Vaccine 26 (2008) 5381–5388

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Live, attenuated influenza virus (LAIV) vehicles are stronginducers of immunity toward influenza B virus

Victor C. Huber1, Loren H. Kleimeyer, Jonathan A. McCullers ∗

Department of Infectious Disease, St. Jude Children’s Research Hospital, 332 N. Lauderdale Street, Memphis, TN 38105-2794, USA

a r t i c l e i n f o

Article history:Received 10 June 2008Received in revised form 21 July 2008Accepted 29 July 2008Available online 15 August 2008

Keywords:InfluenzaDNA vaccineHemagglutinin

a b s t r a c t

Historically, vaccines developed toward influenza viruses of the B type using methodologies developed forinfluenza A viruses as a blueprint have not been equally efficacious or effective. Because most influenzaresearch and public attention concerns influenza A viruses, these shortcomings have not been adequatelyaddressed. In this manuscript, we utilized different influenza vaccine vehicles to compare immunogenicityand protection in mice and ferrets after vaccination against an influenza B virus. We report that plas-mid DNA vaccines demonstrate low immunogenicity profiles and poor protection compared to eitherwhole, inactivated influenza virus (IIV) or, live, attenuated influenza virus (LAIV) vaccines. When mixedprime:boost regimens using LAIV and IIV were studied, we observed a boosting effect in mice after prim-ing with LAIV that was not seen when IIV was used as the prime. In ferrets LAIV induced high antibody

titers after a single dose and provided a boost in IIV-primed animals. Regimens including LAIV as a primedemonstrated enhanced protection, and adjuvantation was required for efficacy using the IIV preparation.Our results differ from generally accepted influenza A virus vaccine models, and argue that strategies for

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. Introduction

Approximately 36,000 deaths and 200,000 hospitalizations arettributed to influenza illness in the US on an annual basis [1,2].accination represents the most promising method for controlling

nfluenza, and vaccines against this virus have a history of moderatefficacy [3–5]. The major factor affecting influenza vaccine efficacynd effectiveness is the ability to accurately predict representativesolates of the circulating subtypes for inclusion in annual vaccines4,6]. Incorrect predictions can result in vaccines that have signif-cantly reduced effectiveness, a problematic situation that mostecently occurred during the 2007–2008 influenza season [7,8].

Seasons where influenza B viruses represent the dominant cir-ulating strain are infrequent, and the resulting excess mortalityttributed to influenza B has typically been less than that due tonfluenza A viruses, specifically those of the H2N2 or H3N2 subtypes

9–11]. In addition, influenza B viruses exhibit a limited host range12], thus precluding development of pandemic-type disease. Theseactors combined have led to less interest in influenza B viruses thann influenza A viruses, and less study has been directed toward this

∗ Corresponding author. Tel.: +1 901 495 3486; fax: +1 901 495 3099.E-mail address: jon.mccullers@stjude.org (J.A. McCullers).

1 Current address: Division of Basic Biomedical Sciences, Sanford School ofedicine, University of South Dakota, Vermillion, SD 57069, USA.

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uld be considered separately from those for influenza A virus.© 2008 Elsevier Ltd. All rights reserved.

ype. Nevertheless, the clinical symptoms of seasonal influenza And B infections are indistinguishable [13–15], and significant mor-idity and mortality [1,2] as well as other complications [16–18], areecorded during influenza B virus epidemics.

Current influenza vaccine strategies were designed withnfluenza A viruses as model antigens [19–21], and it has beenssumed that what works for influenza A will also work fornfluenza B [21]. However, the epidemiology and evolution ofnfluenza A and B viruses differ in several key areas, calling thismplied assumption into question. The HA of influenza B virusesas evolved differently and more gradually than that of influenza Airuses circulating in humans, and the resulting slower rate of anti-enic drift has led to less frequent seasonal changes in antigenicity22–24]. In addition, multiple strains from different lineages ofnfluenza B virus are known to co-circulate, often with differentemporal and geographic patterns [25,26]. In the last decade, strainsxpressing HAs from lineages II and III (generally represented bytrains B/Yamagata/16/88 and B/Victoria/2/87, respectively) haveo-circulated in many parts of the world [25], making predictionf the best strain for inclusion in the vaccine difficult for anyarticular region or season. This was problematic in 2007–2008,

hen 98% of circulating influenza B virus strains were from lineage

I, while the vaccine recommended for the Northern hemisphereas derived from a lineage III virus [7]. Further complicating mat-

ers, inactivated and live attenuated influenza virus (IIV and LAIV,espectively) vaccines in current use show consistently reduced

5382 V.C. Huber et al. / Vaccine 26 (2008) 5381–5388

Table 1Properties of reassortant LAIV expressing BYam98 HA

Temperature-sensitive phenotype of LAIV in vitro

Virus Titer (log10 TCID50/mLa at 37 ◦C) Titer (log10 TCID50/mLa at 33 ◦C)

BYam98ts <3.00 7.125

Attenuated phenotype of BYam98ts in vivob

Weight (g) (change from day 0) Temperature (◦F) (change from day 0) Day 3 viral titersc (log10 TCID50/mL)

Day 0 Day 1 Day 2 Day 3 Day 0 Day 1 Day 2 Day 3 Nasal wash Lung

Ferret 1 1514 1493 (−21) 1526 (+12) 1498 (−16) 100.4 101.5 (+1.1) 100.8 (+0.4) 100.9 (+0.5) 4.375 <1.00Ferret 2 1664 1699 (+35) 1684 (+20) 1697 (+33) 100.9 100.6 (−0.3) 101.4 (+0.5) 101.2 (+0.3) 4.625 <1.00

a TCID50 determined using MDCK cells.

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mmunogenicity for influenza B virus, compared to influenza Airus [27–29].

Based on these differences in evolution and vaccine effective-ess between influenza A and B viruses, we hypothesized thathe most effective vaccines for influenza A viruses are not nec-ssarily the ideal vaccines for influenza B viruses. We thereforeecided to test multiple routes and vehicles for delivery of influenzavirus HA to directly compare regimens for eliciting immunity

gainst this type of influenza. We compared vaccine vehicles thatould induce antibodies exclusively toward the HA component of

nfluenza (DNA) to whole virus preparations that incorporate addi-ional viral proteins (IIV and LAIV). Furthermore, we included alums an adjuvant for relevant groups in order to address immuno-enicity in the presence of this adjuvant, which is the only adjuvanturrently approved for use by the FDA [30]. Our data demonstratehat differences exist between these vehicles in relevant animal

odels of influenza infection. These data are discussed in the con-ext of rational design for vaccines against influenza B virus.

. Materials and methods

.1. Animals

Adult (6–8-week old) female BALB/cJ mice were obtained fromackson Laboratories (Bar Harbor, ME, USA) and housed in groupsf four to five mice as described previously [31]. Young adult fer-ets were obtained from the St. Jude Children’s Research Hospitalreeding program. All animal experiments were performed follow-

ng guidelines established by the Animal Care and Use Committeet St. Jude Children’s Research Hospital (Memphis, TN, USA).

.2. Construction of reassortant viruses

Influenza B virus genes cloned into plasmid pHW2000 wererovided by Drs. Erich Hoffman and Robert G. Webster (St. Judehildren’s Research Hospital). The B/Yamanashi/166/98 (BYam98;

ineage II with an HA related to B/Yamagata/16/88) HA (GenBankccession number AF100355) differed from the published sequencet position N196D (A586G). Viruses were created using reverseenetics as described previously using all eight gene segmentsrom BYam98 [32,33]. Influenza virus, rescued from MDCK:293To-culture, was propagated in the presence of TPCK-trypsin using

onfluent MDCK monolayers, and sequencing was performed toonfirm appropriate genotypes for rescued virus (Hartwell Cen-er, St. Jude Children’s Research Hospital). Phenotypes of rescuediruses were characterized in MDCK cells (TCID50) and in miceMLD50) as described below.

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As an additional vehicle for the delivery of influenza HA toice, viruses demonstrating an LAIV phenotype were created using

lasmids mutated at specific sites in three BYam98 internal genesNP; CY019534, PA; AF102024, and M; AF100392). These previouslydentified mutations in NP (V114A, T341C and G342A and P410H,1229A), PA (V431M, G1291A and Y497H, T1489C), and M (H159Q,477A and M183V, A547G and G549C) [34] were inserted usingite-directed mutagenesis (Stratagene, La Jolla, CA, USA). Geneticnalyses confirmed the genetic makeup of the virus, the ts pheno-ype was confirmed in MDCK cells, and the attenuated phenotypeas confirmed in ferrets (Table 1).

An influenza B virus that is lethal in mice has recently beenescribed [33]. The lethality of this virus is due to a single mutation

n the BYam98 M gene (AF100392) of the virus (N221S, A662G and663C), which we mutated using site-directed mutagenesis (Strata-ene), and the virus was created using reverse genetics as describedbove. This virus stock had a titer of 108.25 TCID50/mL in MDCK cellst 33 ◦C, and had an LD50 of 106.375 TCID50 in mice.

.3. Plasmid DNA inoculation

Plasmid DNA expressing BYam98 HA was maxi-prepped (Qiagennc., Valencia, CA, USA) and bound to 1 micron gold beads (Bio-Rad,ercules, CA, USA) as described [31]. When vector DNA (pHW2000)as delivered as a control, 2.4 �g vector DNA was bound per mg

old. When BYam HA-DNA was delivered, 1.6 �g vector DNA per mgold was mixed with 0.8 �g of the BYam HA-DNA to maintain a totalf 2.4 �g DNA per mg gold. In all instances, the individual DNA com-onents were mixed thoroughly prior to addition to gold particles.NA-coated gold particles were propelled onto the bare abdomenf anesthetized BALB/c mice using a Helios gene gun (Bio-Rad). Twoon-overlapping shots of 0.5 mg gold from the gene gun (2.4 �gNA on 1 mg gold) were administered twice at four-week inter-als. When plasmid DNA was delivered i.m., vector DNA (100 �g perouse) or BYam HA-DNA (100 �g per mouse) was injected into the

eft rear quadriceps in a 100 �L volume twice at four-week intervals.

.4. LAIV inoculation

Mice were inoculated with 5 × 105 TCID50 (33 ◦C) BYam98A-expressing viruses containing ts- and attenuated phenotypes

Table 1), created as described above. LAIV were delivered in

50 �L volume (25 �L per nostril) twice at four-week inter-

als. Ferrets were inoculated with 1 × 107 TCID50 (33 ◦C) BYam98A-expressing viruses containing ts- and attenuated phenotypes,reated as described above, delivered twice in a 1 mL volume500 �L per nostril).

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.5. IIV preparation and inoculation

MDCK-grown wild-type BYam98 virus was concentrated, puri-ed over a sucrose gradient, inactivated with formalin, and HAontent was quantitated as described previously [31]. Mice wereaccinated with 3 �g HA in 100 �L volume i.m. in the right rearuadriceps twice at four-week intervals. When Alum (Reheis,erkeley Heights, NJ, USA) was included as an adjuvant, it wasdded at a concentration of 2 mg/mL. Ferrets were inoculated with5 �g HA in 250 �L volume in the right rear quadriceps twice atour-week intervals.

.6. Serum collection and treatment

Clotted blood collected from either the retro-orbital plexusf anesthetized mice or the internal mammary vein of anes-hetized ferrets 21 days after each vaccination were centrifugedor 10 min at 6000 × g. Serum (100 �L) was treated with 300 �Leceptor-destroying enzyme (RDE) as described by the manufac-urer (Accurate Chemical & Scientific Corp., Westbury, NY, USA).fter addition of equal volumes (300 �L) of 2.5% (v/v) sodium cit-ate and PBS, sera were used in assays to determine vaccine efficacy.

.7. ELISA

96-well plates (Becton Dickinson and Company, Franklin Lakes,J, USA) were coated with concentrated BYam98 wild-type virus

1 �g HA mL−1) and incubated overnight at 4 ◦C. Plates wereashed with PBS containing 0.05% (v/v) Tween-20 (Sigma) (PBST)

nd blocked with 10% FBS in PBST (FBS-PBST) for 2 h at RT.DE-treated sera was serially diluted in FBS-PBST and incubatedvernight at 4 ◦C. Plates were washed and alkaline phosphatase-onjugated goat anti-mouse IgG (�-specific), IgG1, and IgG2aSouthern Biotech, Birmingham, AL, USA) antibodies diluted in FBS-BST were added to the plates and incubated for 2h. Plates wereashed, and 1 mg mL−1 p-nitrophenyl phosphate substrate (Sigma)

n diethanolamine buffer was added. 1 h after substrate addition,D was read at 405 nm using a Multiskan Ascent® plate reader (Lab-

ystems, Helsinki, Finland). Reactivity of ferret sera was determinedimilarly, using individual 2 h incubations with unconjugated goatnti-ferret IgG (H + L) (Bethyl Laboratories, Inc., Montgomery, TX,SA) and alkaline-phosphatase-conjugated rabbit anti-goat IgG

H + L) (Bethyl Laboratories, Inc.), both diluted in FBS-PBST, prior toubstrate addition as described above. Reciprocal serum antibodyiters were calculated at 50% maximal binding.

.8. Virus challenge

Four weeks after receiving the final dose of respective vaccine,ice were challenged i.n. with 7.5 MLD50 (107.25 TCID50) in a 100 �L

olume (50 �L per nostril) reassortant BYam98 viruses containinghe M1 mutation (N221S) created as described above. Four weeksfter final dose of respective vaccine, ferrets were similarly chal-enged i.n. with 105.00 TCID50 in 1 mL (500 �L per nostril). Ferrets

ere monitored daily for signs of clinical illness as described pre-iously [35] and mice were monitored daily for morbidity (weightoss) and mortality (survival). Mice that lost more than 30% of theirnitial body weight were euthanized and recorded as dying on theollowing day.

.9. Nasal wash viral titers

Nasal wash was collected from LAIV-inoculated ferrets on days, 3, 5, and 12 after primary LAIV inoculation and on days 1, 3, andafter secondary LAIV inoculation. Nasal wash was also collected

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6 (2008) 5381–5388 5383

rom all ferrets daily on days 1–6, and on day 9 after challengeith BYam98 virus. Nasal wash was collected as effluvium into

0-mL tubes (Corning) after instillation of 500 �L PBS per nos-ril, and exposed to MDCK cells for virus propagation as describedreviously [35]. Briefly, nasal wash was diluted 10-fold in MDCK

nfection media, and MDCK monolayers (washed twice with ster-le PBS) were incubated with diluted nasal wash samples for 1 h.he inoculum was then removed, MDCK infection media contain-ng 1 �g mL−1 TPCK-trypsin (Worthington) was added, and cells

ere incubated for four days at 33 ◦C, 5% CO2. Cells were observedor cytopathic effect, and infection within individual wells was con-rmed using a standard HA assay. Lung viral titers were calculatedsing the Reed–Muench method. Nasal wash was plated begin-ing with 100 �L undiluted effluvium through a 1:100,000 dilution10−1 through 10−6 TCID50/mL). The minimum titer detectable byhis assay was 101, and the maximum titer was 106.50.

. Results

.1. Comparison of vehicles for HA delivery administered usingwo-dose identical or mixed vaccination regimens

To directly compare immunogenicity of different B HA deliveryehicles in a naïve population, we administered combinations ofve vaccines twice either using identical vaccines (Fig. 1A) or mix-

ng delivery vehicles (Fig. 1B). The choice to expose animals to HAwice was based on the current recommendations for vaccinationf the naïve human population (<9 years of age) [36]. Delivery ofnfluenza B virus HA using identical vehicles demonstrated LAIVnd whole virus IIV vaccines elicited similar influenza-specific IgGntibody titers. Low or negative IgG responses were detected afteraccination with DNA vaccines delivered by two different routes.ddition of adjuvant (Alum) to IIV preparations enhanced immuno-enicity compared to whole virus vaccine without alum. IgG1 andgG2a titers from the IIV + Alum group were of a similar magnitude,

ith no evidence of skewing toward IgG1 as has been reportedor the influenza A virus HA [37]. However, delivery of IIV withoutlum was skewed toward IgG2a, with lower levels of IgG1 detected.imilar to IIV + Alum, LAIV induced both IgG1 and IgG2a antibod-es, whereas HA-DNA delivered via the gene gun (HA-DNA (GG))emonstrated low levels of antibodies, mostly of the IgG1 isotype.A-DNA delivered i.m. (HA-DNA(IM)) did not induce a significant

gG antibody response (Fig. 1A), even in the presence of Alum (dataot shown). Using a mixed delivery regimen (Fig. 1B), IIV followedy LAIV resulted in modest titers, similar to those seen with 2 dosesf IIV alone, while LAIV priming before IIV delivery engenderedmore robust response. LAIV demonstrated an ability to prime

he immune response for a secondary boost by IIV, evidenced asncreases in IgG (�-specific), IgG1, and IgG2a titers, while priming

ith IIV did not allow for a significant boost of the IgG isotypesssayed.

.2. Protection of vaccinated mice from lethal influenza virushallenge

To directly assess the fitness of the these immune responsesn controlling a lethal influenza infection, mice were challenged

ith 7.5 MLD50 of a lethal influenza B virus expressing the BYam98A on its surface (Fig. 2) [33]. The groups most greatly affected by

his challenge dose were those that received either vehicle (PBS or

lum) or HA-DNA (IM), even when Alum was added to the HA-DNAreparation (data not shown). None of these groups had demon-trated high antibody titers after inoculation (Fig. 1A). Seven of theight mice inoculated with Alum exhibited weight loss identical tohe group inoculated with PBS, and these seven mice succumbed

5384 V.C. Huber et al. / Vaccine 26 (2008) 5381–5388

Fig. 1. Vaccination against influenza B virus with identical and mixed delivery vehicles induces IgG immunity in BALB/c mice. Sera taken from mice prior to vaccination (Pre),three weeks after primary (Pri), and three weeks after secondary (Sec) inoculation with influenza B virus HA expressed by identical (A) or mixed (B) vectors were analyzedfor IgG isotypes (IgG (�-specific), IgG1, or IgG2a) by ELISA. ELISA titers are reported as the reciprocal serum dilution representing 50% maximal binding on the titration curve.T t each( , n = 17n ).

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iters are reported as the mean ± standard deviation for individual groups of mice aIM) (�), n = 18; HA-DNA (GG) + HA-DNA (GG) (�), n = 18; IIV (Alum) + IIV (Alum) (�)= 11), except for Pre and Pri titers reported for the group receiving IIV + LAIV (n = 14

o the infection. The remaining mouse in this group did not exhibitorbidity or mortality after inoculation, and is not believed to have

eceived the entire inoculation dose. In agreement with the anti-

ody responses observed, mice that were vaccinated with LAIV asprime, followed by either LAIV or IIV, achieved maximal survival

100%). Alternatively, the groups that were primed with IIV (andoosted with either IIV or LAIV) demonstrated 50–60% survival, cor-esponding with the moderate humoral immunity induced before

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ig. 2. Vaccination against influenza B virus with identical and mixed delivery vehicles inA antigen expressed in multiple delivery vehicles, mice were challenged with 7.5 MLD50 Bf initial body weight) and mortality (percentage of survival) are reported for individual gIM) (�), n = 8; HA-DNA (GG) + HA-DNA (GG) (�), n = 8; IIV (Alum) + IIV (Alum) (�), n = 8= 11).

time-point (Alum + Alum (♦), n = 18; PBS + PBS (�), n = 11; HA-DNA (IM) + HA-DNA; IIV + IIV (�), n = 11; LAIV + LAIV (�), n = 18; IIV + LAIV (�), n = 11; and LAIV + IIV (�),

hallenge, in particular the lower IgG1 titers observed (Fig. 1). Addi-ion of Alum to the IIV preparation led to enhanced survival (100%).he group that received HA-DNA (GG) showed a low level of protec-

ion after challenge (25%), which corresponded with the low level ofmmunity (mostly IgG1) demonstrated after vaccination. The poorrotective efficacy of DNA vaccines in mice is similar to previousata from our lab studying influenza A virus HA-DNA vaccinationy gene gun [31].

duces protective immunity in BALB/c mice. Four weeks after secondary exposure toYam98 HA-expressing virus with a mouse lethal phenotype. Morbidity (percentageroups of mice (Alum + Alum (♦), n = 8; PBS + PBS (�), n = 11; HA-DNA (IM) + HA-DNA; IIV + IIV (�), n = 11; LAIV + LAIV (�), n = 8; IIV + LAIV (�), n = 11; and LAIV + IIV (�),

V.C. Huber et al. / Vaccine 2

Table 2Ferret nasal wash viral titers after LAIV delivery

LAIV primea; NW viral titerb LAIV boosta; NW viral titerb

Day + 1 2.7 × 104 ± 1.2 × 104 4.7 × 104 ± 4.0 × 104

Day + 3 1.1 × 104 ± 1.0 × 104 8.8 × 103 ± 6.7 × 103

Day + 5 1.4 × 103 ± 1.6 × 103 <1.0 × 101

Day + 12 <1.0 × 101 NDc

a LAIV delivered at a dose of 1 × 107 TCID (33 ◦C) in a 1 mL volume (500 �L pern

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.3. Comparison of LAIV shedding between naïve ferrets anderrets primed with IIV

The finding that IIV priming for LAIV resulted in lower secondaryiters in mice than LAIV priming was surprising, and potentially hasmportant implications for humans, since ACIP recommendationsurrently support the use of the two vaccine vehicles in any order38]. Therefore, we decided to test this important point in the fer-et model, which may be more representative of human responses39,40]. We first assessed whether IIV priming limited the growth ofubsequent LAIV exposure (Table 2). When LAIV was administeredo naïve animals, virus shedding was observed on days 1, 3, andafter inoculation. All ferrets were negative when tested 12 days

fter LAIV delivery. If the animals had been exposed to IIV vehicles a prime, shedding of LAIV after secondary exposure was lim-ted to days 1 and 3, with no detectable virus at day 5. This findingndicates immunity induced after IIV inoculation limits LAIV, butoes not completely prevent LAIV shedding. Therefore, neutraliza-ion of virus by pre-existing antibody is unlikely to account for anyifferences in secondary antibody titer.

.4. Humoral immunity induced in ferrets using a mixed vaccineelivery regimen

Three weeks after either primary or secondary inoculation, fer-et sera were analyzed for antibody expression (Fig. 3). An ELISAssay that was used to detect IgG (H + L) demonstrated no HA-pecific antibody detected prior to vaccination. After priming with

ig. 3. Vaccination against influenza B virus using mixed delivery vehicles inducesisparate antibody kinetics. Sera taken from ferrets prior to vaccination (Pre), threeeeks after primary (Pri) inoculation, and three weeks after secondary (Sec) inocu-

ation with influenza B virus HA expressed by mixed vectors were analyzed for IgGH + L) expression by ELISA. ELISA titers are reported as the reciprocal serum dilu-ion representing 50% maximal binding on the titration curve. Titers for individualerrets are reported as individual symbols, with the bar representing the mean forhe (PBS (�), n = 2; IIV + LAIV (©), n = 4; and LAIV + IIV (�), n = 4).

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6 (2008) 5381–5388 5385

AIV, all four ferrets demonstrated high IgG titers, while two of theour ferrets in the IIV-primed group had low antibody titers, withhe other two demonstrating antibody that was below the levelf detection for the assay. Boosting with LAIV enhanced antibodyiters to levels similar to those seen with primary LAIV exposure.oosting with IIV after the LAIV prime did not induce a further

ncrease in titers. Thus, LAIV appears to induce stronger immunitygainst influenza B viruses in the ferret model, as high antibodyiters are elicited after a single LAIV exposure, and similar antibodyevels are not achieved in IIV primed animals until the animalsre boosted with LAIV. Of note, IIV priming did not diminish theoosting effect of LAIV as was seen in the mouse model. Similaratterns of serum antibody levels were detected using the stan-ard HI assay (data not shown). In addition, assays used to detect

nfluenza-specific IgA antibodies in sera and nasal wash, as wells influenza-specific IgG (H + L) antibodies in nasal wash did notetect significant stimulation of antibodies of these isotypes (dataot shown).

.5. Shedding of virus by ferrets after vaccination using a mixedelivery regimen

As a direct correlate of protective immunity induced after vac-ination, ferrets were challenged with BYam98-expressing viruss described above for the murine model. Using clinical scorecombined activity and sneezing ratings), mean body temperature,ercent initial body weight, and protein levels in nasal wash fluid aseadouts of illness [35], challenging either group of ferrets with thisirus did not result in significant clinical illness (data not shown).owever, when nasal washes were collected to assess virus shed-ing after challenge (Fig. 4), the group that was inoculated with PBShed virus at moderately high titers for at least 6 days after chal-enge. The ferrets that received IIV followed by LAIV did not shedetectable levels of virus at any timepoint after challenge, whereaserrets that were in the group primed with LAIV and boosted withIV shed only minimal amounts of virus between days 1 and 5 afterhallenge. All immunized ferrets were negative at day 6 after chal-enge with this virus. Although the antibody data suggest that LAIVs a superior vaccine vehicle to IIV, both were equally efficaciousn the challenge model. Taken together, these data suggest that if

mixed prime:boost regimen is used for influenza B virus IIV and

AIV, an LAIV prime would be favorable in a naïve population (i.e.rst-time vaccine recipients or new vaccine formulation), and LAIVould be better suited for boosting individuals that had alreadyeen primed by either vaccination or natural infection.

ig. 4. Nasal wash viral titers in vaccinated ferrets after challenge with BYam98 HA-xpressing influenza virus. Nasal wash collected daily after challenge was tested forresence of influenza virus. Titers are reported as the mean ± standard deviation forach group at time-points indicated by the symbols (PBS (�), n = 2; IIV + LAIV (©),= 4; and LAIV + IIV (�), n = 4).

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. Discussion

Current vaccines have compromised effectiveness for influenzavirus compared to influenza A virus [27–29]. When coupled withifficulties in strain selection [7], it appears that alternate strategiesor prevention of influenza B virus should be considered. Our dataemonstrate that vaccines based on the LAIV system are more effec-ive against influenza B virus in animal models than other potentiallternatives. LAIV produced a balanced IgG1 and IgG2a response, aactor shown to be important for neutralization of virus, clearancef virus from the lungs, and survival in the influenza A virus model31]. In fact, despite our utilization of a whole-virus preparationor IIV, which is generally more immunogenic than the purified,plit product preparations delivered to humans annually [41,42],djuvantation was necessary to achieve similar protection to thatemonstrated with LAIV. Furthermore, we show that administeringAIV as a prime enhances primary immunity, as well as the capac-ty to respond to either an identical (LAIV) or mixed (IIV) boost,nd we report similar findings in both mouse and ferret models ofnfluenza immunity.

Beginning in the 1960s, vaccine technologies that utilized deter-ents to split or purify virus surface proteins from inactivated viralarticles were employed to reduce the reactogenicity seen after

nactivated, whole virus vaccine delivery [43–47]. Unfortunately,his reduced reactogenicity was associated with reduced immuno-enicity of split product preparations [47–49]. Efforts were made tonhance immunogenicity using adjuvants [50–54], but incorpora-ion of adjuvants was hindered either by a perceived lack of benefitAlum [55]) or development of sterile abscesses at the injection siteArlacel A [56]). Recently the use of Alum as an adjuvant has beenevisited for influenza HAs with pandemic potential that have mod-rate to low immunogenicity in humans [57]. In addition to Alum,djuvants based on oil-in-water formulations have shown successn clinical trials (AS [58,59]) and have achieved clinical approval inlderly populations in Europe (MF59 [60]). The data reported here,hile using vaccine production technologies that differ from thosesed in humans, suggest that adjuvantation for influenza B virusay help achieve optimal immunity against this type of influenza

irus.Historical analysis of influenza B virus vaccines in IIV form

elivered either alone [15,61] or as part of a trivalent prepara-ion [3,27,62–64] have demonstrated reduced immunity comparedo similar formulations of influenza A virus components. Further-

ore, Ohmit et al. [29] recently demonstrated reduced efficacynd effectiveness toward the B virus component of a trivalent vac-ine delivered in LAIV form. Strains from both dominant B lineagesirculated in the United States during the study period, but thetrain(s) infecting subjects in this study were not reported, andnly one influenza B virus strain was subtyped in a follow-up studyn 2005–2006 (it was mis-matched to the vaccine) [65]. Thus, its difficult from the results reported thus far from these studies toecide how much of the perceived poor efficacy of vaccines for B vs.is related to strain mis-match and how much is due to decreased

mmunogenicity. An added confounder is that protection againstnfluenza B virus can be achieved with less than the 1:32 HI titer cur-ently used to correlate with effective protection against influenzaviruses of the H1N1 and H3N2 subtypes [61,64,66,67]. These fac-

ors and our data suggest that, contrary to current practice, utilizingnalogous production techniques and correlates of immunity foroth influenza A and B virus vaccines, as well as incorporating only

single strain each year, yields less than optimal immunity. It is

lear that further study of these issues is needed.Direct comparison of DNA, IIV, and LAIV vehicles for B

A delivery, in unadjuvanted form demonstrated the followingmmunogenicity profile for the different vehicles delivered twice:

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AIV ≥ IIV > HA-DNA (GG) > HA-DNA (IM). Surprisingly, an immuneesponse to HA-DNA delivered using the IM route did not induce anmmune response toward the HA [68], whereas HA-DNA deliveredia the gene gun revealed humoral and protective immunity similaro results we have reported for influenza A virus [31]. With regard toA-DNA delivered IM, literature supports the induction of antibody

esponses of the IgG2a isotype after HA-DNA delivery [69], whichould predict enhanced ability to clear the virus after infection

31,70]. However, the sensitive ELISA assays employed here [31,71]ere unable to detect significant humoral immunity (including

gG2a) or protective responses after HA-DNA (IM) inoculation withdose of 100 �g per mouse (compared to 0.8 �g HA-DNA (GG) perouse) [72], even in the presence of Alum. The lack of a response

n mice inoculated with influenza B HA via the IM route may bessociated with either the route/method of delivery [68,72–74] oreduced antigenicity for influenza B HA [27,29] expressed by thelasmid DNA.

In the current study, high IgG (�-specific and IgG1 for mice andH + L) for ferrets) expression was a strong correlate of protectivemmunity. In addition, groups of mice that demonstrated maxi-

al survival were those that expressed antibodies of both the IgG1nd IgG2a isotypes simultaneously. Corroborating previous datarom our group [31], increased IgG2a expression in mice vaccinatedith IIV alone yielded better protection from lethal challenge overgroup that predominantly expressed IgG1 after vaccination (HA-NA (GG)), even when IgG1 levels were identical for the two groupst the time of challenge. Lower IgG1 titers in the IIV group, how-ver, correlated with poor outcomes compared to adjuvanted IIVnd LAIV despite similar IgG2a titers. A recent study by Bungenert al. [37] reported skewing toward an IgG2a response after vaccina-ion with an unadjuvanted, whole, inactivated influenza A vaccine,nd reported an alternate skewing toward IgG1 when Alum wasncluded in the vaccine preparation. Our vaccine did not exhibit aimilar trend, thus hinting at differences in the type of immunitynduced toward influenza A and B viruses.

Current vaccine recommendations call for administration of twonfluenza vaccine inoculations to naïve human populations (i.e. firstime vaccine recipients under the age of 9) [36]. This is due toimited detection of a humoral response following a single inocu-ation with typical inactivated vaccine formulations, most notablyor the H1N1 and B virus components [27,28]. While humoral cor-elates of immunity after inoculation with trivalent vaccines areiased toward the H3N2 subtype [27–29,35,75], significant pro-ective immunity toward influenza B after a single dose of LAIVas been reported [76–78], and is maintained even when humoral

mmune correlates have not achieved the desired level [79–81].urrent recommendations for influenza vaccination from the ACIP

ndicate that mixed delivery of IIV and LAIV can occur in anyrder [38]. Our data indicate that this assumption on the order ofixed vaccine administration requires more attention, at least for

nfluenza B virus. Specifically, using both the mouse and the ferretodel we show a difference in antibody kinetics after mixed IIV

nd LAIV delivery in both mice and ferrets. The reduced capacityor adequate boosting after IIV delivery is associated with adverseesponse to challenge in the mouse model, but not in ferrets. Thus,hile the scope of this study does not allow for definition of a regi-en for delivery of mixed vehicles, our data urge analyses of human

nfluenza vaccine recipients to see what effects are seen underixed vaccine conditions. If LAIV is the preferred prime, the ear-

ier availability of LAIV compared to IIV (typically 1–2 months lead

ime) may facilitate efforts to administer 2 doses in naïve subjects,omething that has been difficult to achieve with IIV formulationshich are typically not available until October [82]. In addition,e support further attempts to define adjuvants that would boost

mmunity to IIV and/or LAIV, but caution against treating influenza

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and influenza A virus subtypes similarly with regard to formula-ion, immunogenicity, and correlates of protective immunity.

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