

05 0 0

80 160 47 80 ~ 160 686

40 ndash 27 40 ~ 160 286

ndash 16 40 67

ndash ndash 06 0 0

80 80 160 77 80 ~ 1280 5943

60 80 320 77 80 ~ 320 1429

0 80 80 47 40 ~ 80 400

ndash ndash 06 0 0

60 160 640 67 160 ~ 640 2514

60 80 80 67 80 ~ 320 1257

80 40 36 40 ~ 80 333


than 15 Furthermore the recovery of body weight wasmore rapid for mice immunized with vaccine plus adju-vant (the 5th day post challenge) than for mice immu-nized with PBS or vaccine only (often the 7th or 9th day)S-OIV H1N1 vaccine accompanied by an appropriateadjuvant provided a protective effect in a single-doseimmunization regimen

MPLA adjuvant elicited cross-protective immunity to thelethal challenge of homogeneous and heterogeneousS-OIV H1N1 virusMice vaccinated with PBS or S-OIV H1N1 vaccine onlydid not survive the challenge with S-OIV H1N1 influ-enza virus (Figure 3A) Alum as an adjuvant providedsome protection for mice vaccinated with 001 μg (60)and 005 μg (80) NIBRG-14 H5N1 vaccine upon chal-lenged with a heterogeneous S-OIV H1N1 influenzavirus In the presence of MPLA as the adjuvant 001 μgand 005 μg of the NIBRG-14 H5N1 vaccine providedmice complete protection to the challenge of S-OIVH1N1 influenza virus Even vaccination with 0001 μg ofNIBRG-14 H5N1 vaccine plus MPLA as the adjuvant

Figure 5 Adjuvant promoted IgG responses of mice to homologous aleast immunization dose of S-OIV and H5N1 vaccine needed for mice to raH1N1 and NIBRG-14 H5N1 virus mice (n = 3 to 4 per group) immunized w3 weeks of single-dose immunization mice sera were used to evaluate theH5N1 virus (C) H5N1 IgG to H1N1 virus and (D) H1N1 IgG to H5N1 virus imof antibodies in each group of animals

provided 40 survival to the challenge with S-OIVH1N1 influenza virus Furthermnore mice vaccinatedwith PBS or H5N1 vaccine alone died within 7 dayspost challenge with ACalifornia072009 H1N1 virus(Figure 3B lower panel) Mice vaccinated with NIBRG-14 H5N1 plus alum or MPLA as the adjuvant lost 20to 40 or 10 to 30 of their body weight and then re-covered 6 days or 7 to 8 days post challenge respect-ively MPLA not only reduced the vaccine dosagerequired for efficient protection from a homogeneous in-fluenza virus it also raised the survival rate of the lethalchallenge with the heterogeneous ACalifornia072009H1N1 virus A previous study revealed that the additionof MPLA to the original vaccine increased CTL differen-tiation and these memory cells were better equipped torapidly kill infected cells than cells primed with alumalone [28] another study showed that MPLA inducedhigher HAI antibody and IFNγ titers [18] MPLA is aTRIF-biased agonist of TLR4 and induces expression oftype I interferon through TRIF rather than MyD88 [29]These results might be linked to the better protectionof MPLA as an adjuvant however this remains tobe elucidated

nd heterogeneous influenza H1N1 and H5N1 virus To realize theise sufficient protective immune responses to ACalifornia72009ith PBS 001 μg or 005 μg of vaccine with or without adjuvant Afterimmune response type (A) H1N1 IgG to H1N1 virus (B) H5N1 IgG tomune responses The data represent the mean titers plusmn SD (error bars)

Lin et al Journal of Biomedical Science 2013 2019 0 of 13httpwwwjbiomedscicomcontent20119

Adjuvant promoted IgG responses to homologous andheterogeneous influenza H1N1 and H5N1 virusesAs influenza virus has antigen drift and antigen shift ef-fects vaccination with some influenza vaccine might notinduce sufficient immunity to the threat of other influ-enza virus strains In this study we evaluated the crossprotection of S-OIV H1N1 and H5N1 influenza vaccinein mice Both alum and MPLA as adjuvants enhancedH1N1 and H5N1 homologous IgG immune responsesrespectively (Figure 5A and 5B) Furthermore the adju-vants also promoted the production of heterogeneousIgG immune responses H5N1 IgG to S-OIV H1N1

Figure 6 MPLA as the adjuvant significantly reduced lung viral titer Mwith or without different adjuvants For the challenge 106 TCID50 of virus wlungs were homogenized and inoculated into MDCK cells for 48 h Virus titH1N1 virus challenge (B) H5N1 vaccination H5N1 virus challenge (C) H5Nchallenge The TCID50 titers of the virus were calculated by the method ofthe differences in viral titers between two groups of vaccinated mice

influenza virus (Figure 5C) and H1N1 IgG to H5N1 in-fluenza virus (Figure 5D) Alum used as the adjuvantseemed to have a better effect than MPLA did for the in-duction of these IgG immune responses

MPLA as the adjuvant significantly reduced mice lungviral titer post challenge with homologous andheterogeneous influenza virusesTo evaluate the immune responses of immunized miceto influenza virus mice were vaccinated with S-OIVH1N1 or NIBRG-14 H5N1 vaccines in the absence orpresence of different adjuvants For the challenge 106

ice were vaccinated with S-OIV H1N1 or NIBRG-14 H5N1 vaccinesas inoculated into mice nasal cavities Three days post challenge miceers were evaluated using a micro-plaque assay (A) H1N1 vaccination1 vaccination H1N1 virus challenge (D) H1N1 vaccination H5N1 virusReed and Muench [reference 24] P lt 005 indicated the significance

Figure 7 Neutralization antibody response of immunized-mice post challenge with S-OIV H1N1 or NIBRG-14 H5N1 influenza virusNeutralization antibodies of mice post vaccinated with S-OIV H1N1 or NIBRG-14 H5N1 vaccines were evaluated using a microneutralization assayMice sera (pre-immune as negative ACalifornia72009 NIBSC 09152 and antiserum to NIBRG-14 H5N1 HA as positive control) were mixed withviruses (100 TCID50 of NIBRG-121 H1N1 or NIBRG-14 H5N1 virus) at room temperature for 1 hour and were inoculated into 96-well MDCK cells(15 times 104ml) Experiments were performed following the WHO protocol for the microneutralization assay (A) H1N1 vaccination H1N1neutralization antibody (B) H5N1 vaccination H5N1 neutralization antibody (C) H5N1 vaccination H1N1 neutralization antibody (D) H1N1vaccination H5N1 neutralization antibody Error bar is the standard deviation of six serum samples


TCID50 influenza viruses were inoculated into the nasalcavities of the mice The lungs of the mice wereharvested and homogenized three days after inoculationand then inoculated into MDCK cells and incubated for

48 h Virus titers were evaluated using a micro-plaqueassay As shown in Figure 6A 005 μg of S-OIV H1N1vaccine significantly reduced mouse lung H1N1 viral ti-ters the adjuvant promoted vaccine-induced reduction


of lung viral titers For reduction of H5N1 lung viral ti-ters the vaccine alone had no significant effect on theother hand in the presence of either alum or MPLAeven 001 μg of NIBRG-14 H5N1 vaccine significantlyreduced the lung H5N1 viral titer (Figure 6B)To evaluate cross-protection of vaccines to S-OIV

H1N1 and NIBRG-14 H5N1 influenza viruses lung viraltiters were measured in mice vaccinated with S-OIVH1N1 or NIBRG-14 H5N1 vaccines in the absence orpresence of alum or MPLA included as adjuvantsNIBRG-14 H5N1 vaccine alone did not significantly re-duce H1N1 viral titers in the mouse lungs (Figure 6C)where 001 μg of NIBRG-14 H5N1 vaccine plus MPLAsignificantly reduced the lung H1N1 viral titers Con-versely S-OIV H1N1 vaccine even with alum or MPLAadjuvant did not significantly reduce mouse lung H5N1viral titers post challenge (Figure 6D) Thus the avianNIBRG-14 H5N1 might provide some cross-protection toS-OIV H1N1 virus in mice but not vice versa In a similarstudy cross-protection of seasonal influenza virus and2009 pandemic H1N1 influenza virus in mice and ferretsshowed that vaccination with the seasonal influenzavaccines did not confer complete protection in the lowerrespiratory tract in either animal model whereas the ACalifornia72009 vaccine conferred complete protectionin both animal models [30] In our study only the MPLAadjuvant in the NIBRG-14 H5N1 vaccine reduced the het-erogeneous S-OIV H1N1 lung viral titer The underlyingmechanisms remain to be elucidated

Adjuvant elicited homologous but not heterogeneousneutralization antibody against influenza virusNeutralization antibody always plays an important role inthe evaluation of host immunity after vaccination for influ-enza viruses We evaluated the roles of adjuvants in thevaccines to elicit neutralization antibody H1N1 and H5N1vaccines with alum or MPLA adjuvant elicited significantlymore homologous neutralization antibodies to S-OIVH1N1 and H5N1 influenza virus respectively than vaccinealone (Figure 7A and 7C) Alum enhanced the productionof neutralization antibody against homologous influenzavirus but none of adjuvants induced production of hetero-geneous neutralizing antibody (Figure 7B and 7D)Our results indicate that a significant reduction of in-

fluenza viral titer in lungs of mice three days post chal-lenge was a better indicator for survival prediction thanhemagglutination inhibition (HAI) and neutralizationantibody titers Alum and MPLA used as adjuvants re-duce the vaccine dosage requirement and elicit protect-ive immunity to homologous influenza virus MPLAfurther promoted vaccine cross-protection in mice vac-cinated with NIBRG-14 H5N1 to lethal challenge withheterogeneous S-OIV H1N1 influenza virus

ConclusionThe study results showed that a single-dose immunizationin BALBc mice with 01 μg of the candidate vaccine elic-ited complete protective immunity to S-OIV H1N1 virusThis result is consistent with a previous study [16] thisminimal effective vaccine dose in BALBc mice corre-sponds to approximately 30 μg of HA in humans Further-more we also learned that when the vaccine contained anappropriate amount of MPLA adjuvant the vaccine elic-ited protective immunity to S-OIV H1N1 with single-doseimmunization using 1100 to 110 of the original vaccinedosage (corresponding to approximately 03 to 3 μg of HAin humans) Additionally both H1N1 and H5N1 inducedsignificant homologous but not heterogeneous neutra-lization antibody Only in the presence of adjuvant couldthe influenza vaccine protect mice from a lethal challengeof heterogeneous influenza virus MPLA as the adjuvantwith H5N1 vaccine significantly reduced lung H1N1 virustiters post challenge in the mice Results revealed that lungviral titer was a better indicator than IgG HAI andneutralization antibodies titer in predicting survival ratesof mice post influenza virus challenge Results from thisstudy revealed (1) the minimum protective vaccinationdosage (2) the single-dose vaccination regimen inducedprotective immunity in the presence of adjuvant (3)cross-protection evaluation between S-OIV H1N1 andNIBRG-14 H5N1 vaccines (4) adjuvant promoted anti-viral protection and reduce the required vaccinationdosage and (5) MPLA as the adjuvant promoted bettercross-protection of NIBRG-14 H5N1 vaccine to the lethalchallenge with the 2009 pandemic H1N1 influenza viruspossibly through Th1 immunity These data provide im-portant insight for the design and development of vaccineformulas and adjuvants

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsLHT carried out overall research work CCC carried vaccine preparation andanimal experimental WHL carried out animal experimental CDM supervisedthe overall progress WYC is responsible for the overall research All authorshave read and approved the final manuscript

AcknowledgementsThis work was supported by a grant from the Institute of PreventiveMedicine National Defense Medical Center Taiwan awarded to Y-C WangWe are very grateful to the Centers of Disease Control and Prevention (CDCTaiwan) for providing virus strains We also thank members of vaccinemanufacture team Y-H Chang C-H Huang S-C Lai H-C Lin and F-P Linetc for preparation and quality control assay of S-OIV H1N1 and NIBRG-14H5N1 vaccines

Author details1Institute of Preventive Medicine National Defense Medical Center PO Box90048ndash700 San-Hsia Taiwan 2Tri-Service General Hospital National DefenseMedical Center Taipei 100 Taiwan

Received 25 December 2012 Accepted 18 March 2013Published 21 March 2013


References1 WHO Pandemic (H1N1) 2009 - update 85 Available httpwwwwhointcsr

don2010_01_29enindexhtml Accessed 8 February 20102 Hancock KV Veguilla XL Zhong W Butler EN Sun H Liu F Dong L DeVos

JR Gargiullo PM Brammer TL Cox NJ Tumpey TM Katz JM Cross-reactiveantibody responses to the 2009 pandemic H1N1 influenza virus N Engl JMed 2009 3611945ndash1952

3 Itoh Y Shinya K Kiso M Watanabe T Sakoda Y Hatta M Muramoto YTamura D Sakai-Tagawa Y Noda T Sakabe S Imai M Hatta Y Watanabe SLi C Yamada S Fujii K Murakami S Imai H Kakugawa S Ito M Takano RIwatsuki-Horimoto K Shimojima M Horimoto T Goto H Takahashi KMakino A Ishigaki H Nakayama M Okamatsu M Warshauer D Shult PASaito R Suzuki H Furuta Y Yamashita M Mitamura K Nakano K NakamuraM Brockman-Schneider R Mitamura H Yamazaki M Sugaya N Suresh MOzawa M Neumann G Gern J Kida H Ogasawara K Kawaoka Y In vitroand in vivo characterization of new swine-origin H1N1 influenza virusesNature 2009 4601021ndash1025

4 CDC Serum cross-reactive antibody response to a novel influenza A(H1N1) virus after vaccination with seasonal influenza vaccineMMWR Morb Mortal Wkly Rep 2009 58521ndash524

5 Ninomiya A Imai M Tashiro M Odagiri T Inactivated influenza H5N1whole-virus vaccine with aluminum adjuvant induces homologous andheterologous protective immunities against lethal challenge with highlypathogenic H5N1 avian influenza viruses in a mouse modelVaccine 2007 253554ndash3560

6 Philippa J Baas C Beyer W Bestebroer T Fouchier R Smith D SchaftenaarW Osterhaus A Vaccination against highly pathogenic avian influenzaH5N1 virus in zoos using an adjuvanted inactivated H5N2 vaccineVaccine 2007 253800ndash3808

7 Pushko P Tumpey TM Van Hoeven N Belser JA Robinson R Nathan MSmith G Wright DC Bright RA Evaluation of influenza virus-like particlesand Novasome adjuvant as candidate vaccine for avian influenzaVaccine 2007 254283ndash4290

8 Rimmelzwaan GF Claas EC van Amerongen G de Jong JC Osterhaus ADISCOM vaccine induced protection against a lethal challenge with ahuman H5N1 influenza virus Vaccine 1999 171355ndash1358

9 Stephenson I Bugarini R Nicholson KG Podda A Wood JM Zambon MCKatz JM Cross-reactivity to highly pathogenic avian influenza H5N1viruses after vaccination with nonadjuvanted and MF59-adjuvantedinfluenza ADuckSingapore97 (H5N3) vaccine a potential primingstrategy J Infect Dis 2005 1911210ndash1215

10 Wellemans V Laurent S Heacutelie P ElAzhary Y Immunostimulatory propertiesof a novel adjuvant administered with inactivated influenza virusvaccine Vet Res 2007 381ndash14

11 Petrovsky N Aguilar JC Vaccine adjuvants current state and futuretrends Immunol Cell Biol 2004 82488ndash496

12 Miyaki C Quintilio W Miyaji EN Botosso VF Kubrusly FS Santos FL IourtovD Higashi HG Raw I Production of H5N1 (NIBRG-14) inactivated wholevirus and split virion influenza vaccines and analysis of immunogenicityin mice using different adjuvant formulations Vaccine 2010282505ndash2509

13 Chen Z Wang W Zhou H Jr Suguitan AL Shambaugh C Kim L Zhao JKemble G Jin H Generation of live attenuated novel influenza virusACalifornia709 (H1N1) vaccines with high yield in embryonatedchicken eggs J Virol 2010 8444ndash51

14 Kistner O Crowe BA Wodal W Kerschbaum A Savidis-Dacho H Sabarth NFalkner FG Mayerhofer I Mundt W Reiter M Grillberger L Tauer CGraninger M Sachslehner A Schwendinger M Bruumlhl P Kreil TR Ehrlich HJBarrett PN A whole virus pandemic influenza H1N1 vaccine is highlyimmunogenic and protective in active immunization and passiveprotection mouse models PLoS One 2010 51ndash7

15 Maines TR Jayaraman A Belser JA Wadford DA Pappas C Zeng H GustinKM Pearce MB Viswanathan K Shriver ZH Raman R Cox NJ Sasisekharan RKatz JM Tumpey TM Transmission and pathogenesis of swine-origin2009 A (H1N1) influenza viruses in ferrets and mice Science 2009325484ndash487

16 Tamura S Hasegawa H Kurata T Estimation of the effective doses ofnasal-inactivated influenza vaccine in humans from mouse-modelexperiments Jpn J Infect Dis 2010 638ndash15

17 Kistner O Barrett PN Mundt W Reiter M Schober-Bendixen S Eder GDorner F Development of a mammalian cell (Vero) derived candidateinfluenza virus vaccine Vaccine 1998 16960ndash968

18 Quintilio W Kubrusly FS Iourtov D Miyaki C Sakauchi MA Luacutecio F Dias SdeC Takata CS Miyaji EN Higashi HG Leite LC Raw I Bordetella pertussismonophosphoryl lipid A as adjuvant for inactivated split virion influenzavaccine in mice Vaccine 2009 274219ndash4224

19 Caillet C Piras F Bernard MC de Montfort A Boudet F Vogel FRHoffenbach A Moste C Kusters I AF03-adjuvanted and non-adjuvantedpandemic influenza A (H1N1) 2009 vaccines induce strong antibodyresponses in seasonal influenza vaccine-primed and unprimed miceVaccine 2010 283076ndash3079

20 Dormitzer PR Rappuoli R Casini D OrsquoHagan D Runham C Montomoli EBaudner B 3rd Donnelly JJ Lapini G Adjuvant is necessary for a robustimmune response to a single dose of H1N1 pandemic flu vaccine inmice PLOS Curr Influenza PRN 20091025

21 WHO Serological diagnosis of influenza by microneutralization assay In WHOGlobal Influenza Surveillance Network Manual for the laboratory diagnosisand virological surveillance of influenza [World Health Organization 20Avenue Appia 1211 Geneva 27 Switherland 2G ] WHO Press 201163ndash77

22 Wood JM Schild GC Newman RW Seagroatt V An improved singleradial-immunodiffusion technique for the assay of influenza haemagglutininantigen application for potency determinations of inactivated wholevirus and subunit vaccines J Biol Stand 1977 5237ndash247

23 Wood JM Mumford J Schild GC Webster RG Nicholson KG Single-radial-immunodiffusion potency tests of inactivated influenza vaccines for usein man and animals Dev Biol Stand 1986 64169ndash177

24 Reed LJ Muench HA A simple method of estimating fifty per centendpoints Am J Hyg 1938 27493ndash497

25 Nicholson KG Tyrrell DA Harrison P Potter CW Jennings R Clark A SchildGC Wood JM Yetts R Seagroatt V Huggins A Anderson SG Clinicalstudies of monovalent inactivated whole virus and subunit AUSSR77(H1N1) vaccine serological responses and clinical reactions J Biol Stand1979 7123ndash136

26 Eberhardt KR Fligner MA A comparison of two tests for equality of twoproportions Am Stat 1997 31151ndash155

27 Robbins H A fundamental question of practical statistics Am Stat 1977 319728 MacLeoda MKL McKeea AS Davida A Wangb J Masonb R Kapplera JW

Marracka P Vaccine adjuvants aluminum and monophosphoryl lipid Aprovide distinct signals to generate protective cytotoxic memory CD8 Tcells Proc Natl Acad Sci U S A 2011 1087914ndash7919

29 Mata-Haro V Cekic C Martin M Chilton PM Casella CR Mitchell TC Thevaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist ofTLR4 Science 2007 3161628ndash1632

30 Chen GL Min JY Lamirande EW Santos C Jin H Kemble G Subbarao KComparison of a live attenuated 2009 H1N1 vaccine with seasonalinfluenza vaccines against 2009 pandemic H1N1 virus infection in miceand ferrets J Infect Dis 2011 203930ndash936

doi1011861423-0127-20-19Cite this article as Lin et al Characterization of cross protection ofSwine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1influenza vaccine in BALBc mice given a single-dose vaccinationJournal of Biomedical Science 2013 2019

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Abstract

Background

Results

Conclusion

Background

Methods

Vaccine strains and reagents

Vaccine production

Quality control assays of vaccine

Determination of vaccine HA concentration

Ethics statement

Immunization of mice

IgG subclass determination

Cell and virus

Plaque-assay

TCID50

Hemagglutination inhibition (HI) assay

Intranasal influenza challenge

Microneutralization assay

Statistical analysis

Results and discussion

Production of S-OIV H1N1 and NIBRG-14 H5N1 vaccine

Adjuvant promoted IgG responses to homologous S-OIV H1N1 and NIBRG-14 H5N1 influenza viruses

Adjuvant improved S-OIV H1N1 vaccine to elicit a Th1-type antibody response (IgG2a)

Adjuvant enhanced the vaccinersquos hemagglutination inhibition (HAI) antibody response

Minimum dosage of the S-OIV H1N1 single-dose vaccine required for generating protective immunity to a lethal challenge of aCalifornia072009 H1N1

Adjuvants reduced minimum protective dosages of both S-OIV H1N1 and NIBRG-14 H5N1 influenza vaccines

MPLA adjuvant elicited cross-protective immunity to the lethal challenge of homogeneous and heterogeneous S-OIV H1N1 virus

Adjuvant promoted IgG responses to homologous and heterogeneous influenza H1N1 and H5N1 viruses

MPLA as the adjuvant significantly reduced mice lung viral titer post challenge with homologous and heterogeneous influenza viruses

Adjuvant elicited homologous but not heterogeneous neutralization antibody against influenza virus

Conclusion

Competing interests

Authorsrsquo contributions

Acknowledgements

Author details

References

Lin et al Journal of Biomedical Science 2013 2019 of 13httpwwwjbiomedscicomcontent20119

these antibodies (Abs) can provide potent protectionagainst infection andor disease The main deficiency ofthis protection is that it targets highly variable viral de-terminants Failure to anticipate the emergence of anepidemic strain with significant antigenic changes com-pared to the vaccine strain will greatly reduce vaccine-induced protection Several studies have suggested thatproper adjuvant might improve the immunity of influ-enza vaccine and reduce the dose of vaccine [5-10]Aluminum hydroxide (alum) is currently the onlyhuman vaccine adjuvant approved for use in the UnitedStates and although it is effective at boosting antibodyresponses these responses require repeated administra-tion and tend to generate antiparasitic T helper 2 (TH2)rather than antiviral and antibacterial TH1 T cell im-munity [11] As a consequence there is much effort de-voted to develop prospective adjuvant that can establishprotective immunity with fewer vaccinations and lessinjected material through durable antibody and TH1-dependent cytotoxic T cell activity Other potentialimmune adjuvant might be considered and developedAs demonstrated previously Monophosphoryl lipid A(MPLA) is a low-toxicity derivative of LPS with usefulimmunostimulatory properties which is nearing regula-tory approval for use as a human vaccine adjuvant Mostrecently it has been demonstrated that the use of Al(OH)3 with MPLA as an emulsion induced a furtherincrease in HAI titers of AreassortantNIBRG-14VietNam 11942004 times Puerto Rico81934 H5N1 (NIBRG-14) inactivated whole virus and split virion influenzavaccines [12] These finding may have important impli-cations for the development of future vaccine adjuvantAlthough there are studies evaluating immune re-

sponse and protection of influenza H1N1 vaccine to2009 S-OIV in ferrets and mice most recently [13-16]Some studies also approached immune response andprotection of vaccinated animals against other influenzavirus in the absence or the presence of adjuvant In thesestudies some useful information has been revealed Forexample pandemic H1N1 vaccination is effective inmice [17] the adjuvant MPLA can reduce the effectiveimmunization dose of H5N1 and H3N2 influenza vac-cines in mice [18] and some other adjuvant can reducethe effective immunization dose of pandemic vaccines inmice [1920] However most of these studies focus onthe protection of BALBc mice immunized in a two-dose regimen of vaccination Since it is impossible tovaccinate people twice at the emergent time during in-fluenza virus is pandemic worldwide effective vaccinecomponent and dosage of influenza vaccine for singleimmunization is more practical and critical for the pre-vention of influenza virus epidemic In this study single-dose immunization of 2009 H1N1 vaccine based onNIBRG-121 vaccine strain and avian H5N1 vaccine

based on NIBRG-14 vaccine strain were evaluated for itseffectiveness to elicit protective immunity of mice tothe lethal challenge of ACalifornia072009 H1N1 virusMeanwhile the minimum effective dose of single immu-nization in the absence or the presence of adjuvant (eitheralum or MPLA) as well as the cross protection of hetero-geneous vaccine the changes of adjuvant-induced im-mune responses and the reduction of vaccine dose usedin the presence of adjuvant were also elucidated

MethodsVaccine strains and reagentsInfluenza A virus NIBRG-121 (NIBSC ACalifornia72009 H1N1 virus) virus (ACalifornia072009 H1N1CDC 2009712112) hemagglutinin (HA) antigen(ACalifornia72009 NIBSC 09146) and antiserum(ACalifornia72009 NIBSC 09152) were obtainedfrom the Centers for Disease Control and Prevention(CDC Taiwan) or the National Institute for BiologicalStandards and Control (NIBSC UK) NIBRG-121 wasused as vaccine strain and virus ACalifornia072009H1N1 was used as challenge virus

Vaccine productionNIBRG-121 virus strain was amplified in embryonic eggsof 10-day special pathogen free (SPF) for 48 h it wasthen harvested and inactivated with 004 formalin at4degC for overnight Harvested virus was then filteredthrough 045 μm filter and concentrated with Lab scaleTM TFF System treatment with benzonase (cat no1016560001 Merck KGaA Germany) (9 ~ 90 uml at4degC for overnight) and purified with 10-50 sucrosegradient by Alfa Wassermann PKII Pilot-Scale Ultracen-trifuge System Purified virus was dialyzed with PBSfiltered with 045 μm filter and stocked in 15-ml stocktube at minus80degC until use Hemagglutinin (HA) antigenconcentration was determined by single radial immuno-diffusion assay (SRD) [21] for the quantification of vac-cine S-OIV H1N1

Quality control assays of vaccineTo monitor the quality of produced vaccine severalitems of assays were evaluated Vaccine was quantifiedwith SRD method ovalbumin was evaluated withChicken Egg Ovalbumin Elisa Kit (Cat No 6050) fromAlpha Diagnostic International (ADI) endotoxin wasmeasured with Toxin Sensor TM Chromogenic LALEndotoxin Assay Kit (Cat No L00350) obtained fromthe GenScript Corporation Above assays were followedthe protocols of commercial kits Formalin content ofvaccine was evaluated according to the guideline of ver-sion 6th of Pharmacopoeia Chinensis For abnormal tox-icity assay male BALBc mice (body weight 17ndash22 gm)were inoculated intraperitoneal with 05 ml of produced











TCID50
















H5N1 1024


H5N1 314 EUml


H5N1 55 μgml


H5N1 not done






























005 μg 640 1280 80 640 12

001 μg 160 40 80 160 1



005 μg 160 320 320 ndash 1

001 μg 320 80 160 ndash 1







ndash ndash 07 0 0

05 0 0

80 160 47 80 ~ 160 686

40 ndash 27 40 ~ 160 286

ndash 16 40 67

ndash ndash 06 0 0

80 80 160 77 80 ~ 1280 5943

60 80 320 77 80 ~ 320 1429

0 80 80 47 40 ~ 80 400

ndash ndash 06 0 0

60 160 640 67 160 ~ 640 2514

60 80 80 67 80 ~ 320 1257

80 40 36 40 ~ 80 333





































































Abstract

Background

Results

Conclusion

Background

Methods


Vaccine production



Ethics statement



Cell and virus

Plaque-assay

TCID50
















Conclusion

Competing interests


Acknowledgements

Author details

References











TCID50
















H5N1 1024


H5N1 314 EUml


H5N1 55 μgml


H5N1 not done






























005 μg 640 1280 80 640 12

001 μg 160 40 80 160 1



005 μg 160 320 320 ndash 1

001 μg 320 80 160 ndash 1







ndash ndash 07 0 0

05 0 0

80 160 47 80 ~ 160 686

40 ndash 27 40 ~ 160 286

ndash 16 40 67

ndash ndash 06 0 0

80 80 160 77 80 ~ 1280 5943

60 80 320 77 80 ~ 320 1429

0 80 80 47 40 ~ 80 400

ndash ndash 06 0 0

60 160 640 67 160 ~ 640 2514

60 80 80 67 80 ~ 320 1257

80 40 36 40 ~ 80 333





































































Abstract

Background

Results

Conclusion

Background

Methods


Vaccine production



Ethics statement



Cell and virus

Plaque-assay

TCID50
















Conclusion

Competing interests


Acknowledgements

Author details

References















H5N1 1024


H5N1 314 EUml


H5N1 55 μgml


H5N1 not done






























005 μg 640 1280 80 640 12

001 μg 160 40 80 160 1



005 μg 160 320 320 ndash 1

001 μg 320 80 160 ndash 1







ndash ndash 07 0 0

05 0 0

80 160 47 80 ~ 160 686

40 ndash 27 40 ~ 160 286

ndash 16 40 67

ndash ndash 06 0 0

80 80 160 77 80 ~ 1280 5943

60 80 320 77 80 ~ 320 1429

0 80 80 47 40 ~ 80 400

ndash ndash 06 0 0

60 160 640 67 160 ~ 640 2514

60 80 80 67 80 ~ 320 1257

80 40 36 40 ~ 80 333





































































Abstract

Background

Results

Conclusion

Background

Methods


Vaccine production



Ethics statement



Cell and virus

Plaque-assay

TCID50
















Conclusion

Competing interests


Acknowledgements

Author details

References






















005 μg 640 1280 80 640 12

001 μg 160 40 80 160 1



005 μg 160 320 320 ndash 1

001 μg 320 80 160 ndash 1







ndash ndash 07 0 0

05 0 0

80 160 47 80 ~ 160 686

40 ndash 27 40 ~ 160 286

ndash 16 40 67

ndash ndash 06 0 0

80 80 160 77 80 ~ 1280 5943

60 80 320 77 80 ~ 320 1429

0 80 80 47 40 ~ 80 400

ndash ndash 06 0 0

60 160 640 67 160 ~ 640 2514

60 80 80 67 80 ~ 320 1257

80 40 36 40 ~ 80 333





































































Abstract

Background

Results

Conclusion

Background

Methods


Vaccine production



Ethics statement



Cell and virus

Plaque-assay

TCID50
















Conclusion

Competing interests


Acknowledgements

Author details

References





































































Abstract

Background

Results

Conclusion

Background

Methods


Vaccine production



Ethics statement



Cell and virus

Plaque-assay

TCID50
















Conclusion

Competing interests


Acknowledgements

Author details

References

characterization of cross protection of swine-origin influenza virus

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