Broadly neutralizing DNA vaccine with specific mutationalters the antigenicity and sugar-binding activitiesof influenza hemagglutininMing-Wei Chena,b, Hsin-Yu Liaoa,b, Yaoxing Huangc, Jia-Tsrong Janb, Chih-Cheng Huangd, Chien-Tai Renb,Chung-Yi Wub, Ting-Jen Rachel Chengb, David D. Hoc,1, and Chi-Huey Wongb,1

aInstitute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan; bGenomics Research Center, Academia Sinica,Taipei 115, Taiwan; cAaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016; and dAnimal Health Research Institute,Council of Agriculture, Tanshui 25158, Taiwan

Contributed by Chi-Huey Wong, January 4, 2011 (sent for review December 6, 2010)

The rapid genetic drift of influenza virus hemagglutinin is anobstacle to vaccine efficacy. Previously, we found that the consen-sus hemagglutinin DNA vaccine (pCHA5) can only elicit moderateneutralization activities toward the H5N1 clade 2.1 and clade 2.3viruses. Two approacheswere thus taken to improve the protectionbroadness of CHA5. The first one was to include certain surfaceamino acids that are characteristic of clade 2.3 viruses to improvethe protection profiles. When we immunized mice with CHA5 har-boring individual mutations, the antibodies elicited by CHA5 con-taining P157S elicited higher neutralizing activity against the clade2.3 viruses. Likewise, the viruses pseudotyped with hemagglutinincontaining 157S became more susceptible to neutralization. Thesecond approachwas to update the consensus sequence withmorerecent H5N1 strains, generating a second-generation DNA vaccinepCHA5II. We showed that pCHA5II was able to elicit higher cross-neutralization activities against all H5N1 viruses. Comparison ofthe neutralization profiles of CHA5 and CHA5II, and the animalchallenge studies, revealed that CHA5II induced the broadest pro-tection profile. We concluded that CHA5II combined with electro-poration delivery is a promising strategy to induce antibodies withbroad cross-reactivities against divergent H5N1 influenza viruses.

serotype ∣ genotype

DNA vaccines have been considered as an appealing optionagainst pandemic diseases, such as influenza. We have re-

ported previously that a hemagglutinin (HA)-based DNA vaccinecan generate protective immunity against viral challenges in apreclinical model of influenza (1).Many groups have made effortsto develop vaccines and immunotherapeutics based on plasmidDNA (2, 3). For influenza viruses, DNA vaccines based on con-served antigens from viral internal proteins offer the potentialfor eliciting cell-mediated immune responses, which is believedto provide cross-protection and clearance abilities (4, 5).

Influenza A virus remains a threat to humans even though thevaccine against a specific strain provides a useful prophylacticprotection. The virus envelope protein HA is the most abundantprotein and the main player in inducing immune response (6).Therefore, current efforts in the design of flu vaccine ofteninvolve the use of either HA proteins or recombinant virus carry-ing a specific HA. Although the vaccine protection against thehomologous strain is satisfactory, cross-protection against hetero-logous strains is limited; i.e., the vaccine often has low immunityagainst different influenza virus strains. Influenza viruses con-tinuously evolve by increasing the mutations in epitopes (anti-genic drift) or by reconstituting the genome with other strains(antigenic shift) (7). As a result, influenza vaccines need to beupdated annually depending on the circulating strains or uponthe emergence of a pandemic. Understanding the contributionof amino acids on HA to antigenicity would facilitate the designof a universal vaccine that confers broad cross-reactivity tovarious influenza virus strains.

The antigenicity of HA is mainly localized in the globularregion that ranges from amino acid 91–261 (H3 numbering)(8). A low frequency of virus variant that displays multiple pointmutations in the globular region has been found to succeed inescaping vaccinated immunity (9). Furthermore, the globularregion of HA is also the main region recognized by neutralizingantibodies (10). The mutation(s) at antigenic sites may decreasethe antibody-neutralizing ability, which blocks interactionbetween HA and sialosides on the host cell membrane (11, 12).

The HA protein is not only involved in antigenicity, butalso responsible for receptor binding for viral transmission(10). Human influenza strains mainly recognize α2,6 sialosides,whereas the avian strains exhibit high-affinity binding to α2,3 sia-losides (13). Several studies showed that the receptor-bindingspecificity and the avidity of the HA protein can be altered byspecific amino acid substitutions in the globular region. Forexample, for H3 influenza virus, two amino acid substitutionsQ226L and G228S can alter the binding specificity from α2,3 sia-losides to α2,6 sialosides (14, 15). A similar trend was observed inH1 influenza virus; E190D and D225G substitutions shift recep-tor-binding specificity of the 1918 pandemic strain (16, 17). Theantigenicity and receptor-binding activities localize in the sameglobular region of HA. Whether these two characteristics arecorrelated remains to be elucidated. Recent reports suggestedthat specific HA amino acid mutations in the receptor-bindingdomain (RBD) might be related to the sensitivity of the serolo-gical assay (18). These findings suggest that the influenza virustends to induce a small number of amino acid mutations toincrease receptor-binding avidity and reduce the antigenicity ofmutated HA (19), consistent with the studies on recent humanpandemic strains (12, 20), that the amino acid mutations atRBD could affect the receptor-binding avidity and/or specificityofHA,which leads to changes in theHA immunogenicity and anti-genicity and finally the appearance of the immune-escaping strain.Monitoring receptor-binding avidity of circulating viruses mayfacilitate the accurate prediction of the mutants of epidemic po-tential, and further provide references for vaccine strain decision.

We previously reported that a consensus DNA vaccine showedfull protection against most of H5N1 influenza viruses, yet mod-erate protection against certain currently circulating clade 2viruses (1). In this report, we modified CHA5 by incorporatingthe critical residues from clade 2 viruses or by including more

Author contributions: D.D.H. and C.-H.W. designed research; M.-W.C., H.-Y.L., J.-T.J.,C.-C.H., C.-T.R., and C.-Y.W. performed research; C.-T.R. and C.-Y.W. contributed newreagents/analytic tools; M.-W.C., H.-Y.L., Y.H., T.-J.R.C., D.D.H., and C.-H.W. analyzed data;and M.-W.C., H.-Y.L., Y.H., T.-J.R.C., D.D.H., and C.-H.W. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: chwong@gate.sinica.edu.tw ordho@adarc.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1019744108/-/DCSupplemental.

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recently circulating viruses to generate the second-generationconsensus vaccine CHA5II. The broadness of neutralization pro-files of the induced antiserum was evaluated and compared to theantiserum induced by the HA from representative H5N1 strains.The efficacy of the selected candidate was further confirmed withanimal challenge studies.

ResultsDesign of Hemagglutinin Vaccine with Improved Broadness of Protec-tion. Itwaspreviously reported that theantiseruminducedbyCHA5exhibited moderate neutralization activity against A/Indonesia/2005(ID05,clade2.1),A/Anhui/1/2005(AN,clade2.3),andA/duck/Fujian/1734/05 (FJ, clade 2.3). The design was further improvedusing two approaches in order to elicit broader protection activities.

The first approach was to modify CHA5 with changes specificto clade 2.3 viruses. Amino acid alignment analysis of HA RBDin Fig. 1A revealed several clade-specific mutations betweenCHA5 and individual strain(s). For example, A102V, S145L,andK228Rare specific to clade 1 viruses, whereasD110N, S140D,and K205R are specific to clade 2 viruses that are susceptibleto CHA5-induced antiserum. For the insusceptible strains ID05,AN, and FJ, the only common change is S157P. In addition, V190Iand P197S are specific to clade 2.3 viruses.

The second approach was to redesign the consensus sequenceby including the most recently circulating strains. The updatedconsensus DNA vaccine pCHA5II was located closer to the

center of the phylogenetic analysis of HA from H5N1 viruses(Fig. 1B). As shown in Fig. 1A, the pCHA5II has different aminoacids at D110N, S140D, and K205R.

Evaluation of Antigenicity and Immunogenicity in RBD-MutatedHemagglutinin.The impact of the 157th, 190th, and 197th residuesin the HA globular region on immunogenicity and induction ofneutralization activities was evaluated using a pseudotyped-virusplatform. We revised CHA5 to include the S157P, V190I, orP197S mutation and then used these constructs as the vaccineantigens. The vaccine-elicited antisera were then subsequentlyevaluated for their neutralization profile. Compared to CHA5,the CHA5 S157P-induced antiserum can better neutralize clade2.3 viruses as the EC90 values were three times higher. On theother hand, CHA5 S157P antiserum exhibited reduced neutrali-zation activity to clade 1 and clade 2.2 viruses (Table 1). To con-firm the effects of these residues, we also asked whether thereversal of the specific amino acid mutations in HA from theclade 2.3 viruses FJ and AN would make the resulted pseudo-typed virus easier to be neutralized by the serum. Therefore,the 157th, 190th, and 197th residues of HA of FJ and AN weremodified to have the same amino acid present in other virusstrains: i.e., P157S, I190V, and S197P. The modified HA werethen used to produce pseudoviruses; the resulting pseudoviruseswere then analyzed for the neutralization activities of CHA5-induced antiserum. Table 2 shows that CHA5 antiserum can

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Fig. 1. (A) Genetic alignment of the globular region of H5N1 HAs. Protein sequence alignment of the HA-H5N1 globular region. The mutations S157P, V190I,and P197S, highlighted in red, are present in A/Indonesia/5/2005, A/duck/Fujian/1734/05, and A/Anhui/1/2005 HA, but not in pCHA5. Furthermore, D110N,S140D, and K205R substitutions (highlighted in blue) were observed in clade 2 viruses and in pCHA5II. (B) Phylogenetic analysis of the HA used in the study.

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successfully neutralize the pseudotype viruses containing themutations, with two- to threefold increases in titer when com-pared to the activity to neutralize the wild-type pseudoviruses.

The results confirmed that a single amino acid, proline 157,may determine the susceptibility of the circulating clade 2.3 viralstrains to CHA5-induced immunity; that is, the influenza virusmay only need a S157P mutation to change its immunogenicityprofile and thus evade CHA5-induceed immunity. Moreover,proline 157 can dramatically shift the immunogenicity of a vac-cine strain specific to clade 2.3 viruses.

Evaluation of Immunogenicity and Neutralization Activities ofpCHA5II. The activities of pCHA5II were compared parallelly.As shown in Table 1, CHA5II-elicited antisera showed higherneutralization activities against clade 2 viruses than the CHA5-induced antisera did. Indeed, CHA5II showed better activitiesthan CHA5 containing individual mutation that is specific to clade2.3 viruses. Furthermore, the CHA5II-induced antiserum alsoshowed a two- to fourfold increase in neutralization titer againstviruses pseudotyped with modified HA as shown in Table 2.

Receptor-Binding Preference of the Hemagglutinin Variants. Theamino acid mutation in the globular region of HA proteins wasanalyzed to determine receptor-binding specificity and avidity tosialosides in a direct glycan receptor-binding assay (17, 21, 22)(the structure of sialosides is shown in Fig. S1). CHA5, similarto H5 from virus strains, showed preference toward 2,3 sialosides,but not 2,6 sialosides. Glycan binding analyses of CHA5II, whichincludes the three mutations most commonly found in clade 2strains (D110N, S140D, and K205R), exhibited binding specificityand avidity similar to those of CHA5 (Fig. 2A). Interestingly, intro-duction of the 157P mutation to CHA5 (CHA5 S157P) can signifi-cantly increase the receptor-binding avidity (Fig. 2B). CHA5II andCHA5 S157P exhibited binding specificity similar to that of CHA5.

It was suggested that glycosylation of the HA protein is likelyto generate steric hindrance to mask the antigenic site and to

prevent immune system detection (23), resulting in altered virusreceptor binding (24) as well as manipulation of the protein struc-ture. Therefore, whether the increases in the sialosides bindingactivities were caused by the changes in the glycosylation sitesor the secondary structures was further investigated. The resultsconfirmed that CHA5, CHA5II (containing D110N, S140D, andK205R), and the modified CHA5 containing S157P have thesame glycosylation sites (Table S1) and similar secondary struc-tures (Fig. S2). We concluded that S157P substitution increasedthe binding avidity of HA protein for cell-surface glycan receptorsbut did not alter glycosylation or secondary structure of the pro-tein. Thus, the mutations introduced in the HA globular regionhad various effects on receptor binding, antigenicity, and immu-nogenicity among clade 2.3 HAs.

Comparison of CHA5II-induced neutralization activities to the activ-ities induced by H5 from representative viruses. It was clear thatCHA5II could induce broader neutralization activities among

Table 1. Effect of the amino acid mutations in the globular region on CHA5 vaccine immunogenicity

Antisera†

EC90 of HA pseudotyped virus *

VN1194‡ (clade 1)§ ID05 (clade 2.1) TK (clade 2.2) E319-02 (clade 2.3.2) FJ (clade 2.3.4) AN (clade 2.3.4)

CHA5 252 ± 46 89 ± 59 400 ± 67 159 ± 32 63 ± 15 79 ± 18CHA5 S157P 32 ± 9 100 ± 22 25 ± 8 32 ± 9 200 ± 38 173 ± 22CHA5 V190I 178 ± 34 72 ± 41 429 ± 85 122 ± 21 59 ± 18 81 ± 23CHA5 P197S 224 ± 41 63 ± 15 389 ± 62 171 ± 38 63 ± 15 79 ± 18CHA5II 252 ± 46 800 ± 124 1,600 ± 217 800 ± 120 200 ± 90 224 ± 99

TK, A/turkey/Turkey/1/2005.*The geometric means of EC90 neutralization titer from three independent experiments.†The DNA vaccine antigen was used to elicit antiserum.‡The HA of the virus was used to produce HA-pseudotyped virus.§The clade to which the virus belongs.

Table 2. Effect of amino acid residues on the virus susceptibilityof neutralization by CHA5- and CHA5II-induced antiserum

Virus *

EC90 neutralization titers †

CHA5 ‡ CHA5II

FJ 63 ± 15 200 ± 90FJ P157S 200 ± 39 800 ± 132FJ I190V 162 ± 27 598 ± 124FJ S197P 158 ± 31 634 ± 103AN 79 ± 18 224 ± 99AN P157S 200 ± 37 500 ± 81AN I190V 155 ± 32 462 ± 73AN S197P 125 ± 26 317 ± 58

*The HA of the virus was used to produce HA-pseudotyped virus.†The geometric means of EC90 neutralization titer form more than threeindependent experiments.

‡The DNA vaccine antigen was used to elicit antiserum.

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3512 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1019744108 Chen et al.

the artificial HA that we have so far. Whether CHA5II or otherHAs from circulating virus strains can be a better vaccine candi-date remains to be investigated. Therefore, we optimized theHA DNA sequences from all representative strains as DNA vac-cines and analyzed the induced antiserum for their neutralizationactivities with our in vitro neutralization platform. Not surpris-ingly, homologous or intraclade neutralization to the HA fromthe viruses in the same clade was always the strongest (Fig. 3).HAs in clade 1 vs. clade 2 have the most distinct serotype char-acteristics, because the induced antiserum did not have goodcross-clase neutralization activities. The results also showed thatthe clade 2.3 viruses (FJ and AN) appeared to have a tight neu-tralization profile, suggesting that the current vaccine strainsfrom other clades would not have the ability against clade 2.3viruses. Likewise, the antiserum from HA of clade 2.3 virusesshowed little cross-neutralization activity to other clades, evento homologous strains. Recently, the vaccine based on AN alsoshowed lack of protection against most influenza viruses (25).

Consistent with our previous studies, CHA5-induced antiser-um showed lower neutralization activities toward clade 2.1(ID05) and clade 2.3 (FJ and AN) viruses. The updated consen-sus HA-based DNA vaccine CHA5II could induce antiserumthat showed the neutralizing superiority to the antisera inducedby other optimized HA DNA sequences from the representativestrains (Fig. 3). The results indicated that, by shifting the DNAvaccine candidate toward the center position of the phylogenetictree, pCHA5II indeed conferred broader protection profile thanpCHA5, which was located at the outer most of the tree andbetween clade 1 and clade 2. In addition, comparing pCHA5and pCHA5II, there are three mutations (D110N, S140D, andK205R) in the globular region. The introduction of these muta-tions to CHA5 not only enabled the mounting of an effectiveimmune response to clade 2 H5N1 viruses, but also preservedthe cross-neutralization ability to the rest of the viruses.

The pCHA5II Conferred Cross-Clade Protection in Virus-ChallengedMice. To confirm that pCHA5II retained the activities againstclade 1 viruses while overcoming the limited protection of CHA5against A/Indonesia/5/2005/RG2, we challenged the pCHA5II-immunized mice with lethal doses of wild-type A/Vietnam/1194/2004 (Fig. 4 A and B) and RG2 viruses (Fig. 4C andD). As shownin Fig. 4, pCHA5II protected 90% and 100% of the mice fromthe threats of the influenza H5N1 viruses in clade 1 and clade 2

viruses, respectively. Moreover, pCHA5II could better protectmice from body weight loss during the course of A/Indonesia/5/2005/RG2 virus challenges.

DiscussionIn this report, two approaches were initially evaluated for improv-ing the protection profiles of our first-generation DNA vaccine,pCHA5. Based on the alignment of the HA sequence of CHA5immunity-resistant strains, we identified unique amino acidmutation(s) in the HA RBD domain of these insusceptiblestrains. The HA globular region of influenza virus is exposedto the outside environment and described as a viral spike(10, 21, 26). The changes in the HA globular region would thusallow the influenza virus to alter the efficiency for virus entry aswell as for evading host immunity. The phylogenetic comparisonof H5N1 HAs between clade 1 and clade 2 showed that thereare multiple antigenic variations proximal to the RBD (11, 27).Our evaluation using CHA5 harboring individual mutationsconcluded that S157P is the most critical one to influence HAantigenicity. Based on the evidence from a monoclonal anti-body-neutralizing assay, the H5 HA protein is believed to com-prise five distinct neutralizing epitopes (28) and three immuneescape-related antigenic sites (11). The S157P mutation affectsthe group 5 epitope (27) but direct evidence of any correlationwith immune escape was lacking. Herein, we proved that themutation at position 157 of HA in clade 2.3 viruses conferredresistance to CHA5-induced immunity. Furthermore, the 157Pmutation altered CHA5 immunogenicity from cross-clade immu-nity to clade 2.3-specific immunity. The S157P mutation was alsoable to increase the receptor-binding avidity of HA protein, whichis important for influenza virus to evade host immunity. Thisevidence suggests that residue 157 and related epitopes meritfurther investigation.

We hypothesized that certain H5N1 viruses might be able toincrease receptor avidity and alter specificity through N-linkedglycosylation. This phenomenon is especially true for H5N1 clade2 strains; viruses in clade 2.2 have evolved to possess receptor-binding specificity to α2,6 sialic acid, due to the loss of glycosyla-tion at residue 158 and acquisition of the 193R residue (29). Someevidence suggested that shorter glycan chain on the HA proteincould increase receptor binding and immunogenicity (30). Inthis report, however, we showed that the 157P residue in recentcirculating clade 2.3 viruses was critical for immune escape but

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was presented by the heatmap that was generatedusing the web tool of HIV databases. The green spotmeans that the EC90 endpoint titer elicited by specificHA was significantly higher than 95% confidence in-terval (>95% C.I.) compared to other antisera againstthe same pseudovirus. The EC90 endpoint titer lowerthan 25, i.e., no neutralization activities, was indicatedas “<25.”

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did not modulate N-linked glycosylation or secondary proteinstructure.

The second approach is to redesign the consensus HA. Werevised our prototype DNA vaccine pCHA5 to pCHA5II afterrededucing the consensus sequence from 1,192 full-length H5sequences. Because pCHA5II was deduced from all HA sequencesby 2007, rather thanMay 2006 for pCHA5, these residues representthe new mutations that emerged in circulating clade 2.3 strains.There are five mutations from pCHA5 to CHA5II (F8L, D110N,S140D, K205R, and I529T). Among the five mutations, three ofthe mutations are in the receptor-binding subdomain (S140D andK205R) or proximity vestigial esterase domain (D110N). S140Dappears to be in the H5-escape-related antigenic site 1, an exposedloop (Ca2 of H1/HA3 140 to 145/H5 136 to 141) for antibodybinding (31, 32). Another mutation, K205R, corresponding tothe antigenic siteD ofH3,may influence class I cytotoxic T lympho-cyte recognition and facilitate viral escape from CHA5 immunity(33, 34). Furthermore, D110N is located in a well-defined MHC-II-presenting epitope in H1 HA (S1; amino acids 107–119) (35);mutation of this residue may increase antigenicity. Indeed, ourserotyping results showed that CHA5II could confer broader neu-tralizing activity than CHA5. It was surmised that the amino acidmutations surrounding the RBD correlated not only to T-cell epi-topes but also to B-cell epitopes, as well as to vaccine immunogeni-city. Further research to evaluate the effects of these distinctivemutations on vaccine immunogenicity is ongoing.

To identify potentialDNAvaccine candidates for anH5N1 influ-enza epidemic, one can also screen for the best candidate that showsthe broadest protection profile among the circulating virus strains.Our screening results confirmed that our pCHA5II can elicit anti-serum that provides the broadest protection profile. It is not surpris-ing to observe significant intraclade protection, but it is ratherexciting to learn that some specific HAs have cross-clade neutrali-zation activities. It was noticeable that ID05, categorized to clade2.1, can confer cross-neutralization activity to clade 2.2 and 2.3viruses. Furthermore, HA from Turkey (pTK), which was in clade2.2 and close to the central position in HA phylogenetic tree, could

induce a similar cross-neutralization pattern as pCHA5 did. Theresults shown in Fig. 3 are consistent with the World Health Orga-nization (WHO) suggestion and the cross-neutralization resultsfrom many groups, in which ID05 and TK in clade 2.2 viruses weresuggested to be vaccine strains for an H5N1 epidemic (36, 37). TheID05-based virus-like particles were also proven to induce protec-tive immunity against other clade 1 viruses (38, 39).

The neutralization trend shown in Fig. 3, where the pCHA5II,compared to pCHA5, can elicit higher cross-neutralization activ-ities against all clade 2 viruses and retain admirable neutralizationactivity for clade 1 viruses, correlated well with the animal chal-lenge results in Fig. 4. Thus, the neutralization assay against HA-pseudotyped virus provides a reliable platform for determiningH5N1 virus antigenicity and can be an effective platform for iden-tification of adequate vaccine strains for a flu pandemic. TheWHO as well as the Food and Drug Administration criteria forassessing pandemic influenza vaccines in immunological naïvepopulations (40) are based on nonrandomized noncontrol HIantibody studies, though they still have some significant implica-tions. Furthermore, the HI assay may underestimate humanimmune responses to influenza viruses (41). To improve the pre-diction of the protection profiles of a given vaccine, a convenient,sensitive, and quantitative assay that enables wide-spectrum ana-lysis to probe the cross-protection profiles could facilitate thevaccine development process. Using the pseudotyped-virus neu-tralization analysis platform, we have successfully evaluated thecorrelation of genotypes and genotypes ofH5N1 viruses. The plat-form is flexible to deal with any viruses without the safety issuesand can be easily standardized; therefore, our pseudovirus neutra-lization platform may provide a better alternative for evaluatingthe immune response generated by influenza vaccine as well as forevaluating the cross-neutralization activities critical for vaccinedevelopment. In addition, pseudovirus-based neutralization plat-form like this will also provide clade information of a suspectedvirus strain. As a matter of fact, our result based on serologicalanalysis using pseudotyped-virus assay implied that ID05 is anti-gentically related to clade 2.2 viruses, rather than clade 2.1 viruses.A similar suggestion has indeed been claimed by WHO.

The results in Fig. 3 also provided an answer to a long-termquestion, whether the codes for cross-neutralization lied in thesequences of HA itself or cross-neutralization was elicited bythe great enhancement in immunogenicity by the electroporationstrategy. As shown in Fig. 3, various optimized HAs can induceantiserum with differential neutralization activities, confirmingthat the sequence of HA indeed dominated the immunogenicity.Our results also showed that CHA5II might provide a promisingand facile approach for a prophylactic that needs minimal predic-tion and annually updating.

Materials and MethodsViruses. The attenuated reassortant H5N1 influenza viruses A/Indonesia/5/2005 (RG2) was obtained from the Center for Disease Control in Indonesia.All viruses were cultivated in the allantoic cavity of specific-pathogen-free(SPF) embryonated eggs, titered in Madin–Darbey canine kidney (MDCK)cells, and expressed as 50% tissue culture infective dose (TCID50). The LD50

in mice was determined for each virus before use in challenge experiments.

Vaccine and Plasmid Construction. CHA5II was generated from1,192 full-lengthHAsequencesbytheendof2007,usingthesamemethodasdescribedpreviously(1). Using pCHA5 as a template, CHA5II and HAs from various H5N1 viruseswere constructedby site-directedmutagenesis (Multi Site-DirectedMutagenesisKit, Stratagene). Furthermore, the neuraminidase gene from influenza virusA/Vietnam/1194/2004 was also optimized, synthesized, and cloned into pVAX(pNA) for use in the production of HA-pseudotyped viruses.

Vaccination. Five- to 6-wk-old female BALB/c mice were immunized withendotoxin-free pCHA5II or other HA constructs, prepared with GenElute™HP Endotoxin-Free Plasmid Maxiprep Kit (Sigma-Aldrich). The DNA vaccinewas given 30 μg per mouse at weeks 0 and 3 by an intramuscular adminis-tration of plasmids followed by instantaneous electrical stimulation (TriGrid

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Fig. 4. Vaccine protection against lethal challenge by wild-type A/Vietnam/1194/2004 H5N1 virus (A and B) or Indonesia/5/2005/RG2 virus (C and D).BALB/c mice were immunized with two injections of pCHA5, pCHA5II, orthe control plasmid (pVAX) using intramuscular immunization with electro-poration. The immunized mice were intranasally challenged with the wild-type (A/Vietnam/1194/2004, clade 1) or reassortant (RG2, clade 2.1) H5N1viruses (n ¼ 10 per group). After virus challenge, survival (A and C) and bodyweight (B and D) were recorded for 14 d.

3514 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1019744108 Chen et al.

Delivery System, Ichor) (1, 42). The spacing of the TriGrid electrode array was2.5 mm, and the electrical field was applied at an amplitude of 250 V∕cm ofelectrode spacing for a duration of 40 ms three times over a 400 ms interval.After immunization, the mice were housed in the SPF animal facility at theInstitute of Cell Biology, Academia Sinica, Taiwan. Two weeks after the sec-ond immunization, the immunized mice were bled for HA-specific antibodyanalysis and for neutralization assay to assess vaccine efficacy. All animalexperiments were evaluated and approved by the Institutional Animal Careand Use Committee of Academia Sinica, Taiwan.

HA-Pseudotyped-Virus Assay.All the procedures were described previously (1).Briefly, human 293T cells were cotransfected with three plasmids: pNA, pNL4-3.Luc.R-E- (National Institute of Allergy and Infectious Diseases, National In-stitutes of Health) (43), and HA of interest. After overnight incubation, thetransfected cells were washed with PBS and incubated with fresh culturemedium for another 24 h. The supernatant was then harvested and was usedfor neutralization assay. The mixtures containing 50 × TCID50 of HA-pseudo-typed viruses and various dilutions of the antiserum were incubated at 37 °Cfor 30 min and added to MDCK cells at 37 °C. The plates were washed after4 h incubation and replenished with fresh medium. After 48 h, luciferase ac-tivity in the cells was determined by the Luciferase Assay System (Promega).The relative luminescence values determined in the wells containing cells andHA-pseudotyped virus were defined as 0% neutralization; the values in thecells-only wells were defined as 100% neutralization. The maximum antiser-um dilution fold for 90% neutralization was defined as EC90 endpoint titer.

Generation of a Heatmap for HA Serotyping Using Virus Neutralization Titers.The HA serotyping analysis was generated based on the EC90 endpoint titerdetermined in pseudotyped-virus neutralization assays. Geometric mean titer

and 95% C.I. for given pseudoviruses or antiserum were calculated from theEC90 titers determined in three independent experiments. The EC90 titer datawere displayed by the heatmap using the web tool available in HIV databases(http://www.hiv.lanl.gov/content/sequence/HEATMAP/heatmap.html).

Virus Challenge Experiments. Two weeks after the second immunization, theimmunizedmicewere anesthetized and intranasally challengedwith a 50LD50

of the reassortant RG2 and 100LD50 of the wild-type A/Vietnam/1194/2004H5N1 virus. After infection, themicewere observed daily for 14 d, and survivaland clinical parameters such as body weight were recorded. The challengeexperiments were performed under biosafety level-2-plus (for RG2) andlevel-3 (for wild-type Vietnam/1194 H5N1 virus) enhancement conditions.

Statistical Analysis. The animal experiments to evaluate neutralization activ-ities of the serum were repeated at least three times (n ¼ 3 per group). Thevirus challenge experiments were conducted with n ¼ 10 per group. Aminoacid ClustalW multiple alignment and the threshold frequency for inclusionin consensus sequences were conducted by BioEdit Sequence AlignmentEditor version 7.0.5.3 (44).

ACKNOWLEDGMENTS. We thank Professor Kwok-Yung Yuen at The Universityof Hong Kong for the wild-type A/Vietnam/1194/2004 H5N1 viruses. Wethank the Taiwan Centers for Disease Control (CDC) for providing the RG2virus and for propagating the wild-type H5N1 viruses. We also thank DrewHanneman for help with the electroporation instrument, Shih-Gi Wang forhis assistance with mouse immunizations, and Dr. Chung-Hsuan Chen for hishelps in glycosylation site determination using mass spectrometry. This workwas supported by Academia Sinica and the Taiwan Pandemic InfluenzaVaccine Research and Development Program from the Taiwan CDC.

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