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Vaccine 27 (2009) 5740–5747

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

hort communication

mmunoinformatic comparison of T-cell epitopes contained in novelwine-origin influenza A (H1N1) virus with epitopes in 2008–2009onventional influenza vaccine

nne S. De Groot a,b,c,∗, Matt Ardito a, Elizabeth M. McClaine a,eonard Moise a,b, William D. Martin a

EpiVax, 146 Clifford Street, Providence, RI 02903, USAInstitute for Immunology and Informatics, University of Rhode Island, 80 Washington Street, Providence, RI 02903, USAAlpert Medical School, Brown University, Providence, RI 02903, USA

r t i c l e i n f o

rticle history:eceived 6 June 2009eceived in revised form 6 July 2009ccepted 10 July 2009vailable online 4 August 2009

eywords:1N1

nfluenza-cell epitopemmunoinformaticslgorithmell-mediated

a b s t r a c t

In March 2009 a novel swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the West-ern United States. Vaccination with conventional influenza vaccine (CIV) does not result in cross-reactiveantibodies, however, the disproportionate number of cases (37%) occurring among persons younger than50 years old suggested that adaptive immune memory might be responsible for the relative lack of vir-ulence in older, healthy adults. Using EpiMatrix, a T-cell epitope prediction and comparison tool, wecompared the sequences of the three hemagglutinin (HA) and neuraminidase (NA) proteins contained in2008–2009 CIV to their counterparts in A/California/04/2009 (H1N1) looking for cross-conserved T-cellepitope sequences. We found greater than 50% conservation of T helper and CTL epitopes between novelS-OIV and CIV HA for selected HLA. Conservation was lower among NA epitopes. Sixteen promiscuoushelper T-cell epitopes are contained in the S-OIV H1N1 HA sequence, of which nine (56%) were 100%conserved in the 2008–2009 influenza vaccine strain; 81% were either identical or had one conserva-tive amino acid substitution. Fifty percent of predicted CTL epitopes found in S-OIV H1N1 HA were also

found in CIV HA sequences. Based on historical performance, we expect these epitope predictions to be93–99% accurate. This in silico analysis supports the proposition that T-cell response to cross-reactiveT-cell epitopes, due to vaccination or exposure, may have the capacity to attenuate the course of S-OIVH1N1 induced disease—in the absence of cross-reactive antibody response. The value of the CIV or live-attenuated influenza vaccine containing the 2008–2009 vaccine strains, as defense against H1N1, could

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be further tested by evalufrom individuals infected

. Introduction

The emergence of swine-origin influenza A (H1N1) virus (S-OIV)1] as a global epidemic [2] is due in part to differences betweenhe hemagglutinin (HA) sequence of the S-OIV and conventionalnfluenza vaccine (CIV) strains and the susceptibility of mostuman populations to this new strain of influenza. The differencesetween the hemagglutinin (HA) sequence of the S-OIV and the CIV

accine strains became clear when individuals vaccinated against1N1 viral strains, by CIV, failed to produce cross-reactive anti-odies to the new H1N1 influenza [3]. This lack of cross-reactivityuggested that the existing vaccine might not provide effective

∗ Corresponding author at: EpiVax, 146 Clifford Street, Providence RI 02903, USA.el.: +1 401 272 2123; fax: +1 401 272 7562.

E-mail address: AnnieD@EpiVax.com (A.S. De Groot).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.07.040

human immune responses to the conserved T-cell epitopes using PBMCH1N1 and from CIV vaccinees.

© 2009 Elsevier Ltd. All rights reserved.

cross-protection, setting up a “perfect storm” in terms of potentialfor widespread disease and significant economic impact. Com-pounding this concern was the fact that that novel S-OIV H1N1 isresistant to amantadine and rimantadine [4]. Resistance to the tworemaining licensed antivirals, oseltamivir and zanamivir, followingpotential spread of the H1N1 epidemic in the southern hemisphere,has been an additional concern raised by the WHO and the CDC [5].

The 2008–2009 seasonal CIV was composed from the HA andNA proteins of three types of viruses: A/Brisbane/59/2007 H1N1,A/Brisbane/10/2007 H3N2 and B/Florida/4/2006. HA and NA fromthese three viruses were also used to develop the southern hemi-sphere influenza vaccine for 2008–2009. More than 130 million

individuals in the United States have had access to the currentvaccine and more than 1 billion doses were manufactured for dis-tribution worldwide, with an estimated 500 million doses used [6].

The 2008–2009 seasonal CIV contained an H1N1 virus(A/Brisbane/59/2007). Novel S-OIV A/California/04/09 shares only

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A.S. De Groot et al. / Va

2–73% amino acid identity [7]. In contrast, the amino acidequence identity in the HA portion among seasonal vaccinetrains (2007–2008 and 2008–2009) is 97% and 98%, respec-ively. The amino acid sequence divergence between HA-H1 of/California/04/09 and HA-H1 of A/Brisbane/59/2007 probablyontributes to the lack of antibody cross-reactivity detected amongndividuals immunized with 2008–2009 CIV [3]. While overall HAnd NA sequence changes are of great significance in terms of anti-ody response, immunological differences are also expressed onhe level of T-cell epitopes, short peptide sequences recognized bycells in the context of HLA molecules.

T-cell responses to conserved epitopes may be particularlymportant when new strains of influenza emerge. In mice and inumans, memory T cells to conserved epitopes have been showno confer protection to heterotypic infection [8,9]. The activationf Th cells is also critically important to the magnitude, qualitynd kinetics of antibody response [10]. In the absence of func-ional (memory) CD4+ T cells, studies in mice have shown that theate of viral clearance upon secondary infection slows consider-bly, beyond the degree seen in the primary response [11–13]. Alson mice, cross-reactive memory T-helper cells have been shown toontribute to cross-strain antibody responses [14]. In human pop-lations, cross-reactive T-cell responses have been demonstratedetween circulating strains of influenza and epidemic strains (suchs H5N1) in the absence of cross-reactive antibodies [15]. Bothross-reactive CTL and T helper cells have been identified by a num-er of investigators [16,17].

A number of mechanisms could contribute to the relative lackf severe disease among older adults in areas where pandemic S-IV is circulating. One potential explanation for protection against

evere disease in the absence of cross-reactive antibodies mighte the presence of cross-reactive T-cell response. Therefore, weecided to determine whether cross-conserved T-cell epitopesight be present in the novel A/California/04/09 sequence. So as

o get the most immediate estimate of the potential for the exist-ng CIV to protect against the emerging S-OIV, we examined therotein sequences of S-OIV and CIV 2008–2009 in silico, usingell validated immunoinformatics tools. Herein, we describe a set

f cross-reactive T-cell epitopes that are relatively well conservedetween the novel S-OIV and the existing seasonal influenza vac-ine strains. We provide a list of potential cross-reactive T-cellpitopes (and epitopes unique to the CIV and epidemic strains) thatay be synthesized and used to confirm cross-reactivity between

IV and the epidemic strain in vitro, using peripheral white bloodells (PBMC) from exposed and vaccinated donors.

The overall average predictive efficacy of a number of epitopeapping algorithms, considering a range of viral and bacterial

athogens, has been shown to be as high as 93% for Class IIpitopes and higher for Class I epitopes [18,19]. As shown here,any of the conserved influenza epitopes defined by our tools

ave been previously published. Thus, computationally identi-ed cross-reactive epitopes are a good starting point for further

mmunological and immunoprophylactic studies. We expect thathe epitopes described will be confirmed in standard in vitro assaysnd that they might also be useful for the development of a novelpitope-based “cross-strain” influenza vaccine. Production of suchvaccine may be more rapid and efficient than egg- or cell culture-ased vaccines in the context of an emerging epidemic.

. Methods

.1. Epitope mapping

EpiMatrix, a T-cell epitope mapping algorithm developed byhe principal scientists at EpiVax, screens protein sequences for

27 (2009) 5740–5747 5741

9–10 amino acid long peptide segments predicted to bind to one ormore MHC alleles [20,21]. EpiMatrix uses the pocket profile methodfor epitope prediction, which was first described by Sturniolo andHammer in 1999 [22]. For reasons of efficiency and simplicity, pre-dictions are limited to the eight most common HLA Class II allelesand six “supertype” HLA Class I alleles [23]. EpiMatrix raw scores arenormalized with respect to a score distribution derived from a verylarge set of randomly generated peptide sequences. Any peptidescoring above 1.64 on the EpiMatrix “Z” scale (approximately thetop 5% of any given peptide set) has a significant chance of bindingto the MHC molecule for which it was predicted. Peptides scoringabove 2.32 on the scale (the top 1%) are extremely likely to bind; thescores of most well known T-cell epitopes fall within this range ofscores [24–26]. EpiMatrix has been successfully applied to the anal-ysis of previously published epitopes [27], and in the prospectiveselection of epitopes from HIV [28], Mycobacterium tuberculosis[29], Tularemia [30] and vaccinia virus [31]. An ancillary algorithm,ClustiMer, identifies “clustered” or promiscuous epitopes [29,30].Conservatrix compares similar sequences (between strains) [32]and BlastiMer identifies homologies between the putative epitopesidentified by EpiMatrix and any protein sequence on file at GenBank[23].

2.2. Influenza sequences

HA and NA sequences were obtained from the NationalCenter for Biotechnology Information (NCBI). The downloadedsequences were from A/California/04/2009 H1N1 GENBANK:NA ACP41107; HA ACP41105), A/Brisbane/59/2007 H1N1 (GEN-BANK: NA ACA28847; HA ACA28844), A/Brisbane/10/2007 H3N2(GENBANK: NA ACO95273; HA ACO95270), and B/Florida/4/2006(GENBANK: NA ACA33351; HA ACA33493). The novel S-OIV H1N1sequences analyzed were selected from among those posted toGenBank on April 29th, 2009.

2.3. Conservation

We used the Conservatrix algorithm to parse and compare9-mers for segments that were highly conserved between the epi-demic strain and CIV. In assessing HLA specific conservation weallowed up to two amino acids out of any 9-mer frame to vary, aslong as the variant peptide was still predicted to bind to the tar-get HLA allele. This allowance is supported by studies that indicatethat there may be even greater flexibility in the binding of T-cellreceptors to peptides that bind in the HLA groove than previouslyrecognized [33,34].

2.4. HLA restriction

The HLA alleles selected for this analysis were selected tomaximize cross-HLA coverage [32,35]. Both DeLisi and Sette haveaddressed the issue of HLA coverage for epitope predictionsby demonstrating that epitope-based vaccines that contain epi-topes restricted by selected “supertype” HLA can provide thebroadest possible coverage of the human population [36]. Theset of six, HLA Class I alleles used in this analysis on average,cover over 99% of the population when considering Caucasian,North American Black, Japanese, Chinese, and Hispanics. The eight“supertype” Class II used also cover over 99% of the human

tope predictions on “promiscuous epitopes’—epitopes that arerecognized in the context of more than one HLA [37–39]. Wefocuses our analysis on regions of the HA and NA sequencesthat contained more than four potential Class II HLA bindingmotifs.

5742 A.S. De Groot et al. / Vaccine 27 (2009) 5740–5747

Table 1The number of HLA Class I restricted epitopes, per allele, in each of the four sequences (epidemic strain H1N1 California and the three strains included in CIV). The numberof epitopes that are conserved, for each of the strains, in the epidemic strain, is shown in parentheses.

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. Results

.1. HA and NA sequence conservation

We aligned the full-length HA and NA sequences of novel-OIV and those contained in the 2008–2009 CIV strains. Wend that the amino acid sequence of A/California/04/20091N1 HA is 79.5% conserved in A/Brisbane/59/2007 H1N1,2.4% conserved in A/Brisbane/10/2007 H3N2, and 30.9% con-erved in B/Florida/4/2006. A/California/04/2009 H1N1 NA is0.6% conserved in A/Brisbane/59/2007 H1N1, 44.2% con-erved in A/Brisbane/10/2007 H3N2, and 32.0% conserved in/Florida/4/2006. We also considered conservation at the level ofhe 9-mer sequence ‘frame’ which fits into the MHC binding groove.A and NA sequences were parsed into 4,088 distinct 9-mer pep-

ides and compared using Conservatrix. A total of 281 peptides wereound to be 100% conserved in all four flu strains. Of these, 167 areound in HA and 114 in NA. We expanded the list to include peptides80% conserved (at least seven out of nine identities). 327 peptidesere greater than 80% conserved in at least two of the input strains.

hese 327 peptides were retained for T-cell epitope analysis.

.2. Epitope analysis

Employing the EpiMatrix system, we screened the four input

trains for the presence of Class I and Class II restricted T-cell epi-opes as previously described [20,21]. EpiMatrix Class II predictionsave a record of 93% accuracy in prospective studies carried out bypiVax [24–26,30,32]. The EpiMatrix algorithm has 95% accuracyverall with regard to CTL epitopes for the Class I supertypes [26].

3.3. Class I epitope analysis

On average almost 45% of the predicted CTL epitopes found inA/California/04/2009 H1N1 HA are conserved in the HA sequencescontained in the 2008–2009 CIV. Conservation varies by HLA allele.For example, twelve (12) of the twenty-two (22) epitopes restrictedby HLA A*2401 were conserved between the novel flu and CIV (55%),but only eleven (11) out of thirty-four (34) HLA A*0301 restrictedepitopes were conserved (32%). Both the degree of cross-reactivityof Class I epitopes and the absolute number of CTL epitopes areimportant because the repertoire of possible HLA-restricted epi-topes recognized by an individual’s T cells has been shown to differ,even between HLA-matched individuals [40–42].

Very few predicted A/California/04/2009 H1N1 NA Class II (Thelper) and Class I (CTL) epitopes are conserved in the NA sequencesof the 2008–2009 CIV. This lower level of conservation betweenstrains is not surprising given the highly variable nature of the NAprotein. Table 1 provides a summary of the CTL epitope conser-vation analysis and Table 2 lists the CTL epitopes defined for theA/California/04/2009 H1N1 sequence.

3.4. Identifying Class II epitope clusters

After screening for putative Class II epitopes we used the Clus-tiMer algorithm to identify regions of high T-cell epitope density.

We have found that epitope clusters tend to be more immunogenicthan isolated Class II epitopes and for that reason have found that93% of the epitopes predicted using this approach are validated inprospective studies [24–26,30,32]. The ClustiMer algorithm identi-fied sixteen (16) HA and seven (7) NA Class II epitope clusters in the

A.S. De Groot et al. / Vaccine 27 (2009) 5740–5747 5743

Table 2The California/2009 (H1N1) peptides listed in this table were predicted to bind to the corresponding alleles (at top) and are at least 78% conserved in the Brisbane/59 strain(H1N1). Those peptide sequences that are predicted to bind to multiple Class I alleles are underlined (these would be promiscuous Class I epitopes) and those that are boldare conserved in the Brisbane/10 strain (H3N2).

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ew S-OIV H1N1 virus. Sixteen (16) HA epitope clusters were foundn the sequence of A/Brisbane/59/2007 H1N1, thirteen (13) within/Brisbane/10/2007 H3N2, and sixteen (16) within B/Florida/2006.en (10) NA epitopes were found within A/Brisbane/59/2007 H1N1,ve (5) within A/Brisbane/10/2007 H3N2, and eight (8) within/Florida/2006.

.5. Conservation among Class II epitope clusters

We examined the exact sequences of the predicted Class II

pitope clusters, and found that nine out of sixteen (56%) of the T-elper epitopes within A/California/04/2009 H1N1 HA were 100%onserved with the corresponding sequence in A/Brisbane/59/20071N1. An additional four out of the sixteen (25%) HA epitope clus-

ers were 90% conserved with their corresponding sequences in

A/Brisbane/59/2007 H1N1. Within A/California/04/2009 H1N1 NA,only one of seven epitope clusters (15%) was 100% conserved inA/Brisbane/59/2007 H1N1 and an additional three clusters (43%)were 90% conserved between S-OIV and H1N1 Brisbane. An exam-ple of a 100% conserved, promiscuous Class II epitope is shownin Fig. 1. A detailed comparison of conserved Class II epitopes isprovided in Tables 1-S and a list of the epitopes can be found inTable 2-S [supplemental materials].

3.6. Predicted and conserved epitopes compared to published

So as to obtain an estimate of the validity of these predictions,we compared the CTL epitopes that were conserved between thenovel S-OIV H1N1 HA and NA and the HA and NA sequences con-tained in the 2008–2009 CIV with epitopes published in the IEDB

5744 A.S. De Groot et al. / Vaccine 27 (2009) 5740–5747

Fig. 1. One example of a MHC Class II epitope that is 100% conserved between the HA A-BRISBANE-59-2007-H1N1 sequence used in the FLUVAX 2008-09 and the HAsequence of the current circulating strain, A-CALIFORNIA-04-2009-H1N1. The EpiMatrix Scores for each of the most common Class II DR alleles are shown.

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43]. Among the conserved HA sequences between the novel S-OIV1N1 and A/Brisbane/59/2007 H1N1 strains, thirteen of the seven-

een epitopes predicted to be restricted by HLA A*0201 have beenested; ten (77%) of which are confirmed, published epitopes. Sixf the eleven epitopes predicted to be restricted by HLA A*0301ave been tested, yielding five (83%) of which are confirmed andublished. With regards to the epitopes that are predicted to beestricted to HLA A*2401, six of the eight epitopes tested (75%) areonfirmed and published. Ten of the eleven (91%) epitopes pre-icted and tested against HLA B*4403 are confirmed and published.smaller percentage (<60%) of the predicted HLA A*0101 and HLA

*0701 epitopes have been tested and published, based on recordsvailable at the IEDB [Supplemental Table 3-S].

Among the conserved NA sequences between the novel S-OIV1N1 and A/Brisbane/59/2007 H1N1 strains, five of the nine epi-

opes predicted to be restricted by HLA A*0201 have been tested;our (80%) of which are confirmed, published epitopes. Of the fourpitopes predicted to be restricted by HLA A*2401 to have beenested, all four (100%) are confirmed, published epitopes. Withegards to the epitopes that are predicted to be restricted to HLA*0701 and HLA B*4403 respectively, 75% are confirmed and pub-

ished. A smaller percentage (50%) of the predicted HLA A*0101 and*0301 epitopes have been tested and published [Supplementalable 3-S].

The Class II epitope clusters identified by the EpiMatrix sys-em were also compared to those in the IEDB. Eight of the sixteenredicted epitopes found in the A/California/04/2009 H1N1 HAequence have been tested. Seven of those eight (88%) that wereested are confirmed epitopes. Four of the seven epitope clustersdentified in the A/California/04/2009 H1N1 NA sequence haveeen tested, yielding only one (25%) that was actually confirmed asn epitope and published in the IEDB [Supplemental Table 4-S]. Epi-opes derived from the A/California/04/2009 HA and NA sequenceshat are not published may still be immunogenic; confirmationould be performed using PBMC from exposed or vaccinated donorsFig. 2).

. Discussion

Early reports suggested that S-OIV may be more virulentmong younger individuals; greater than 35% of cases have beendentified among individuals age 19–49. Antibody responses

uence of A-CALIFORNIA-04-2009-H1N1 and the HA sequence of the A-BRISBANE-An adjacent epitope (HQNEQGSGY) is not conserved and was not included in the

resulting from previous vaccination of children with any of fourseasonal trivalent, inactivated influenza vaccines (CIV) or with live,attenuated influenza vaccine (LAIV) are not cross-reactive with thenovel influenza A (H1N1) virus [3]. A slightly higher prevalence ofcross-reactive antibodies was found among immunized adults inthe same study; this was due in part to antibodies that may haveexisted prior to vaccination with CIV. We postulated that cross-reactive T-cell responses might have contributed to diminishedreports of influenza-like illnesses and confirmed S-OIV infectionamong older adults, who are more likely to be either vaccinated(because of vaccine risk group recommendations) or previouslyexposed to circulating influenza H1N1. We therefore set out todetermine whether there were any T-cell epitopes conservedbetween S-OIV and vaccine strain HA and NA. These epitopesmight explain the limited virulence of emerging H1N1 among theolder exposed or vaccinated adults. If a link between conservedepitopes and attenuated disease course could be established, itwould suggest that vaccination with 2008–2009 CIV could helpreduce the public health and economic impacts of a widespreadepidemic of A/California/04/2009 H1N1 influenza.

Our results can be summarized as follows: Based on EpiMa-trix analysis of conserved 9-mers, (1) there is good cross-reactivityfor promiscuous Class II T helper epitopes; (2) there is fair togood cross-reactivity in terms of HA CTL epitopes; (3) thereis less cross-reactivity in the NA epitopes; and (4) there maybe sufficient cross-reactivity in T-cell epitopes between the epi-demic H1N1 strain and CIV 2008–2009 that cross-reactive T-cellresponses could contribute to protection following vaccination.A similar comparison of influenza strains has been performedusing T-cell epitope sequences deposited in the IEDB [44]. Thatpreliminary study reported 38 T-cell (combined CD4+ and CD8+)epitopes in HA-H1 seasonal flu with only four (11%) conservedin novel S-OIV and five epitopes in NA-N1 seasonal flu withonly one (20%) conserved. These data may underestimate thenumber of conserved epitopes as the study considered only pre-viously characterized epitopes. Depending on the design of thepeptides that are tested in vitro (length of the peptide sequence,

extent of overlap if overlapping peptides were tested), experi-mental studies do not necessarily identify all immunoreactiveepitopes. Compared to studies that use published epitopes as astarting point, we may have found a larger number of conservedepitopes due to our more focused approach (performing a com-

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rehensive in silico analysis first and a review of the literatureecond).

To be effective, an influenza vaccine must be designed andanufactured prior to the beginning of an influenza season or

andemic, and must induce immune responses that recognizehe circulating strains of influenza A and B [45]. Conventionalrivalent vaccines are developed based on annual WHO strain rec-mmendations. Hemagglutinin and neuraminidase are the majorurface glycoproteins of the influenza virus [46]. Humoral immu-ity against these two proteins is believed to be the primaryeans of resistance to and recovery from influenza virus infection

47,48], therefore these two antigens are included in the annualaccine.

Humoral immunity is the arm of the immune system involved inrotection from influenza. Secretory IgA and IgM provide protectiongainst the establishment of initial infection, however, IgG neutral-zes newly replicating virus once infection has been established49,50]. In addition, IgG prevents viral pathology in the (murine)ung. Humoral immunity to influenza is not, however, completelyrotective. Antibodies to HA can neutralize viral infectivity; butntigenic variation in this molecule is responsible for occasionalailure of the CIV and for the poor control of infection after immu-ization [48]. Antibodies to NA do not block infection, but theyesult in the reduction of pulmonary virus titers below a pathogenichreshold [48,51,52].

There is a great deal of evidence that T-cell epitopes are alsoxtremely important for protection against influenza. T helper cellslay a critical role in isotype-switching (the generation of IgG) and

n the generation of higher affinity antibodies [53]. The generationf effective antibody titers and CTL memory is not possible withoutognate T helper (Th) cells [54–56]. In humans, hemagglutinin-pecific T cells represent a major subset of proliferating CD4+ Telper cells following influenza vaccination [57] and can assistith the development of qualitatively distinct heterotypic influenza

ntibody responses [14,58]—in fact, the importance of cognate Telp to the elaboration of B cell responses was first characterized

n influenza [59–62]. Furthermore, T-cell response has a dramaticmpact on the manifestations of influenza, because the rate of virallearance depends on the presence of CD4+ T cells [63]. In additiono T helper immune responses, cytotoxic T cells (CD8+ cells) haveeen shown to be critically important for the clearance of influenza

nfection [64–66].Due to the absence of any evidence that cross-reactive antibod-

es were responsible for protecting older individuals from influenza,e reasoned that T-cell epitope analysis of the sequence of H1N1alifornia might reveal other mechanisms for the relative paucity ofisease among older, potentially vaccinated, or exposed individuals.he results demonstrate good cross-reactivity, suggesting that the

ack of pathology in older subjects may be due to cross-reactive T-ell response. Immunogenicity and cross-reactivity of the epitopesdentified here could easily be confirmed using blood samples from-OIV-exposed humans and CIV- or LAIV- vaccinated subjects whoave not yet been exposed to the novel virus. The T-cell epitoperedictions presented here are expected to be accurate based onrevious published experience [26,32].

Another means of testing this observation would be to evaluatehe effect of vaccination with synthetic versions of the epitopesescribed here against S-OIV challenge, in HLA transgenic mice.utbred murine models and other non-humanized animal modelsave been used to evaluate immunogenicity, however, animal MHCre not equivalent to human MHC (HLA). A number of humanized

ouse strains have been developed that express the most commonLA A, HLA B and HLA DR molecules [67]. A direct correlation hassually been found between T-cell responses in infected humansnd T-cell responses induced in immunized HLA transgenic mice68–71]. HLA transgenic mice are now routinely used to assay and

27 (2009) 5740–5747 5745

optimize (human) epitope-driven vaccines in pre-clinical studies[67,72,73].

4.1. Closing thoughts

There is an urgent unmet need for an influenza vaccine withgreater potency, durability of antibody response, and strain-cross-reactivity that can be developed more rapidly than CIV. The currentepidemic spread of H1N1 underscores the need to design vac-cines that are more effective against new strains. Influenza vaccinesdeveloped using the existing model are susceptible to failure sincesignificant HA and NA antigenic variation can occur in the time thatelapses from selection of the vaccine candidate strain and virusexposure [74]. Vaccine that have built-in cross-subtype efficacycould prevent significant spread of an emerging or re-emergingstrain. A cross-subtype vaccine containing immunogenic consensussequence (ICS) epitopes could achieve this goal.

Immunoinformatics tools such as the tools used for this reportcan also be used to design improved epitope-based vaccinesfor influenza. Highly immunogenic, cross-conserved epitopes canbe designed by carefully overlapping conserved and immuno-genic 9-mer sequences found in the influenza strains of interest.We used immunogenic consensus sequence” (ICS) epitopes todevelop an HIV vaccine and a smallpox vaccine and found that theimmune responses generated by these epitopes surpassed any pre-vious immune responses to non-ICS peptides [unpublished EpiVaxdata,32]. We recently tested the smallpox epitopes in the context ofmucosal vaccinia challenge, and found that ICS epitopes, conservedbetween Vaccinia and Variola, completely protected mice—in theabsence of any discernable antibody raised by vaccination.

We therefore believe that ICS epitopes derived from the novelpandemic H1N1 strain and other previously circulating strains (vac-cine or other) could be developed and used, in the short term, forprotective effect, in the case of a shortage of CIV vaccine supply. Or,alternatively, until such time as a new influenza vaccine containingHA and NA from the circulating H1N1 is available, it may be suffi-cient to vaccinate against influenza using one of the versions of theexisting flu vaccine. Our preference would be for live-attenuatedvaccination (in the absence of a matched CIV for S-OIV), since therewould be a higher chance of cross-protection by CTL. And in themeantime, the current epidemic provides a lesson for the future:the process of CIV vaccine development is too slow for a seriouslyvirulent pandemic challenge. It would be wise to use this pandemicto design better, safer, more effective influenza vaccines.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.vaccine.2009.07.040.

References

[1] Centers for Disease Control and Prevention (CDC). Swine Influenza A (H1N1)infection in two children—Southern California. MMWR Morb Mortal Wkly Rep2009;58(April (15)):400–2. March–April 2009.

[2] Centers for Disease Control and Prevention (CDC). Update: infections witha swine-origin influenza A (H1N1) virus—United States and other countries.MMWR Morb Mortal Wkly Rep 2009;58(May (16)):431–3. April 28, 2009.

[3] Centers for Disease Control and Prevention. Serum antibody response to a novelinfluenza A (H1N1) virus after vaccination with seasonal influenza vaccine.MMWR Morb Mortal Wkly Rep 2009;58(19):521–4.

[4] Centers for Disease Control and Prevention (CDC). Update: drug susceptibilityof swine-origin influenza A (H1N1) viruses, April 2009. MMWR Morb Mortal

Wkly Rep 2009;58(May (16)):433–5.

[5] World Health Organization. Outbreak news. Wkly Epidemiol Rec2009;84(18):149–60.

[6] Centers for Disease Control and Prevention (CDC). Prevention & control ofinfluenza—Recommendations of the Advisory Committee on ImmunizationPractices (ACIP) 2008. MMWR 2008;57(August (RR07)):1–60.

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746 A.S. De Groot et al. / Va

[7] Gallaher WR. Towards a sane and rational approach to management of InfluenzaH1N1 2009. Virol J 2009;6:51 [Epub ahead of print].

[8] Boon AC, de Mutsert G, van Baarle D, Smith DJ, Lapedes AS, Fouchier RA, etal. Recognition of homo- and heterosubtypic variants of influenza A viruses byhuman CD8+ T lymphocytes. J Immunol 2004;172:2453–60.

[9] Kreijtz JH, Bodewes R, van Amerongen G, Kuiken T, Fouchier RA, Osterhaus AD,et al. Primary influenza A virus infection induces cross-protective immunityagainst a lethal infection with a heterosubtypic virus strain in mice. Vaccine2007;25:612–20.

10] Kamperschroer C, Dibble JP, Meents DL, Schwartzberg PL, Swain SL. SAPis required for Th cell function and for immunity to influenza. J Immunol2006;177:5317–27.

11] Belz GT, Wodarz D, Diaz G, Nowak MA, Doherty PC. Compromised influenzavirus-specific CD8(+)-T-cell memory in CD4(+)-T-cell deficient mice. J Virol2002;76:12388–93.

12] Cardin RD, Brooks JW, Sarawar SR, Doherty PC. Progressive loss of CD8+ T cell-mediated control of a gamma-herpesvirus in the absence of CD4+ T cells. J ExpMed 1996;184:863–71.

13] Brooks JW, Hamilton-Easton AM, Christensen JP, Cardin RD, Hardy CL, DohertyPC. Requirement for CD40 ligand, CD4(+) T cells, and B cells in an infectiousmononucleosis-like syndrome. J Virol 1999;73:9650–4.

14] Marshall D, Sealy R, Sangster M, Coleclough C. TH cells primed during influenzavirus infection provide help for qualitatively distinct antibody responses tosubsequent immunization. J Immunol 1999;163:4673–82.

15] Lee LY, Ha do LA, Simmons C, de Jong MD, Chau NV, Schumacher R, et al. MemoryT cells established by seasonal human influenza A infection cross-react withavian influenza A (H5N1) in healthy individuals. J Clin Invest 2008;118:3478–89.

16] Townsend AR, Skehel JJ. The influenza A virus nucleoprotein gene controls theinduction of both subtype specific and cross-reactive cytotoxic T cells. J ExpMed 1984;160:552–63.

17] Assarsson E, Bui HH, Sidney J, Zhang Q, Glenn J, Oseroff C, et al. Immunomicanalysis of the repertoire of T-cell specificities for influenza A virus in humans.J Virol 2008;82(24):12241–51.

18] Lin HH, Zhang GL, Tongchusak S, Reinherz EL, Brusic V. Evaluation of MHC-II peptide binding prediction servers: applications for vaccine research. BMCBioinformatics 2008;9(December (Suppl. 12)):S22.

19] Lin HH, Ray S, Tongchusak S, Reinherz EL, Brusic V. Evaluation of MHC classI peptide binding prediction servers: applications for vaccine research. BMCImmunol 2008;9(March):8.

20] De Groot AS, Jesdale BM, Szu E, Schafer JR. An interactive web site providing MHCligand predictions: application to HIV research. AIDS Res Hum Retroviruses1997;13:539–41.

21] Schafer JA, Jesdale BM, George JA, Kouttab NM, De Groot AS. Prediction of well-conserved HIV-1 ligands using a Matrix-based Algorithm, EpiMatrix. Vaccine1998;16(19):1880–4.

22] Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O, Sahin U, et al. Generation oftissue-specific and promiscuous HLA ligand databases using DNA microarraysand virtual HLA class II matrices. Nat Biotechnol 1999;17:555–61.

23] De Groot AS, Knopf PM, Martin B. De-immunization of therapeutic proteins byT cell epitope modification. In: Mire-Sluis, A, editor. State of the art analyti-cal methods for the characterization of biological products and assessment ofcomparabilitiy. Dev Biol. Karger: Basel; 2005; 122. p. 137–160.

24] De Groot AS, McMurry J, Marcon L, Franco J, Rivera D, Kutzler M, et al. Developingan epitope-driven tuberculosis (TB) vaccine. Vaccine 2005;23:2121–31.

25] Moise L, McMurry JA, Buus S, Frey S, Martin WD, De Groot AS. In silico-accelerated identification of conserved and immunogenic variola/vacciniaT-cell epitopes. Vaccine (David Weiner, special editor); 2009. [Epub ahead ofprint].

26] De Groot AS, Martin W. Reducing risk, improving outcomes: bioengineering lessimmunogenic protein therapeutics. Clin Immunol 2009;131(May (2)):189–201.

27] Meister GE, Roberts CG, Berzofsky JA, De Groot AS. Two novel T cell epitopeprediction algorithms based on MHC-binding motifs; comparison of predictedand published epitopes from Mycobacterium tuberculosis and HIV proteinsequences. Vaccine 1995;(6):581–91.

28] Bond KB, Sriwanthana B, Hodge TW, De Groot AS, Mastro TD, Young NL, et al. AnHLA-directed molecular and bioinformatics approach identifies new HLA-A11HIV-1 subtype E cytotoxic T lymphocyte epitopes in HIV-1-infected Thais. AIDSRes Hum Retroviruses 2001;17(8):703–17.

29] Dong Y, Dimaria S, Sun X, Jesdale BM, De Groot AS, Rom WN, et al. HLA-A2-restricted CD8+ cytotoxic T cell responses to novel Mycobacterium tuberculosistargets superoxide dismutase and alanine dehydrogenase. Infect Immun2004;72(4):2412–5.

30] McMurry JA, Gregory SH, Moise L, Rivera D, Buus S, De Groot AS. Diversity ofFrancisella tularensis Schu4 antigens recognized by T lymphocytes after naturalinfections in humans: identification of candidate epitopes for inclusion in arationally designed tularemia vaccine. Vaccine 2007;25:3179–91.

31] Otero M, Calarota SA, Dai A, De Groot AS, Boyer JD, Weiner DB. Efficacy ofnovel plasmid DNA encoding vaccinia antigens in improving current smallpoxvaccination strategy. Vaccine 2006;24:4461–70.

32] De Groot AS, Rivera DS, McMurry JA, Buus S, Martin W. Identification of

immunogenic HLA-B7 “Achilles’ heel” epitopes within highly conserved regionsof HIV. Vaccine 2008;26:3059–71.

33] Lichterfeld M, Williams KL, Mui SK, Shah SS, Mothe BR, Sette A, et al. Tcell receptor cross-recognition of an HIV-1 CD8+ T cell epitope presented byclosely related alleles from the HLA-A3 superfamily. Int Immunol 2006;18(July(7)):1179–88.

[

[

27 (2009) 5740–5747

34] Frahm N, Korber BT, Adams CM, Szinger JJ, Draenert R, Addo MM, et al. Consis-tent cytotoxic-T-lymphocyte targeting of immunodominant regions in humanimmunodeficiency virus across multiple ethnicities. J Virol 2004;78:2187–200.

35] De Groot AS, Berzofsky JA. From genome to vaccine—new immunoinformaticstools for vaccine design. Methods 2004;34:425–8.

36] Sette A, Sidney J. Nine major HLA class I supertypes account for the vast prepon-derance of HLA-A and -B polymorphism. Immunogenetics 1999;50:201–12.

37] Sette A, Sidney J. HLA supertypes and supermotifs: a functional perspective onHLA polymorphism. Curr Opin Immunol 1998;10:478–82.

38] Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, LanzavecchiaA. Universally immunogenic T cell epitopes: promiscuous recognition by T cells.Eur J Immunol 1989;19:2237–42.

39] De Groot AS, Clerici M, Hosmalin A, Hughes SH, Barnd D, Hendrix CW, et al.Human Immunodeficiency virus reverse transcriptase T helper epitopes iden-tified in mice and humans: correlation with a cytotoxic T cell epitope. J InfectDis 1991;164:1058–65.

40] Betts MR, Ambrozak DR, Douek DC, Bonhoeffer S, Brenchley JM, Casazza JP, etal. Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) andCD8(+) T cell responses: relationship to viral load in untreated HIV infection. JVirol 2001;75(24):11983–91.

41] Gianfrani C, Oseroff C, Sidney J, Chesnut RW, Sette A. Human memory CTLresponse specific for influenza A virus is broad and multispecific. Hum Immunol2000;61:438–52.

42] Jameson J, Cruz J, Ennis FA. Human cytotoxic T-lymphocyte repertoire toinfluenza A viruses. J Virol 1998;72:8682–9.

43] Zhang Q, Wang P, Kim Y, Haste-Andersen P, Beaver J, Bourne PE, et al. Immuneepitope database analysis resource (IEDB-AR). Nucleic Acids Res 2008;36(July(Web Server issue)):W513–8.

44] Fleri, W. (2009). Swine Flu Analysis. Retrieved August 4, 2009, from ImmuneEpitope Database Knowledgebase and Forums. Website: http://iedb.zendesk.com/forums/21519/entries/39712.

45] Kilbourne ED. What are the prospects for a universal influenza vaccine? NatMed 1999;5(October (10)):1119–20.

46] Choppin P, Tamm I. Studies of two kinds of virus particles which compriseinfluenza A2 virus strains. Reactivity with virus inhibitors in normal sera. J ExpMed 1960;112:921.

47] Epstein SL, Misplon JA, Lawson CM, Subbarao EK, Connors M, Murphy BR. Beta 2-microglobulin-deficient mice can be protected against influenza A infection byvaccination with vaccinia-influenza recombinants expressing hemagglutininand neuraminidase. J Immunol 1993;150(June (12)):5484–93.

48] Johansson B, Bucher D, Kilbourne E. Purified influenza virus hemagglutinin andneuraminidase are equivalent in stimulation of antibody response but inducecontrasting types of immunity to infection. J Virol 1989;63:1239.

49] Murphy BR, Nelson DL, Wright PF, Tierney EL, Phelan MA, Chanock RM. Secretoryand systemic immunological response in children infected with live attenuatedinfluenza A virus vaccines. Infect Immun 1982;36:1102–8.

50] Burlington DB, Clements ML, Meiklejohn G, Phelan M, Murphy BR.Hemagglutinin-specific antibody responses in immunoglobulin G, A, and Misotypes as measured by enzyme-linked immunosorbent assay after primaryor secondary infection of humans with influenza A virus. Infect Immun1983;41:540–5.

51] Compans R, Klenk H-D, Caliguiri L, Choppin P. Influenza virus proteins. Analysisof polypeptides of the virion and identification of spike glycoproteins. Virology1970;42:880.

52] Kilbourne ED. Comparative efficacy of neuraminidase-specific and conventionalinfluenza virus vaccines in induction of antibody to neuraminidase in humans.J Infect Dis 1976;134:385.

53] Paul WE, Benacerraf B. Functional specificity of thymus-dependent lympho-cytes. Science 1977;195:1293.

54] Shedlock DJ, Shen H. Requirement for CD4 T cell help in generating functionalCD8 T cell memory. Science 2003;300:337–9.

55] Ngo-Giang-Huong N, Candotti D, Goubar A, Autran B, Maynart M, Sicard D, et al.HIV type 1-specific IgG2 antibodies: markers of helper T cell type 1 response andprognostic marker of long-term nonprogression. AIDS Res Hum Retroviruses2001;17:1435–46.

56] Heeney JL. Requirement of diverse T-helper responses elicited by HIV vaccines:induction of highly targeted humoral and CTL responses. Expert Rev Vaccines2004;3(August (4 Suppl.)):S53–64.

57] Novak EJ, Liu AW, Nepom GT, Kwok WW. MHC class II tetramers identifypeptide-specific human CD4(+) T cells proliferating in response to influenzaA antigen. J Clin Invest 1999;104(12):R63–7.

58] Kamperschroer C, Dibble JP, Meents DL, Schwartzberg PL, Swain SL. SAP IsRequired for Th Cell Function and for Immunity to Influenza. J Immunol2006;177:5317–27.

59] Scherle PA, Gerhard W. Functional analysis of influenza-specific helper Tcell clones in vivo. T cells specific for internal viral proteins provide cog-nate help for B cell responses to hemagglutinin. J Exp Med 1986;164(4):1114–28.

60] Scherle PA, Gerhard W. Differential ability of B cells specific for external vs.internal influenza virus proteins to respond to help from influenza virus-specific

T-cell clones in vivo. Proc Natl Acad Sci USA 1988;85:4446–50.

61] Russell SM, Liew FY. T cells primed by influenza virion internal compo-nents can cooperate in the antibody response to haemagglutinin. Nature1979;280(5718):147–8.

62] Johansson BE, Moran TM, Kilbourne ED. Antigen-presenting B cells and helperT cells cooperatively mediate intravirionic antigenic competition between

ccine

[

[

[

[

[

[

[

[

[

[

A.S. De Groot et al. / Va

influenza A virus surface glycoproteins. Proc Natl Acad Sci USA 1987;84(October(19)):6869–73.

63] Belz GT, Wodarz D, Diaz G, Nowak MA, Doherty PC. Compromised influenzavirus-specific CD8+-T-cell memory in CD4+-T-cell-deficient mice. J Virol2002;76:12388–93.

64] Yap KL, Braciale TJ, Ada GL. Role of T-cell function in recovery from murineinfluenza infection. Cell Immunol 1979;43(2):341–51.

65] Yap KL, Ada GL, McKenzie IF. Transfer of specific cytotoxic T lymphocytes pro-tects mice inoculated with influenza virus. Nature 1978;273(5659):238–9.

66] McMichael AJ, Gotch FM, Noble GR, Beare PA. Cytotoxic T-cell immunity toinfluenza. NEJM 1983;309:13–7.

67] Charo J, Sundback M, Geluk A, Ottenhoff T, Kiessling R. DNA immunization ofHLA transgenic mice with a plasmid expressing mycobacterial heat shock pro-

tein 65 results in HLA class I- and II-restricted T cell responses that can beaugmented by cytokines. Hum Gene Ther 2001;12(14):1797–804.

68] Shirai M, Arichi T, Nishioka M, Nomura T, Ikeda K, Kawanishi K, et al.CTL responses of HLA-A2.1-transgenic mice specific for hepatitis C viralpeptides predict epitopes for CTL of humans carrying HLA-A2. J Immunol1995;154:2733–42.

[

[

27 (2009) 5740–5747 5747

69] Le A-XT, Bernhard EJ, Holterman MJ, Strub S, Parham P, Lacy E, et al. Cytotoxic Tcell responses in HLA-A2.1 transgenic mice: recognition of HLA alloantigens andutilization of HLA-A2. 1 as a restriction element. J Immunol 1989;142:1366–71.

70] Didierlaurent A, Ramirez JC, Gherardi M, Zimmerli SC, Graf M, Orbea HA,et al. Attenuated poxviruses expressing a synthetic HIV protein stimu-late HLA-A2-restricted cytotoxic T-cell responses. Vaccine 2004;22(25–26):3395–403.

71] Man S, Newberg MH, Crotzer VL, Luckey CJ, Williams NS, Chen Y, et al. Definitionof a human T cell epitope from influenza A non-structural protein 1 using HLA-A2.1 transgenic mice. Int Immunol 1995;7:597–605.

72] Ishioka GY, Fikes J, Hermanson G, Livingston B, Crimi C, Qin M, et al. Utilization ofMHC class I transgenic mice for development of minigene DNA vaccines encod-ing multipleHLA-restricted CTL epitopes. J Immunol 1999;162(7):3915–25.

73] Livingston BD, Newman M, Crimi C, McKinney D, Chesnut R, Sette A. Opti-mization of epitope processing enhances immunogenicity of multiepitope DNAvaccines. Vaccine 2001;19(September (32)):4652–60.

74] Besselaar TG, Botha L, McAnerney JM, Schoub BD. Antigenic and molecularanalysis of influenza A (H3N2) virus strains isolated from a localised influenzaoutbreak in South Africa in 2003. J Med Virol 2004;73:71–8.