Oral Fluids as a Live-Animal Sample Source for Evaluating Cross-Reactivity and Cross-Protection following Intranasal Influenza A VirusVaccination in Pigs

Holly R. Hughes,a Amy L. Vincent,a Susan L. Brockmeier,a Phillip C. Gauger,b Lindomar Pena,c Jefferson Santos,c

Douglas R. Braucher,a* Daniel R. Perez,c* Crystal L. Lovinga

Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Services, U.S. Department of Agriculture, Ames, Iowa, USAa;Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa, USAb; Department of Veterinary Medicine, University ofMaryland, College Park, College Park, Maryland, USAc

In North American swine, there are numerous antigenically distinct H1 influenza A virus (IAV) variants currently circulating,making vaccine development difficult due to the inability to formulate a vaccine that provides broad cross-protection. Experi-mentally, live-attenuated influenza virus (LAIV) vaccines demonstrate increased cross-protection compared to inactivated vac-cines. However, there is no standardized assay to predict cross-protection following LAIV vaccination. Hemagglutination-inhib-iting (HI) antibody in serum is the gold standard correlate of protection following IAV vaccination. LAIV vaccination does notinduce a robust serum HI antibody titer; however, a local mucosal antibody response is elicited. Thus, a live-animal samplesource that could be used to evaluate LAIV immunogenicity and cross-protection is needed. Here, we evaluated the use of oralfluids (OF) and nasal wash (NW) collected after IAV inoculation as a live-animal sample source in an enzyme-linked immu-nosorbent assay (ELISA) to predict cross-protection in comparison to traditional serology. Both live-virus exposure and LAIVvaccination provided heterologous protection, though protection was greatest against more closely phylogenetically related vi-ruses. IAV-specific IgA was detected in NW and OF samples and was cross-reactive to representative IAV from each H1 cluster.Endpoint titers of cross-reactive IgA in OF from pigs exposed to live virus was associated with heterologous protection. WhileLAIV vaccination provided significant protection, LAIV immunogenicity was reduced compared to live-virus exposure. Thesedata suggest that OF from pigs inoculated with wild-type IAV, with surface genes that match the LAIV seed strain, could be usedin an ELISA to assess cross-protection and the antigenic relatedness of circulating and emerging IAV in swine.

Influenza A virus (IAV), of the family Orthomyxoviridae, infectsmany species, including humans, pigs, horses, sea mammals,

and birds. The structural proteins hemagglutinin (HA), neur-aminidase (NA), and matrix 2 (M2), along with the cellular lipidbilayer, form the envelope of IAV. The HA and NA proteins playessential roles in virus entry and release of progeny, respectively,and are primary immunogens involved in a protective antibodyresponse (reviewed in reference 1), while internal genes such asnucleoprotein (NP), polymerase subunit PB1, and matrix protein(M)1 are also important for a protective immune response (2).

Pigs are possible “mixing vessels” for the emergence of anti-genically distinct IAV isolates since the respiratory tracts of pigscontain receptors for both avian and mammalian IAV (3, 4). In-terspecies transmission of IAV is a real concern and has been welldocumented, with transmission between pigs and humans (5–7)as well as between pigs and birds or poultry (8–10). The relation-ship between human IAV and swine IAV is an animal and publichealth concern because of noted spillover events. Routine IAVvaccination is an important tool in the management of animalhealth to reduce economic loss associated with IAV infection (11)and for pigs displayed at agricultural fairs (12) in terms of bothpublic and animal health.

One of the main roadblocks for swine IAV vaccine develop-ment is the presence of multiple antigenic variants within H1 andH3 subtypes, which results in the need for vaccines that providecross-protection. Classical H1N1 (cH1N1) was the predominantIAV in U.S. swine until the introduction of the novel H3N2 strainin 1998 (13). H3N2 quickly became endemic in U.S. swine and

reassorted with extant cH1N1, resulting in genes from human,avian, and swine IAV combining into a single IAV strain (13, 14).Through introductions of human seasonal IAV into swine andgenetic evolution of existing strains, there are currently six phylo-genetic clusters of H1 IAV circulating in U.S. herds that are des-ignated beta (�), gamma (�), �-2, delta-1 (�1), delta-2 (�2), andpandemic (15–19). There is limited hemagglutination-inhibiting(HI) cross-reactivity between H1 clusters when using pig hyper-immune serum (derived through vaccination with adjuvanted,

Received 1 July 2015 Returned for modification 21 July 2015Accepted 13 August 2015

Accepted manuscript posted online 19 August 2015

Citation Hughes HR, Vincent AL, Brockmeier SL, Gauger PC, Pena L, Santos J,Braucher DR, Perez DR, Loving CL. 2015. Oral fluids as a live-animal sample sourcefor evaluating cross-reactivity and cross-protection following intranasal influenzaA virus vaccination in pigs. Clin Vaccine Immunol 22:1109 –1120.doi:10.1128/CVI.00358-15.

Editor: D. W. Pascual

Address correspondence to Crystal L. Loving, crystal.loving@ars.usda.gov.

* Present address: Douglas R. Braucher, Boehringer Ingelheim Vetmedica Inc.,Ames, Iowa, USA; Daniel R. Perez, Department of Population Health, College ofVeterinary Medicine, University of Georgia, Athens, Georgia, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/CVI.00358-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00358-15

October 2015 Volume 22 Number 10 cvi.asm.org 1109Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

monovalent whole-inactivated virus [WIV] vaccine) to evaluateantigenic relatedness (17, 18).

The majority of currently licensed IAV vaccines for swine areadjuvanted, WIV vaccines given by the intramuscular (i.m.) route.This vaccine formulation induces IAV-specific antibody in serumas measured by the HI assay or a whole-virus enzyme-linked im-munosorbent assay (ELISA) (20). WIV vaccines primarily provideprotection against homologous or antigenically related strains butprovide limited cross-protection against heterologous strains (i.e.,strains with the same subtype but limited antigenic cross-reactiv-ity) (21, 22). Notably, differences in the immune response follow-ing vaccination through the i.m. route compared to natural infec-tion include minimal mucosal IAV-specific IgA and cell-mediatedimmune responses (21). To overcome limitations associated withWIV vaccines, several next-generation IAV vaccines, includinglive-attenuated influenza virus (LAIV) vaccines and replication-defective adenovirus vectors that encode IAV genes, have beenexperimentally tested for use in pigs (23). LAIV and adenovirus-vectored vaccines have been shown experimentally to provide bet-ter cross-protection than WIV vaccines (24–28) and to elicit cel-lular immunity (24, 29), and IAV-specific maternal immunitydoes not interfere with adenovirus-vectored or LAIV vaccine effi-cacy in piglets (29–31).

Nonstructural protein 1 (NS1) of IAV is a virulence factor as aconsequence of its type I interferon (IFN) antagonist activity (32).Introducing nucleotide mutations into the NS1 gene causes theloss of type I IFN inhibition; thus, the host IFN response inhibitsgrowth of the virus (33). Through truncation of the last 93 aminoacids of the NS1 IAV protein, an LAIV was generated that wasshown to induce a robust IFN response and to be attenuated inpigs (34). This truncated NS1 LAIV with H3 surface genes wasfurther shown to elicit protection against homologous and heter-ologous H3 challenge viruses, as well as partial heterosubtypicprotection when vaccinated pigs were challenged with H1 virus(35).

Human replication-defective adenovirus serotype 5 encodingHA (Ad5-HA) has been shown experimentally to provided ho-mologous protection (24, 31, 36) when administered by either theintramuscular or intranasal (i.n.) route and to overcome the prob-lem of interference by maternal-derived immunity (31). Addi-tionally, Ad5-HA IAV vaccines in swine were shown to providepartial heterologous protection when administered intranasally(24).

Despite advantages of LAIV- and Ad5-based vaccines, theseplatforms have yet to come to market. A live-animal sample andassay to predict vaccine efficacy and cross-protection, as well asadditional cross-protection studies using contemporary viruses,will aid in the continued development of these platforms. SerumHI titer is the established gold-standard method to predict IAVcross-reactivity and, subsequently, the cross-protective efficacy ofa particular vaccine. A reciprocal serum HI titer of �40 is usuallyconsidered protective (correlated with a 50% reduction in the riskof IAV infection [37]), and a greater than 4-fold reduction in HItiter between viruses is considered to represent significant anti-genic drift with a predicted loss in cross-protection (37). LAIVvaccination elicits modest serum HI antibody responses to ho-mologous antigens, but LAIV also provides protection againstheterologous IAV in which LAIV antibodies did not cross-react(25, 38). Thus, HI titers following LAIV vaccination may be usefulfor evaluating vaccine immunogenicity and predicting homolo-

gous protection, but predictions of cross-protection using thisgold standard method are unreliable.

Since intranasal LAIV vaccination elicits IAV-specific antibodyat mucosal surfaces (29, 39), it is possible that a live-animal mu-cosal sample would be better for evaluating intranasal IAV vaccineimmunogenicity and cross-protective efficacy. To test this hy-pothesis, a study was completed in which groups of pigs wereexposed to wild-type (WT) IAV, LAIV, or Ad5-HA, all with apandemic lineage surface gene(s), for collection of mucosal andserum samples postexposure. Vaccinated pigs were then chal-lenged with either a heterologous �-cluster or �-cluster IAV toevaluate the cross-protective efficacy of the vaccines and to evalu-ate IAV-specific antibody levels in prechallenge samples in asso-ciation with protective efficacy. Taking the results together, WTIAV inoculation or LAIV or Ad5-HA vaccination provided somelevel of cross-protection (depending on the challenge strain), andimmunogenicity likely impacted the extent of cross-protective ef-ficacy. IAV-specific IgA in oral fluids was found to be associatedwith cross-protective efficacy; the data may serve as predictiveindices, and additional work is warranted to further validate thesefindings.

MATERIALS AND METHODSEthics statement. Animal experiments were approved by the InstitutionalAnimal Care and Use Committee of the National Animal Disease Centerin Ames, IA. Animals were housed in animal biosafety level 2 (ABSL-2)conditions for the entirety of the study.

Vaccines and viruses. The LAIV vaccine expressed the 2009 pandemicH1N1 (H1N1pdm09) (pdm) surface genes from A/New York/18/2009(NY/09) with the A/turkey/Ohio/313053/2004 (H3N2) internal genes, inwhich a truncated NS1 gene (34) was carried (see Fig. S1 in the supple-mental material). The reverse-engineered, genetically matched parent vi-rus was derived by rescuing the HA and NA genes from A/New York/18/2009 with the A/turkey/Ohio/313053/2004 internal genes (no attenuationmutations introduced) as previously described (40, 41) and is referred toas RG NY/09. The replication-defective adenovirus type 5 vector HA(Ad5-HA) vaccine was derived using an AdEasy system (Agilent, SantaClara, CA) by cloning the A/California/04/2009 HA gene into thepShuttle-cytomegalovirus (CMV) vector, and adenovirus rescue was per-formed as previously described (42). Heterologous challenge viruses wereH1N1 �-cluster A/swine/Minnesota/03012/2010 virus (MN/10) andH1N2 �-cluster A/swine/Illinois/3134/2010 IAV (IL/10) obtainedthrough submission of clinical samples to the University of MinnesotaVeterinary Diagnostic Laboratory (kindly provided by Marie Culhane).All vaccines and viruses were propagated in Madin-Darby canine kidney(MDCK) cells in serum-free Opti-MEM media (Gibco, Grand Island,NY) supplemented with tosylsulfonyl phenylalanyl chloromethyl ketone(TPCK)-trypsin (Sigma, St. Louis, MO), L-glutamine, and antibiotics,with the exception of Ad5-HA, which was grown in AD-HEK293 cellswith high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco,Grand Island, NY) supplemented with 10% fetal bovine serum. Ad5-HAwas purified using a double discontinuous cesium chloride gradient perthe manufacturer’s instructions and dialyzed as previously described (42).All vaccines and viruses were diluted in phosphate-buffered saline (PBS)to the desired concentration immediately prior to administration.

Experimental design. Three-week-old pigs were obtained from ahigh-health-status herd free from IAV and porcine reproductive and re-spiratory syndrome virus. Pigs were administered enrofloxacin (Baytril;Bayer, Pittsburgh, PA) and ceftiofur crystalline free acid (Excede; Zoetis,Florham Park, NJ) upon arrival according to the recommendations of themanufacturers. IAV antibody status was confirmed to be negative usingthe IDEXX Multi-Screen Ab test (IDEXX, Westbrook, ME). In part I ofthe experiment (attenuation), piglets were randomly assigned to groups

Hughes et al.

1110 cvi.asm.org October 2015 Volume 22 Number 10Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

of eight and immunized i.n. at 4 weeks of age. Groups received LAIV, WTNY/09, or RG NY/09 parent virus or were not vaccinated (NV). Pigs werehumanely euthanized at 3 days postvaccination (dpv) to assess the atten-uation and pathogenicity of the LAIV compared to the wild-type parentvirus. In part II of the experiment (immune measures of cross-protec-tion), piglets were randomly assigned to groups of eight, immunized i.n.at 4 weeks of age, and boosted 3 weeks later by the same route with thesame dose (Table 1). LAIV vaccine and WT NY/09 virus were targeted at106 50% tissue culture infective doses (TCID50)/ml and at 2 ml per dose.The back titer of the inoculum after administration indicated that the WTNY/09 dose was 105.5 TCID50 per pig, whereas the LAIV dose was 104.2

TCID50 per pig. Pigs in the Ad5-HA group received 1010 TCID50 in 2 mli.n. Nonvaccinated (NV) controls received 2 ml PBS i.n. Three weeksfollowing the boost, all pigs except for the nonvaccinated/nonchallenged(NV/NC) controls were challenged i.n. with 105.8 TCID50 MN/10(�) orIL/10(�). Pigs were humanely euthanized at 5 days postinfection (dpi) forcollection of samples to evaluate vaccine efficacy.

Sample collection. For postvaccination sample collection, nasal swabs(NS) were taken at 0 to 3 dpv for the subset of pigs necropsied on day 3postpriming. Serum, nasal washes (NW), and oral fluids (OF) were col-lected from all remaining pigs every 7 days following primary immuniza-tion. Blood was collected by venous puncture and stored in BD Vacu-tainer serum separator tubes (BD, Franklin Lakes, NJ). Serum wascollected by centrifugation at 800 � g for 20 min, divided into aliquots,and frozen at �80°C. NW were collected by instilling 5 ml PBS into anaris and collecting effluent as previously described (43), divided intoaliquots, and frozen at �80°C. Typically, 0.5 to 2 ml of NW was collected.OF were obtained following previously described methods (44) with fewmodifications. Briefly, cotton ropes were hung in each room for approx-imately 30 min for the pigs to chew. Ropes from each group were placedinto separate ziplock bags. The rope was manually squeezed inside the bagand liquid sample decanted into a 50-ml conical tube. The tubes werecentrifuged at 800 � g for 20 min, and the supernatant was filteredthrough 0.45-�m-pore-size syringe filters and immediately frozen at�80°C. For postchallenge sample collection, nasal swabs were collecteddaily beginning on the day of challenge (0 dpi) through necropsy (5 dpi)using polyester-tipped swabs (Puritan, Guilford, ME) prewetted in 2 mlminimal essential media (MEM) and stored frozen. At necropsy, tracheawash was obtained by removing the trachea prior to lung lavage. Thetrachea, from 1 cm below the larynx to 2 cm above the bifurcation, wasremoved and submerged in 3 ml MEM and vigorously agitated for 15 s.The trachea was then removed, and the medium was aliquoted and frozenat �80°C. Lung lavage samples were obtained by lavage performed with50 ml MEM as previously described (38). An aliquot of lung lavage fluidwas plated on blood agar and Casman’s agar plates containing 0.01%(wt/vol) NAD and 5% horse serum for routine aerobic culture to rule outbacterial infection. All postchallenge samples were stored on ice until theywere divided into aliquots and frozen within 2 h of collection.

Pathological examination. At necropsy, the percentage of lung af-flicted with the purple-red consolidation typical of IAV infection wasevaluated (45). The total percentage of pneumonia was calculated on thebasis of the weighted proportions of each lobe with respect to the totallung volume (46). A portion of the right middle lobe was fixed in 10%buffered formalin for 48 h, processed by routine histopathologic proce-dures, and stained with hematoxylin and eosin. Microscopic lesions wereevaluated and scored by a veterinary pathologist blinded to the treatmentgroups using parameters previously described (47).

Antibody evaluation. The HI assay was performed as recommendedin the WHO animal influenza training manual using turkey red bloodcells and WT NY/09 virus or H1 challenge virus as the target antigen aspreviously described (41). The HI assay using OF and NW was performedfollowing the same protocol as the serum HI assay, beginning with aninitial dilution of 1:10. Data are expressed as the reciprocal titer. Theserum neutralization assay was performed by generating serial 2-fold di-lutions of heat-inactivated sera in MEM supplemented with 5% bovineserum albumin and antibiotics and by incubating with 100 TCID50 beforeinoculation of confluent MDCK monolayers in 96-well plates as previ-ously described (48). Cells were incubated for 48 h, fixed, and stained forviral nucleoprotein (NP) antigen by immunocytochemistry (49). OF andNW neutralization assays were performed similarly except that the NWsamples were not heat inactivated prior to use in the neutralization assay.Reciprocal titers were Log2 transformed and used for statistical analysis ofHI and neutralization titer data. IAV-specific IgA and IgG levels in the OFand the NW were recorded as the optical density (OD) value using anindirect whole-virus ELISA and the viruses listed in Table 2 as previouslydescribed (38), with the exception that the NW and OF samples werediluted 1:2 in PBS for use in the assay. Antibody levels were reported as themean OD at 405 nm for each vaccine group. Endpoint antibody titers wereobtained by initially diluting OF and NW samples (pooled by treatmentgroup) 1:2 in PBS and titrating 2-fold in duplicate before performing theELISA. The resulting OD data were modeled as a nonlinear function of theLog10 dilution using the GraphPad Prism (GraphPad software Inc., LaJolla, CA) Log (agonist) versus response-variable slope four-parameterlogistic model. Endpoints were interpolated by using 2� the average ODof the nonvaccinated control as the cutoff. Endpoints for each virus wereorganized by cluster, and average cluster endpoint dilutions are expressedfor both OF and NW. Total IgA and IgG ELISAs were performed using apig IgA or IgG quantification kit (Bethyl Laboratories, Montgomery, TX)following the manufacturer’s protocol.

Virus titration. To determine viral loads, nasal swab, trachea wash,and lung lavage samples were titrated on MDCK cells to determine theTCID50/ml as previously described (38). Briefly, frozen samples werethawed and filtered through 0.45-�m-pore-size syringe filters, and 10-fold serial titrations were made in triplicate in serum-free media contain-ing TPCK-trypsin (Sigma, St. Louis, MO) (1 �g/ml) and added to conflu-ent MDCK monolayers in 96-well plates. Cells were incubated for 48 h,fixed, and stained for NP by immunocytochemistry (49). For each titra-

TABLE 1 Experimental design for evaluation of IAV vaccineimmunogenicity and cross-protective efficacya

Group (n 8) Vaccine Challenge virus

NV/NC None NoneNV/CH None MN/10(�)WT NY/09 MN/10(�)LAIV LAIV MN/10(�)Ad5-HA Ad5-HA MN/10(�)NV/CH None IL/10(�)WT NY/09 IL/10(�)LAIV LAIV IL/10(�)Ad5-HA Ad5-HA IL/10(�)a n, number of pigs per group; NV, nonvaccinated; NC, nonchallenged; WT, wild type;LAIV, truncated nonstructural protein 1 live-attenuated influenza virus vaccine; MN/10(�), A/swine/MN/03012/2010; IL/10(�), A/swine/IL/3134/2010.

TABLE 2 IAV isolates used to evaluate antibody responses followingintranasal immunization

Cluster Virus name Abbreviation Subtype

Pandemic A/NY/18/2009 NY/09 H1N1Pandemic A/CA/04/2009 CA/09 H1N1Pandemic A/swine/IL/5265/2010 IL/10 H1N1� A/swine/IL/3134/2010 IL/10 H1N2� A/swine/IN/3062/2010 IN/10 H1N1� A/swine/OH/511445/2007 OH/07 H1N1� A/swine/MN/03012/2010 MN/10 H1N1�-2 A/swine/MO/03013/2010 MO/10 H1N2�-1 A/swine/MN/02011/2008 MN/08 H1N2H3 A/turkey/OH/313053/2004 OH/04 H3N2

LAIV Vaccines and Correlates of Protection

October 2015 Volume 22 Number 10 cvi.asm.org 1111Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

tion, the Log10 transformed TCID50/ml was calculated for each sampleusing the method of Reed and Muench (50).

Statistical analysis. Reciprocal HI and neutralization titers were Log2

transformed and viral titers were Log10 transformed for analysis and ex-pressed as reciprocal titer values. ELISA data are presented as the averageoptical density at 405 nm. Statistical analysis was completed with GraphPad prism version 6 using the Kruskal-Wallis test followed by Dunn’smultiple comparison or using two-way analysis of variance (ANOVA)and Tukey’s multiple comparison where appropriate.

RESULTSAttenuation characteristics of H1 LAIV vaccine. Pigs were i.n.inoculated with WT NY/09, H1 LAIV, or the reverse-genetics-rescued isogenic parent virus (RG NY/09) to assess the attenua-tion characteristics of the H1 LAIV vaccine. Macroscopic lunglesions (pneumonia) at 3 dpv were significantly reduced in pigsinoculated with LAIV compared to those in pigs inoculated withRG NY/09 (Fig. 1A). Amounts of WT NY/09 virus were detectedin nasal swabs by 1 dpv, peaked at 2 dpv, and began to decline by3 dpv. The amount of virus detected in nasal swabs from pigsinoculated with LAIV was significantly reduced at each time pointtested compared to the amount of virus detected in nasal swabsfrom pigs exposed to WT NY/09 and was significantly reduced at3 dpv compared to the amount detected in pigs exposed to RGNY/09 (average, 0.03 versus 1.8 Log10 TCID50/ml) (Fig. 1B). Pigsinoculated with LAIV had significantly reduced mean viral titersin the trachea (0.2 Log10 TCID50/ml) compared to animals ex-posed to WT NY/09 and RG NY/09 (4.5 and 3.9 Log10 TCID50/ml,respectively) (Fig. 1C). Additionally, LAIV pigs had reduced meanviral titers in the lung lavage fluid compared to animals exposed to

WT NY/09 (P 0.01) (Fig. 1D). The back titer of the challengeindicated that each pig in the RG NY/09 groups received 105.9

TCID50, each pig in the WT NY/09 group received 105.5 TCID50,and each pig in the LAIV group received 104.2 TCID50.

IAV-specific antibody in serum, nasal wash, and oral fluids.Serum, NW, and OF samples were collected weekly following pri-mary exposure (to WT NY/09, LAIV, and Ad5-HA) for evaluationof IAV-specific antibodies. At 42 dpv, serum HI antibodies againstWT NY/09 virus were detected for all immunization groups,though there were differences in titers between treatment groups(Table 3). Average serum HI titers against homologous NY/09virus were 180, 55, and 31 in animals immunized with WT NY/09,LAIV, and Ad5-HA, respectively. Average serum HI titers againstthe challenge viruses were below the limit of detection (10), withthe exception of the WT NY/09 group, which had a low but de-tectable (12 � 2) serum HI titer against the �-cluster (MN/10)virus. NW and OF samples collected at 42 dpv were also evaluatedas a sample source in the HI assay, but there was no measureableHI activity using these samples, even at a dilution of 1:10. Allsamples from nonvaccinated animals (NV) had no detectable HItiters (10).

Functional IAV-specific antibody levels were also measuredusing a virus neutralization assay (Table 3). WT NY/09 and LAIVexposure induced production of serum neutralizing antibodyagainst NY/09 virus (titers, 168 � 27 and 74 � 27, respectively),and titers against IL/10(�) virus were similar. However, serumneutralization titers against the MN/10(�) challenge virus com-pared to neutralization of NY/09 virus were significantly reduced(54 � 6.7 [P 0.005] for WT NY/09-immunized animals and

FIG 1 Attenuation characteristics of H1 NS1-truncated LAIV vaccine. Groups of pigs were inoculated with WT NY/09 virus, LAIV, or the reverse-genetics(RG)-rescued NY/09 parent virus by the intranasal route or were left nonvaccinated (NV) and samples collected at 3 days postvaccination to evaluate attenuation.(A) Percent macroscopic lung lesions. (B) Viral shedding in nasal swabs collected 0 to 3 days postimmunization. (C and D) Viral load in trachea wash (C) andlung lavage (D) fluids. Each dot represents a single animal in that respective treatment group, with the means � standard errors of the means (SEM) for n 8shown for the group indicated. Data were analyzed with the Kruskal-Wallis test and Dunn’s posttest. P values of 0.05 were considered significant. *, P 0.05;**, P 0.01.

Hughes et al.

1112 cvi.asm.org October 2015 Volume 22 Number 10Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

25 � 6.3 [P 0.03] for LAIV-immunized animals). Ad5-HA im-munization did not result in detectable serum neutralization an-tibody. Moreover, OF samples from animals exposed to WTNY/09 were able to neutralize WT NY/09 virus (titer 8 � 1) butnot the heterologous challenge viruses, while NW samples wereunable to neutralize any virus used in the assay.

NW and OF samples were used in an indirect whole-virusELISA to detect antibody specific to H1N1pdm09 lineage virusthroughout the course of the vaccine regimen (Fig. 2). By 14 dpv,significant levels of IAV-specific IgA were measured in the NW(Fig. 2A) and OF (Fig. 2B) from pigs in the WT NY/09 (NW andOF, P 0.0001) and LAIV (NW, P 0.001; OF, P 0.0001)groups and were maintained throughout the experiment.H1N1pdm09 IAV-specific IgG was detected only in OF samplesbeginning at 28 dpv (1 week postboost) and was maintained abovebackground levels (NV group) only in samples from pigs in theWT NY/09 group (Fig. 2C). The total IgA and IgG antibody levelsin each sample were determined, and amounts never varied morethan 2-fold between time points (data not shown), suggesting thatsample collection was consistent throughout the experiment. TheAd5-HA vaccine did not elicit detectable levels of H1N1pdm09-specific IgA or IgG in NW or OF samples (see Fig. S2A in thesupplemental material). Due to an inability to measureH1N1pdm09-specific IgA in NW or OF collected from Ad5-HA-vaccinated animals, additional heterologous viruses were not eval-uated using samples from Ad5-HA vaccinates.

Macroscopic and microscopic lung pathology. Macroscopicand microscopic lung lesions were evaluated at 5 dpi followingchallenge with heterologous H1 �-cluster MN/10 or �-clusterIL/10 virus (Fig. 3). Pigs in the nonvaccinated, nonchallenged(NV/NC) control group had negligible macroscopic pneumonia

(score, 0.16% � 0.07). Pigs in the nonvaccinated challenged (NV/CH) groups challenged with IL/10(�) or MN/10(�) had minimalmacroscopic pneumonia (3.5% � 1.1 and 2.1% � 0.3, respec-tively). Pigs with prior exposure to WT NY/09 virus, challengedwith MN/10(�), did not have reduced pneumonia compared toNV/CH pigs due to overall low scores at 5 dpi (Fig. 3A); however,these animals did not have significantly increased pneumoniascores compared to the NV/NC controls (P 0.63). Prior WTNY/09 inoculation did reduce macroscopic lung lesions followingIL/10(�) challenge (0.63%, P 0.001) (Fig. 3B). While LAIV vac-cination did not statistically significantly reduce pneumonia fol-lowing challenge with either virus, there was a trend for reducedmacroscopic pneumonia following challenge and the percentageof pneumonia for the LAIV groups was not significantly increasedover that seen with NV/NC pigs (P 0.41). While LAIV-vacci-nated pigs challenged with MN/10(�) had microscopic pathologyscores greater than those seen with NV/NC controls (1.1 and 1.3versus 0.3, respectively), these scores were not significantly in-creased over NV/CH pig scores (0.79) (Fig. 3C). Pigs challengedwith IL/10(�) demonstrated mild microscopic lesions and did nothave scores greater than those determined for the NV/NC controls(Fig. 3D). Ad5-HA vaccination did not protect against the devel-opment of lung lesions following heterologous IL/10(�) or MN/10(�) challenge (see Fig. S2B and C in the supplemental material).

Viral load in the respiratory tract. Following challenge, viraltiters in nasal swabs (NS), trachea washes, and lung lavage fluidwere measured to evaluate protection against virus replication. NSwere collected daily from 0 dpi through necropsy at 5 dpi (Fig. 4Aand D). Pigs exposed to WT NY/09 virus and subsequently chal-lenged with MN/10(�) had reduced average viral titers in NS ondpi 1 through 4 (P 0.0001), but the average viral titers in NS

TABLE 3 Serum hemagglutination inhibition and virus neutralization titers measured at 42 days postvaccination

Vaccine

Serum titer of indicated target virusa

HI Neutralization

NY/09 (pdm) IL/10(�) MN/10(�) NY/09 (pdm) IL/10(�) MN/10(�)

WT NY/09 180 � 32.9 10 12.5 � 1.6 168 � 26.8 152 � 24 54 � 6.7**LAIV 55 � 7.3 10 10 74 � 26.8 72 � 17 25 � 6.3*Ad5-HA 31 � 10 10 10 2 2 2NV 10 10 10 2 2 2a *, P 0.03; **, P 0.005 (compared to NY/09 neutralization). Statistical significance was measured by the Kruskal-Wallis test (n 8).

FIG 2 Kinetics of IAV-specific mucosal IgA and IgG levels following immunization. Groups of pigs were intranasally inoculated with WT NY/09 or LAIV on day0 and boosted on day 21. NW and OF were collected weekly to day 42 to evaluate IAV H1N1pdm09 (CA/09)-specific mucosal antibody by whole-virus ELISAcompared to nonvaccinated (NV) animals. (A and B) IAV-specific IgA detected in nasal wash (A) and oral (B) fluids. (C) IAV-specific IgG detected in OF. Dataare expressed as means � SEM of OD determined for n 8 for the indicated group.

LAIV Vaccines and Correlates of Protection

October 2015 Volume 22 Number 10 cvi.asm.org 1113Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

collected on dpi 5 were similar to the titers measured in NS col-lected from NV/CH animals (Fig. 4A). Virus was not recoveredfrom any NS collected from pigs exposed to WT NY/09 and chal-lenged with IL/10(�) (Fig. 4D). LAIV vaccination limited theamount of MN/10(�) challenge virus in NS only at dpi 1 (P 0.0001), and NS mean viral titers were similar to the titers mea-sured in NS from NV/CH pigs on dpi 2 to 5. However, LAIVvaccination significantly limited the amount of IL/10(�) challengevirus shed and the length of time during which the virus was shedin nasal passages (P 0.005) (Fig. 4D). Pigs vaccinated withAd5-HA also had reduced amounts of challenge virus in NS on dpi1 to 2, but the average titers were the same as those in the respec-tive NV/CH groups on dpi 3 to 5 (see Fig. S2D in the supplementalmaterial).

Mean titers of the MN/10(�) challenge virus were significantlyreduced in trachea washes from pigs previously exposed to WTNY/09 virus compared to NV/CH pigs (1.5 Log10 TCID50/ml and4.6 Log10 TCID50/ml, respectively) (Fig. 4B). LAIV-vaccinated an-imals had mean titers of MN/10(�) virus in the trachea on dpi 5similar to those measured from NV/CH pigs (Fig. 4B). Addition-ally, mean viral titers in the lung lavage fluid of all immunizationgroups challenged with MN/10(�) did not differ from the levelsseen with NV/CH pigs (Fig. 4C). Pigs exposed to WT NY/09 orLAIV and challenged with IL/10(�) did not have any virus recov-ered from trachea wash or lung lavage fluid (Fig. 4E and F) on dpi5. Ad5-HA-vaccinated animals also exhibited increased cross-

protection against IL/10(�) (see Fig. S2E and F in the supplemen-tal material).

Measures of IAV-specific cross-reactive immunity. Sinceboth live-virus exposure and LAIV vaccination provided in-creased cross-protection against the �-cluster H1 variant and lesscross-protection against the �-cluster H1 variant, we evaluatedwhether levels of IAV-specific IgA in a live-animal sample sourcewere predictive of cross-protection, given that the standard HIassay was unable to measure cross-reactivity to either challengevirus (Table 3). Using NW and OF samples collected from all pigsat 42 dpv, the cross-reactivity of IgA to eight H1 viruses fromdifferent phylogenetic clusters (listed in Table 2) and one H3 virus(Fig. 5) was determined. Mucosal IAV-specific IgA reactive to ho-mologous, H1N1pdm09 lineage viruses as well as IgA cross-reac-tive to representative viruses from each H1 cluster and an H3 viruswas detected in NW from WT NY/09 virus-immunized animals(P 0.05), while LAIV-immunized animals did not have detect-able IgA reactive to all representative clusters (Fig. 5A). OF sam-ples from WT NY/09- and LAIV-immunized animals had signif-icantly higher levels of IAV-specific IgA than those from the NVcontrols (P 0.0001 for all clusters and vaccine groups) (Fig. 5B).IgA in NW from LAIV-vaccinated animals had significant reduc-tions in OD values for �-cluster viruses (P 0.003), �-2 clusterviruses (P 0.0006), and H3 viruses (P 0.01) compared to theOD values for homologous NY/09 antigen. However, IgA in OFfrom LAIV-vaccinated animals had lower OD values across all

FIG 3 Pneumonia following heterologous IAV-challenged, IAV-vaccinated pigs. Groups of pigs were inoculated with the indicated vaccine and subsequentlychallenged intranasally with heterologous virus, and lung pathology was evaluated at 5 dpi. (A and B) Macroscopic lesion scores of animals following challengewith MN/10(�) (A) or IL/10(�) (B) IAV compared to those calculated for nonvaccinated and challenged (NV/CH) or nonvaccinated and nonchallenged(NV/NC) animals. (C and D) Microscopic lesion scores following challenge with (C) MN/10(�) or (D) IL/10(�) IAV. Each dot represents a single animal in thatrespective treatment group, with the mean � SEM for n 8 shown for the group indicated. Data were analyzed with the Kruskal-Wallis test and Dunn’s posttest.P of 0.05 was considered significant. *, P 0.05.

Hughes et al.

1114 cvi.asm.org October 2015 Volume 22 Number 10Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

heterologous viruses compared to those seen with viruses of thepandemic cluster (P 0.001).

Since reductions in OD values compared to those of homolo-gous IAV antigen were similar across tested heterologous H1 vari-ants, average endpoint titers of IgA specific to each H1 cluster weredetermined to further elucidate differences in cross-reactive IgA.IAV-specific IgA endpoint titers in NW and OF against heterolo-gous H1 cluster viruses were reduced compared to the homolo-gous H1 endpoint titers (Fig. 5C and D). Differences between theOF IgA endpoint titer against the NY/09 vaccine virus and meanIgA endpoint titers in OF from WT NY/09-exposed pigs were anaverage of 1.8-fold for similar H1N1pdm09 lineage viruses, 4-foldfor �-cluster viruses, 4.8-fold for �-cluster viruses, 6.7-fold for�-cluster viruses, and 8.4-fold for H3 virus (Fig. 5D). IgA endpointtiters from OF collected from LAIV-vaccinated animals were re-duced by an average of 1.6-fold compared to those seen with sim-ilar H1N1pdm09 lineage viruses, 2.5-fold compared to those seenwith �-cluster viruses, 2-fold compared to those seen with �-clus-ter viruses, 3.8-fold compared to those seen with �-cluster viruses,and 2.1-fold compared to those seen with H3 viruses.

DISCUSSION

In this study, we evaluated the ability of H1N1pdm09 cluster H1LAIV and a replication-defective viral vector encoding IAVH1N1pdm09 HA to provide cross-protection against representa-tive heterologous �- and �-cluster H1 IAV infections compared tolive virus infections and investigated predictive immune measuresof cross-protection in samples collected from a live animal. Inevaluations of viral loads, vaccination with LAIV or exposure to

wild-type virus resulted in significant cross-protection against the�-cluster challenge IL/10 IAV; however, protection against�-cluster MN/10 was more limited. The H1N1pdm09 cluster and�-cluster viruses are more closely phylogenetically and antigeni-cally related than the �-cluster and H1N1pdm09 cluster viruses(17, 18); thus, the increased protection against IL/10(�) comparedto MN/10(�) was somewhat anticipated. Together, these data sug-gest that a multivalent next-generation swine IAV vaccine may benecessary to protect against the diverse H1 viruses cocirculating inU.S. swine.

Although cold-adapted, temperature-sensitive LAIV has beenapproved for use in humans (51) and in horses (52), LAIV vac-cines have not been approved for use in pigs. Original work on theLAIV vaccine focused on H3N2 surface genes (25, 34, 35). Here wedescribe the first use of an H1N1 NS1-truncated virus as an LAIVvaccine and compared the levels of immunogenicity and efficacyfollowing exposure to WT NY/09 virus, which shares the same HAand NA as the LAIV. Similarly to previous reports of H3 NS1-truncated LAIV vaccination in pigs (35) and H1 NS1-truncatedLAIV vaccination in mice (33), the H1 NS1-truncated LAIV inpigs was attenuated, with significantly less virus detected in thenose, trachea, and lungs as well as reduced levels of macroscopicpneumonia (Fig. 1). A previous study used a lower dose of chal-lenge WT virus (3 Log10 TCID50 less than was used in the currentstudy) (27), and it was as pathogenic as the typical dose of WTvirus used in the current study. This suggests that, regardless ofdose, WT IAV induces significant pathology, and although LAIVwas administered at a level 1.8 Log10 TCID50 lower than that of theWT virus, the LAIV was attenuated. In addition, there are signif-

FIG 4 IAV vaccination impacted IAV burden in the respiratory tract following heterologous challenge. Groups of pigs were inoculated with the indicated vaccineand subsequently challenged intranasally with heterologous virus. Viral titers in NS (A and D), trachea wash fluid (B and E), and lung lavage fluid (C and E) ofpigs challenged with MN/10(�) (A to C) or IL/10(�) (D to F) are indicated. Each dot represents a single animal in that respective treatment group, with themean � SEM for n 8 shown for the group indicated. The Kruskal-Wallis test and Dunn’s multiple-comparison test were used for analysis. P of 0.05 wasconsidered significant. ***, P 0.001; ****, P 0.0001.

LAIV Vaccines and Correlates of Protection

October 2015 Volume 22 Number 10 cvi.asm.org 1115Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

icant reports that truncating the NS1 gene of IAV leads to attenu-ation (33, 34, 53, 54) Despite the dose of LAIV vaccine adminis-tered and the minimal evidence of LAIV replication in vivo, theLAIV elicited a measureable IAV-specific antibody response, sim-ilarly to previous studies (33, 35). Administering a higher dose ofLAIV may increase the degree of cross-protection, similarly to thecross-protection observed with live virus; however, this may nottranslate into a substantial increase in immune responses. In afollow-up study using a 1.5-Log10-higher dose of LAIV adminis-tered i.n. to 21-day-old pigs, minimal LAIV was recovered fromNS and IAV-specific mucosal IgA endpoint titers were not in-creased over the titers observed in the current study (data notshown).

Direct and indirect transmission of IAV is a major animalhealth as well as public health concern. Direct pig-to-pig transmis-sion has been shown with the 2009 pandemic H1N1 virus (55), aswell as indirect aerosol transmission with emerging H3N2 variantviruses (38). Agricultural fairs present a unique interface for in-terspecies transmission of IAV between pigs and people (6, 7, 12).WIV vaccines for IAV have been shown to limit disease but do notalways limit virus shedding (21, 38). An IAV vaccine that limitsnasal shedding of virus would likely have the ability to limit fur-ther transmission; therefore, we evaluated nasal shedding as ameasure of cross-protection following challenge. All animals chal-lenged with MN/10(�) had detectable virus in NS as early as 1 dpi.All immunized animals had reduced viral shedding of MN/10(�)in nasal swabs compared to NV/CH controls at 1 dpi, though all

animals had similar levels of shedding by the end of the study (5dpi). In contrast, LAIV-vaccinated pigs challenged with IL/10(�)demonstrated reduced virus replication in the nose that wascleared by 4 dpi. These reductions in nasal shedding in vaccinated,challenged animals suggests that LAIV vaccination could limit thetransmission of heterologous virus to naive or vaccinated contactanimals (38, 56, 57), thus slowing or breaking the transmissioncycle. Studies are under way to investigate this hypothesis.

A common parameter used to evaluate IAV vaccine efficacy isthe prevention or significant reduction of lung lesions followingchallenge compared to nonvaccinated challenged animal results(45). LAIV-vaccinated animals challenged with IL/10(�) hadpneumonia scores that were not significantly different from thescores calculated for NV/CH controls. However, pneumoniascores following challenge were relatively low across all treatmentgroups, with NV/CH pigs averaging 3.5%, making significant re-ductions in pneumonia difficult to assess. Our observation of lowpneumonia scores following challenge is consistent with previousstudies of H1 IAV intranasal challenge in pigs and is not uncom-mon in 10-week-old pigs (24, 58). Despite the low pneumoniascores, virus was recovered from the lungs of all NV/CH pigs, andIL/10(�) was not recovered from lung lavage fluid or tracheawashes of LAIV-vaccinated pigs, indicating protection against vi-rus replication. The observation of lung pathology in the absenceof virus in the respiratory tract is consistent with previous workevaluating LAIV or Ad5-HA efficacy in pigs (24, 29, 53). WhereasAd5-HA-vaccinated animals had increased macroscopic lung le-

FIG 5 LAIV vaccination elicits mucosal IgA cross-reactive to heterologous H1 and H3 IAV. Nasal wash and oral fluid samples were collected at 42 dpv and used tomeasure levels of cross-reactive IAV-specific IgA by whole-virus ELISA. Samples were tested against representative viruses from each H1 cluster and an H3 virus. (A andB) Cross-reactive IgA data expressed as means � SEM at an optical density (OD) of 405 nm in nasal wash (A) and oral (B) fluids. ns, no significant difference from NVcontrol. (C and D) Reciprocal endpoint titers of IAV-specific IgA averaged by cluster in nasal wash samples pooled by respective group (C) and oral-fluid group (D). Eachdot represents a viral antigen in the indicated cluster with the mean endpoint dilution for the cluster indicated (see Materials and Methods).

Hughes et al.

1116 cvi.asm.org October 2015 Volume 22 Number 10Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

sions following MN/10(�) challenge compared to the NV/CHcontrols (see Fig. S2B in the supplemental material), the micro-scopic lesion characteristics were not consistent with previous de-scriptions of lesions associated with vaccine-associated enhancedrespiratory disease (47). Collectively, these observations suggestthat intranasal LAIV and/or Ad5-HA vaccination primes a muco-sal cell-mediated immune response that is rapidly activated uponheterologous challenge and that this activation may control viralburden but result in immunopathology (59). Future work evalu-ating the mucosal IAV-specific cell-mediated immune responsefollowing intranasal IAV vaccination may provide insight into themechanism associated with pneumonia following heterologouschallenge; however, such an evaluation is unlikely to provide auseful live-animal sample that could be readily used for evaluatingLAIV immunogenicity and cross-protection. While lung lesionsare often used to evaluate IAV vaccine efficacy, together, thesedata indicate that some vaccines may significantly limit virus rep-lication but that lung lesions may not be reduced.

The HI assay is commonly used to evaluate WIV vaccine im-munogenicity and efficacy, and hyperimmune antisera (generatedthrough WIV vaccination) are often used to evaluate antigenicrelatedness between swine IAV strains (17, 60). Previous studieshave shown that LAIV or Ad5-HA vaccination results in lowerserum HI titers to homologous IAV and undetectable cross-reac-tive HI titers to IAV (24–26, 38). Consistent with previous reports,LAIV and Ad5-HA vaccination resulted in low HI titers to homol-ogous IAV and did not elicit cross-reactive HI titers to heterolo-gous IAV in the current study. These data reemphasize that nei-ther LAIV vaccination nor Ad5-HA vaccination induces a robustperipheral HI antibody response and that the commonly usedserum HI assay may not be suitable for predicting LAIV orAd5-HA vaccine efficacy. In contrast, serum virus neutralizationantibody was detected following LAIV vaccination and neutraliza-tion titers may be a useful measure of LAIV immunogenicity andefficacy. The greater sensitivity of the neutralization assay than theHI assay has made it an important tool in the development of IAVvaccines that are poorly immunogenic (37, 61, 62). Serum fromLAIV vaccinates neutralized homologous H1N1pdm09 virus andIL/10(�) virus equally. Pigs exposed to WT NY/09 or LAIV hadsignificant reductions in serum neutralization against MN/10(�)compared to homologous NY/09 virus neutralization. Higher se-rum neutralization titers measured against IL/10(�) virus thanagainst MN/10(�) virus were associated with control of virus rep-lication in the lungs and trachea. These data suggest that the serumneutralization assay may be an effective tool to predict cross-pro-tection of LAIV vaccines in pigs, though further work is warrantedto evaluate cross-reactive titers against the panel of H1 viruses,particularly as it relates to cross-protection.

While serum can be an easy sample source for evaluating vac-cine immunogenicity, compartmentalization of immune re-sponses may limit the utility of peripheral blood and antibodyassays to predict immunogenicity and efficacy associated with in-tranasally delivered vaccines. Previous studies have shown thatIAV antigen-specific antibody can be detected by ELISA in NWand OF samples collected following vaccination or infection (24,38, 39, 63). Experimental IAV infection elicits an IAV-specificantibody response detectable in OF (63), and OF have become arapidly evolving diagnostic sample source for several human andanimal pathogens (63, 64). We detected IAV-specific antibody inboth NW and OF following WT NY/09 exposure and LAIV im-

munization using whole-virus ELISA. OF samples from immu-nized animals had increased IAV-specific antibody compared toNW samples as determined by a standard OD reading performedwith a single dilution of sample or by sample endpoint titration.Previous studies have shown that intranasal LAIV vaccination in-duces a local IAV-specific IgA response measureable by ELISA andthat the response was associated with protection from homolo-gous challenge in humans (65) as well as in pigs (21, 38). Althoughwe did not evaluate protection from homologous challenge, thehigher antibody response observed in OF than in NW followingvaccination would suggest that OF could serve as a sample sourceto evaluate LAIV immunogenicity and homologous protective ef-ficacy as well as provide a measure of herd immune status (63).

Cross-reactive IAV-specific IgA in NW following LAIV vacci-nation has been shown to correlate with improved heterologouscross-protection in humans and in animal models of human IAVinfection (26, 65, 66). Given the reductions in heterologous virusnasal shedding and in measureable IAV-specific IgA levels in mu-cosal samples prechallenge (42 dpv), we evaluated the associationof cross-reactive mucosal IgA in immunized pigs with the level ofcross-protection observed. Both NW and OF had measurablecross-reactive IgA; however, using ELISA OD levels as an associa-tion value, a broadly cross-reactive response to all H1 cluster vi-ruses as well to as an H3 virus was observed, with OD values nearlythe same across heterologous phylogenetic clusters. Using this as-sociation alone, we would have predicted complete protectionagainst both challenge viruses, since mucosal IAV-specific IgA wascross-reactive with both MN/10(�) and IL/10(�) antigens at sim-ilar OD values; however, this was not the case. Endpoint titrationsof IAV-specific IgA in NW and OF from WT NY/09-exposed pigsseparated the data showing cross-reactivity between virus clusters,and a general trend for decreasing IgA endpoint titers was evident,as phylogenetically distant viruses were used as the test antigen(pdm��-cluster��-cluster��-cluster) (19), and these levelswere associated with cross-protection. Due to the reduced immu-nogenicity of the LAIV vaccine, there was no clear associationbetween IgA endpoint titers and cross-protection. Performing thisanalysis with OF samples from WT NY/09-inoculated animals,which had higher IAV-specific IgA titers, did show an associationwith antigenic relatedness, cross-reactive IgA titers, and cross-protection (Fig. 4 and 5B). Collectively, these data suggest thatIAV-specific IgA in OF could be used to assess antigenic related-ness and cross-protection of circulating and emerging IAVs butthat it may require that pigs be inoculated with wild-type virusesto elicit a response such that sufficient levels of mucosal IAV-specific IgA are induced. Vaccine or surveillance researcherswould be able to curate a collection of OF samples from IAV-infected pigs and measure the antigenic relatedness of the IAVimmunogen to currently circulating IAV strains and update andmodify vaccine strains as necessary. This approach is somewhatsimilar to that of human seasonal IAV vaccine selection, whereferrets are infected with live IAV to elicit a high serum HI titer inorder to assess the antigenic relatedness of circulating IAV isolates(67, 68).

ACKNOWLEDGMENTS

We thank Zahra Olson, Lilia Walther, and Steven Kellner for technicalassistance and Jason Huegel, Tyler Standley, and Jason Crabtree for assis-tance with animal work.

Mention of trade names or products is solely for the purpose of pro-

LAIV Vaccines and Correlates of Protection

October 2015 Volume 22 Number 10 cvi.asm.org 1117Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

viding specific information and does not imply endorsement by theUSDA. USDA is an equal opportunity provider and employer.

The findings and conclusions in the manuscript are ours and do notnecessarily represent the views of the USDA.

Funding for this work is provided in part by The National Pork Board(no. 13-122) and Boehringer-Ingelheim Vetmedica, Inc.

REFERENCES1. Kreijtz JH, Fouchier RA, Rimmelzwaan GF. 2011. Immune responses to

influenza virus infection. Virus Res 162:19 –30. http://dx.doi.org/10.1016/j.virusres.2011.09.022.

2. Sridhar S, Begom S, Bermingham A, Hoschler K, Adamson W, CarmanW, Bean T, Barclay W, Deeks JJ, Lalvani A. 2013. Cellular immunecorrelates of protection against symptomatic pandemic influenza. NatMed 19:1305–1312. http://dx.doi.org/10.1038/nm.3350.

3. Ito T. 2000. Interspecies transmission and receptor recognition of influ-enza A viruses. Microbiol Immunol 44:423– 430. http://dx.doi.org/10.1111/j.1348-0421.2000.tb02516.x.

4. Matrosovich MN, Gambaryan AS, Teneberg S, Piskarev VE, YamnikovaSS, Lvov DK, Robertson JS, Karlsson KA. 1997. Avian influenza Aviruses differ from human viruses by recognition of sialyloligosaccharidesand gangliosides and by a higher conservation of the HA receptor-bindingsite. Virology 233:224 –234. http://dx.doi.org/10.1006/viro.1997.8580.

5. Bowman AS, Nelson SW, Page SL, Nolting JM, Killian ML, SreevatsanS, Slemons RD. 2014. Swine-to-human transmission of influenzaA(H3N2) virus at agricultural fairs, Ohio, USA, 2012. Emerg Infect Dis20:1472–1480. http://dx.doi.org/10.3201/eid2009.131082.

6. Feng Z, Gomez J, Bowman AS, Ye J, Long LP, Nelson SW, Yang J,Martin B, Jia K, Nolting JM, Cunningham F, Cardona C, Zhang J, YoonKJ, Slemons RD, Wan XF. 2013. Antigenic characterization of H3N2influenza A viruses from Ohio agricultural fairs. J Virol 87:7655–7667.http://dx.doi.org/10.1128/JVI.00804-13.

7. Vincent AL, Swenson SL, Lager KM, Gauger PC, Loiacono C, Zhang Y.2009. Characterization of an influenza A virus isolated from pigs during anoutbreak of respiratory disease in swine and people during a county fair inthe United States. Vet Microbiol 137:51–59. http://dx.doi.org/10.1016/j.vetmic.2009.01.003.

8. Altmüller A, Kunerl M, Muller K, Hinshaw VS, Fitch WM, ScholtissekC. 1992. Genetic relatedness of the nucleoprotein (NP) of recent swine,turkey, and human influenza A virus (H1N1) isolates. Virus Res 22:79 –87. http://dx.doi.org/10.1016/0168-1702(92)90091-M.

9. Choi YK, Lee JH, Erickson G, Goyal SM, Joo HS, Webster RG, WebbyRJ. 2004. H3N2 influenza virus transmission from swine to turkeys,United States. Emerg Infect Dis 10:2156 –2160. http://dx.doi.org/10.3201/eid1012.040581.

10. Choi YK, Nguyen TD, Ozaki H, Webby RJ, Puthavathana P, BuranathalC, Chaisingh A, Auewarakul P, Hanh NT, Ma SK, Hui PY, Guan Y,Peiris JS, Webster RG. 2005. Studies of H5N1 influenza virus infection ofpigs by using viruses isolated in Vietnam and Thailand in 2004. J Virol79:10821–10825. http://dx.doi.org/10.1128/JVI.79.16.10821-10825.2005.

11. Wideman G. 2013. Impact of influenza A in pork production, p 191–193.Proceedings of the London Swine Conference, London, Ontario, Canada.

12. Bowman AS, Nolting JM, Nelson SW, Slemons RD. 2012. Subclinicalinfluenza virus A infections in pigs exhibited at agricultural fairs, Ohio,USA, 2009 –2011. Emerg Infect Dis 18:1945–1950. http://dx.doi.org/10.3201/eid1812.121116.

13. Vincent AL, Ma W, Lager KM, Janke BH, Richt JA. 2008. Swineinfluenza viruses: a North American perspective. Adv Virus Res 72:127–154. http://dx.doi.org/10.1016/S0065-3527(08)00403-X.

14. Zhou NN, Senne DA, Landgraf JS, Swenson SL, Erickson G, Rossow K,Liu L, Yoon K, Krauss S, Webster RG. 1999. Genetic reassortment ofavian, swine, and human influenza A viruses in American pigs. J Virol73:8851– 8856.

15. Vincent AL, Ma W, Lager KM, Gramer MR, Richt JA, Janke BH. 2009.Characterization of a newly emerged genetic cluster of H1N1 and H1N2swine influenza virus in the United States. Virus Genes 39:176 –185. http://dx.doi.org/10.1007/s11262-009-0386-6.

16. Ma W, Vincent AL, Lager KM, Janke BH, Henry SC, Rowland RR, HesseRA, Richt JA. 2010. Identification and characterization of a highly virulenttriple reassortant H1N1 swine influenza virus in the United States. VirusGenes 40:28–36. http://dx.doi.org/10.1007/s11262-009-0413-7.

17. Lorusso A, Vincent AL, Harland ML, Alt D, Bayles DO, Swenson SL,

Gramer MR, Russell CA, Smith DJ, Lager KM, Lewis NS. 2011. Geneticand antigenic characterization of H1 influenza viruses from United Statesswine from 2008. J Gen Virol 92:919 –930. http://dx.doi.org/10.1099/vir.0.027557-0.

18. Nfon CK, Berhane Y, Hisanaga T, Zhang S, Handel K, Kehler H,Labrecque O, Lewis NS, Vincent AL, Copps J, Alexandersen S, Pasick J.2011. Characterization of H1N1 swine influenza viruses circulating inCanadian pigs in 2009. J Virol 85:8667– 8679. http://dx.doi.org/10.1128/JVI.00801-11.

19. Anderson TK, Campbell BA, Nelson MI, Lewis NS, Janas-Martindale A,Killian ML, Vincent AL. 2015. Characterization of co-circulating swineinfluenza A viruses in North America and the identification of a novel H1genetic clade with antigenic significance. Virus Res 201:24 –31. http://dx.doi.org/10.1016/j.virusres.2015.02.009.

20. Gauger PC, Loving CL, Vincent AL. 2014. Enzyme-linked immunosor-bent assay for detection of serum or mucosal isotype-specific IgG and IgAwhole-virus antibody to influenza A virus in swine. Methods Mol Biol1161:303–312. http://dx.doi.org/10.1007/978-1-4939-0758-8_25.

21. Heinen PP, van Nieuwstadt AP, de Boer-Luijtze EA, Bianchi AT. 2001.Analysis of the quality of protection induced by a porcine influenza Avaccine to challenge with an H3N2 virus. Vet Immunol Immunopathol82:39 –56. http://dx.doi.org/10.1016/S0165-2427(01)00342-7.

22. Vincent AL, Ciacci-Zanella JR, Lorusso A, Gauger PC, Zanella EL,Kehrli ME, Jr, Janke BH, Lager KM. 2010. Efficacy of inactivated swineinfluenza virus vaccines against the 2009 A/H1N1 influenza virus in pigs.Vaccine 28:2782–2787. http://dx.doi.org/10.1016/j.vaccine.2010.01.049.

23. Chen Q, Madson D, Miller CL, Harris DL. 2012. Vaccine developmentfor protecting swine against influenza virus. Anim Health Res Rev 13:181–195. http://dx.doi.org/10.1017/S1466252312000175.

24. Braucher DR, Henningson JN, Loving CL, Vincent AL, Kim E, Steitz J,Gambotto AA, Kehrli ME, Jr. 2012. Intranasal vaccination with replica-tion-defective adenovirus type 5 encoding influenza virus hemagglutininelicits protective immunity to homologous challenge and partial protec-tion to heterologous challenge in pigs. Clin Vaccine Immunol 19:1722–1729. http://dx.doi.org/10.1128/CVI.00315-12.

25. Vincent AL, Ma W, Lager KM, Janke BH, Webby RJ, Garcia-Sastre A,Richt JA. 2007. Efficacy of intranasal administration of a truncated NS1modified live influenza virus vaccine in swine. Vaccine 25:7999 – 8009.http://dx.doi.org/10.1016/j.vaccine.2007.09.019.

26. Gustin KM, Maines TR, Belser JA, van Hoeven N, Lu X, Dong L,Isakova-Sivak I, Chen LM, Voeten JT, Heldens JG, van den Bosch H,Cox NJ, Tumpey TM, Klimov AI, Rudenko L, Donis RO, Katz JM.2011. Comparative immunogenicity and cross-clade protective efficacy ofmammalian cell-grown inactivated and live attenuated H5N1 reassortantvaccines in ferrets. J Infect Dis 204:1491–1499. http://dx.doi.org/10.1093/infdis/jir596.

27. Gauger PC, Loving CL, Khurana S, Lorusso A, Perez DR, Kehrli ME,Jr, Roth JA, Golding H, Vincent AL. 2014. Live attenuated influenzaA virus vaccine protects against A(H1N1)pdm09 heterologous chal-lenge without vaccine associated enhanced respiratory disease. Virol-ogy 471– 473:93–104.

28. Masic A, Booth JS, Mutwiri GK, Babiuk LA, Zhou Y. 2009. Elastase-dependent live attenuated swine influenza A viruses are immunogenic andconfer protection against swine influenza A virus infection in pigs. J Virol83:10198 –10210. http://dx.doi.org/10.1128/JVI.00926-09.

29. Sandbulte MR, Platt R, Roth JA, Henningson JN, Gibson KA, Rajao DS,Loving CL, Vincent AL. 2014. Divergent immune responses and diseaseoutcomes in piglets immunized with inactivated and attenuated H3N2swine influenza vaccines in the presence of maternally-derived antibodies.Virology 464 – 465:45–54.

30. Vincent AL, Ma W, Lager KM, Richt JA, Janke BH, Sandbulte MR,Gauger PC, Loving CL, Webby RJ, Garcia-Sastre A. 2012. Live attenu-ated influenza vaccine provides superior protection from heterologousinfection in pigs with maternal antibodies without inducing vaccine-associated enhanced respiratory disease. J Virol 86:10597–10605. http://dx.doi.org/10.1128/JVI.01439-12.

31. Wesley RD, Lager KM. 2006. Overcoming maternal antibody interfer-ence by vaccination with human adenovirus 5 recombinant viruses ex-pressing the hemagglutinin and the nucleoprotein of swine influenza vi-rus. Vet Microbiol 118:67–75. http://dx.doi.org/10.1016/j.vetmic.2006.07.014.

32. Wang X, Li M, Zheng H, Muster T, Palese P, Beg AA, Garcia-Sastre A.2000. Influenza A virus NS1 protein prevents activation of NF-kappaB and

Hughes et al.

1118 cvi.asm.org October 2015 Volume 22 Number 10Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

induction of alpha/beta interferon. J Virol 74:11566 –11573. http://dx.doi.org/10.1128/JVI.74.24.11566-11573.2000.

33. Talon J, Salvatore M, O’Neill RE, Nakaya Y, Zheng H, Muster T,Garcia-Sastre A, Palese P. 2000. Influenza A and B viruses expressingaltered NS1 proteins: a vaccine approach. Proc Natl Acad Sci U S A 97:4309 – 4314. http://dx.doi.org/10.1073/pnas.070525997.

34. Solórzano A, Webby RJ, Lager KM, Janke BH, Garcia-Sastre A, RichtJA. 2005. Mutations in the NS1 protein of swine influenza virus impairanti-interferon activity and confer attenuation in pigs. J Virol 79:7535–7543. http://dx.doi.org/10.1128/JVI.79.12.7535-7543.2005.

35. Richt JA, Lekcharoensuk P, Lager KM, Vincent AL, Loiacono CM,Janke BH, Wu WH, Yoon KJ, Webby RJ, Solorzano A, Garcia-Sastre A.2006. Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine. J Virol 80:11009 –11018. http://dx.doi.org/10.1128/JVI.00787-06.

36. Wesley RD, Tang M, Lager KM. 2004. Protection of weaned pigs byvaccination with human adenovirus 5 recombinant viruses expressing thehemagglutinin and the nucleoprotein of H3N2 swine influenza virus. Vac-cine 22:3427–3434. http://dx.doi.org/10.1016/j.vaccine.2004.02.040.

37. Katz JM, Hancock K, Xu X. 2011. Serologic assays for influenza surveil-lance, diagnosis and vaccine evaluation. Expert Rev Anti Infect Ther9:669 – 683. http://dx.doi.org/10.1586/eri.11.51.

38. Loving CL, Lager KM, Vincent AL, Brockmeier SL, Gauger PC, Ander-son TK, Kitikoon P, Perez DR, Kehrli ME, Jr. 2013. Efficacy in pigs ofinactivated and live attenuated influenza virus vaccines against infectionand transmission of an emerging H3N2 similar to the 2011–2012 H3N2v.J Virol 87:9895–9903. http://dx.doi.org/10.1128/JVI.01038-13.

39. Clements ML, Murphy BR. 1986. Development and persistence of localand systemic antibody responses in adults given live attenuated or inacti-vated influenza A virus vaccine. J Clin Microbiol 23:66 –72.

40. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. 2000.A DNA transfection system for generation of influenza A virus from eightplasmids. Proc Natl Acad Sci U S A 97:6108 – 6113. http://dx.doi.org/10.1073/pnas.100133697.

41. Pena L, Vincent AL, Ye J, Ciacci-Zanella JR, Angel M, Lorusso A,Gauger PC, Janke BH, Loving CL, Perez DR. 2011. Modifications in thepolymerase genes of a swine-like triple-reassortant influenza virus to gen-erate live attenuated vaccines against 2009 pandemic H1N1 viruses. J Virol85:456 – 469. http://dx.doi.org/10.1128/JVI.01503-10.

42. Loving CL, Kehrli ME, Jr, Brockmeier SL, Bayles DO, Michael DD,Schlink SN, Lager KM. 2013. Porcine granulocyte-colony stimulatingfactor (G-CSF) delivered via replication-defective adenovirus induces asustained increase in circulating peripheral blood neutrophils. Biologicals41:368 –376. http://dx.doi.org/10.1016/j.biologicals.2013.07.001.

43. Loving CL, Brockmeier SL, Vincent AL, Palmer MV, Sacco RE, Nich-olson TL. 2010. Influenza virus coinfection with Bordetella bronchisep-tica enhances bacterial colonization and host responses exacerbating pul-monary lesions. Microb Pathog 49:237–245. http://dx.doi.org/10.1016/j.micpath.2010.06.004.

44. Ramirez A, Wang C, Prickett JR, Pogranichniy R, Yoon KJ, Main R,Johnson JK, Rademacher C, Hoogland M, Hoffmann P, Kurtz A,Kurtz E, Zimmerman J. 2012. Efficient surveillance of pig populationsusing oral fluids. Prev Vet Med 104:292–300. http://dx.doi.org/10.1016/j.prevetmed.2011.11.008.

45. Richt JA, Lager KM, Janke BH, Woods RD, Webster RG, Webby RJ.2003. Pathogenic and antigenic properties of phylogenetically distinct re-assortant H3N2 swine influenza viruses cocirculating in the United States.J Clin Microbiol 41:3198 –3205. http://dx.doi.org/10.1128/JCM.41.7.3198-3205.2003.

46. Halbur PG, Paul PS, Frey ML, Landgraf J, Eernisse K, Meng XJ, LumMA, Andrews JJ, Rathje JA. 1995. Comparison of the pathogenicity oftwo US porcine reproductive and respiratory syndrome virus isolates withthat of the Lelystad virus. Vet Pathol 32:648 – 660. http://dx.doi.org/10.1177/030098589503200606.

47. Gauger PC, Vincent AL, Loving CL, Henningson JN, Lager KM, JankeBH, Kehrli ME, Jr, Roth JA. 2012. Kinetics of lung lesion developmentand pro-inflammatory cytokine response in pigs with vaccine-associatedenhanced respiratory disease induced by challenge with pandemic (2009)A/H1N1 influenza virus. Vet Pathol 49:900 –912. http://dx.doi.org/10.1177/0300985812439724.

48. Vincent AL, Lager KM, Ma W, Lekcharoensuk P, Gramer MR, Loi-acono C, Richt JA. 2006. Evaluation of hemagglutinin subtype 1 swine

influenza viruses from the United States. Vet Microbiol 118:212–222.http://dx.doi.org/10.1016/j.vetmic.2006.07.017.

49. Kitikoon P, Nilubol D, Erickson BJ, Janke BH, Hoover TC, Sornsen SA,Thacker EL. 2006. The immune response and maternal antibody inter-ference to a heterologous H1N1 swine influenza virus infection followingvaccination. Vet Immunol Immunopathol 112:117–128. http://dx.doi.org/10.1016/j.vetimm.2006.02.008.

50. Reed LJ, Muench H. 1938. A simple method for estimating fifty percentendpoints. Am J Hyg (Lond) 27:493– 497.

51. Belshe RB. 2004. Current status of live attenuated influenza virus vaccinein the US. Virus Res 103:177–185. http://dx.doi.org/10.1016/j.virusres.2004.02.031.

52. Paillot R, Hannant D, Kydd JH, Daly JM. 2006. Vaccination againstequine influenza: quid novi? Vaccine 24:4047– 4061. http://dx.doi.org/10.1016/j.vaccine.2006.02.030.

53. Kappes MA, Sandbulte MR, Platt R, Wang C, Lager KM, HenningsonJN, Lorusso A, Vincent AL, Loving CL, Roth JA, Kehrli ME, Jr. 2012.Vaccination with NS1-truncated H3N2 swine influenza virus primes Tcells and confers cross-protection against an H1N1 heterosubtypic chal-lenge in pigs. Vaccine 30:280 –288. http://dx.doi.org/10.1016/j.vaccine.2011.10.098.

54. Wacheck V, Egorov A, Groiss F, Pfeiffer A, Fuereder T, Hoeflmayer D,Kundi M, Popow-Kraupp T, Redlberger-Fritz M, Mueller CA, Cinatl J,Michaelis M, Geiler J, Bergmann M, Romanova J, Roethl E, MorokuttiA, Wolschek M, Ferko B, Seipelt J, Dick-Gudenus R, Muster T. 2010. Anovel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonistNS1. J Infect Dis 201:354 –362. http://dx.doi.org/10.1086/649428.

55. Lange E, Kalthoff D, Blohm U, Teifke JP, Breithaupt A, Maresch C,Starick E, Fereidouni S, Hoffmann B, Mettenleiter TC, Beer M, Vahl-enkamp TW. 2009. Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1 after experimental infection of pigs. J GenVirol 90:2119 –2123. http://dx.doi.org/10.1099/vir.0.014480-0.

56. Price GE, Lo CY, Misplon JA, Epstein SL. 2014. Mucosal immuni-zation with a candidate universal influenza vaccine reduces virus trans-mission in a mouse model. J Virol 88:6019 – 6030. http://dx.doi.org/10.1128/JVI.03101-13.

57. Houser KV, Pearce MB, Katz JM, Tumpey TM. 2013. Impact of priorseasonal H3N2 influenza vaccination or infection on protection andtransmission of emerging variants of influenza A(H3N2)v virus in ferrets.J Virol 87:13480 –13489. http://dx.doi.org/10.1128/JVI.02434-13.

58. Vincent AL, Lager KM, Janke BH, Gramer MR, Richt JA. 2008. Failureof protection and enhanced pneumonia with a US H1N2 swine influenzavirus in pigs vaccinated with an inactivated classical swine H1N1 vaccine.Vet Microbiol 126:310 –323. http://dx.doi.org/10.1016/j.vetmic.2007.07.011.

59. La Gruta NL, Kedzierska K, Stambas J, Doherty PC. 2007. A question ofself-preservation: immunopathology in influenza virus infection. Immu-nol Cell Biol 85:85–92. http://dx.doi.org/10.1038/sj.icb.7100026.

60. Lewis NS, Anderson TK, Kitikoon P, Skepner E, Burke DF, Vincent AL.2014. Substitutions near the hemagglutinin receptor-binding site deter-mine the antigenic evolution of influenza A H3N2 viruses in U.S. swine. JVirol 88:4752– 4763. http://dx.doi.org/10.1128/JVI.03805-13.

61. Cox RJ, Madhun AS, Hauge S, Sjursen H, Major D, Kuhne M, HoschlerK, Saville M, Vogel FR, Barclay W, Donatelli I, Zambon M, Wood J,Haaheim LR. 2009. A phase I clinical trial of a PER.C6 cell grown influ-enza H7 virus vaccine. Vaccine 27:1889 –1897. http://dx.doi.org/10.1016/j.vaccine.2009.01.116.

62. Keitel WA, Atmar RL. 2009. Vaccines for pandemic influenza: summaryof recent clinical trials. Curr Top Microbiol Immunol 333:431– 451.

63. Panyasing Y, Goodell CK, Gimenez-Lirola L, Kittawornrat A, Wang C,Schwartz KJ, Zimmerman JJ. 2013. Kinetics of influenza A virus nucleo-protein antibody (IgM, IgA, and IgG) in serum and oral fluid specimensfrom pigs infected under experimental conditions. Vaccine 31:6210 –6215. http://dx.doi.org/10.1016/j.vaccine.2013.10.040.

64. Nokes DJ, Enquselassie F, Nigatu W, Vyse AJ, Cohen BJ, BrownDW, Cutts FT. 2001. Has oral fluid the potential to replace serum forthe evaluation of population immunity levels? A study of measles,rubella and hepatitis B in rural Ethiopia. Bull World Health Organ79:588 –595.

65. Clements ML, Betts RF, Tierney EL, Murphy BR. 1986. Serum and nasalwash antibodies associated with resistance to experimental challenge withinfluenza A wild-type virus. J Clin Microbiol 24:157–160.

LAIV Vaccines and Correlates of Protection

October 2015 Volume 22 Number 10 cvi.asm.org 1119Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

66. Asahi-Ozaki Y, Yoshikawa T, Iwakura Y, Suzuki Y, Tamura S, Kurata T,Sata T. 2004. Secretory IgA antibodies provide cross-protection againstinfection with different strains of influenza B virus. J Med Virol 74:328 –335. http://dx.doi.org/10.1002/jmv.20173.

67. Russell CA, Jones TC, Barr IG, Cox NJ, Garten RJ, Gregory V, Gust ID,Hampson AW, Hay AJ, Hurt AC, de Jong JC, Kelso A, Klimov AI,Kageyama T, Komadina N, Lapedes AS, Lin YP, Mosterin A, Obuchi M,Odagiri T, Osterhaus AD, Rimmelzwaan GF, Shaw MW, Skepner E,

Stohr K, Tashiro M, Fouchier RA, Smith DJ. 2008. Influenza vaccinestrain selection and recent studies on the global migration of seasonalinfluenza viruses. Vaccine 26(Suppl 4):D31–D34. http://dx.doi.org/10.1016/j.vaccine.2008.07.078.

68. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF,Osterhaus AD, Fouchier RA. 2004. Mapping the antigenic and geneticevolution of influenza virus. Science 305:371–376. http://dx.doi.org/10.1126/science.1097211.

Hughes et al.

1120 cvi.asm.org October 2015 Volume 22 Number 10Clinical and Vaccine Immunology

on October 7, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from