Reactivity and Cross-Protection following Intranasal Influenza A Virus
Vaccination in Pigs
Holly R. Hughes,aAmy L. Vincent,aSusan L. Brockmeier,aPhillip C. Gauger,bLindomar Pena,cJefferson 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 of
Maryland, 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 not induce a robust serum HI antibody titer; however, a local mucosal antibody response is elicited. Thus, a live-animal sample source that could be used to evaluate LAIV immunogenicity and cross-protection is needed. Here, we evaluated the use of oral fluids (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 LAIV vaccination 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. While LAIV vaccination provided significant protection, LAIV immunogenicity was reduced compared to live-virus exposure. These data suggest that OF from pigs inoculated with wild-type IAV, with surface genes that match the LAIV seed strain, could be used in an ELISA to assess cross-protection and the antigenic relatedness of circulating and emerging IAV in swine.
I
nfluenza A virus (IAV), of the familyOrthomyxoviridae, infects many 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 lipid bilayer, form the envelope of IAV. The HA and NA proteins play essential roles in virus entry and release of progeny, respectively, and are primary immunogens involved in a protective antibody response (reviewed in reference1), while internal genes such as nucleoprotein (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 pigs contain receptors for both avian and mammalian IAV (3,4). In-terspecies transmission of IAV is a real concern and has been well documented, 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 public health concern because of noted spillover events. Routine IAV vaccination is an important tool in the management of animal health to reduce economic loss associated with IAV infection (11) and for pigs displayed at agricultural fairs (12) in terms of both public and animal health.
One of the main roadblocks for swine IAV vaccine develop-ment is the presence of multiple antigenic variants within H1 and H3 subtypes, which results in the need for vaccines that provide cross-protection. Classical H1N1 (cH1N1) was the predominant IAV in U.S. swine until the introduction of the novel H3N2 strain in 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 and genetic 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), and pandemic (15–19). There is limited hemagglutination-inhibiting (HI) cross-reactivity between H1 clusters when using pig hyper-immune serum (derived through vaccination with adjuvanted,
Received1 July 2015 Returned for modification21 July 2015 Accepted13 August 2015
Accepted manuscript posted online19 August 2015
CitationHughes 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 source for evaluating cross-reactivity and cross-protection following intranasal influenza A 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, [email protected].
*Present address: Douglas R. Braucher, Boehringer Ingelheim Vetmedica Inc., Ames, Iowa, USA; Daniel R. Perez, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA.
Supplemental material for this article may be found athttp://dx.doi.org/10.1128 /CVI.00358-15.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/CVI.00358-15
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monovalent whole-inactivated virus [WIV] vaccine) to evaluate antigenic relatedness (17,18).
The majority of currently licensed IAV vaccines for swine are adjuvanted, WIV vaccines given by the intramuscular (i.m.) route. This vaccine formulation induces IAV-specific antibody in serum as measured by the HI assay or a whole-virus enzyme-linked im-munosorbent assay (ELISA) (20). WIV vaccines primarily provide protection against homologous or antigenically related strains but provide 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-mediated immune responses (21). To overcome limitations associated with WIV vaccines, several next-generation IAV vaccines, including live-attenuated influenza virus (LAIV) vaccines and replication-defective adenovirus vectors that encode IAV genes, have been experimentally 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 immunity does 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 a consequence of its type I interferon (IFN) antagonist activity (32). Introducing nucleotide mutations into the NS1 gene causes the loss of type I IFN inhibition; thus, the host IFN response inhibits growth of the virus (33). Through truncation of the last 93 amino acids of the NS1 IAV protein, an LAIV was generated that was shown to induce a robust IFN response and to be attenuated in pigs (34). This truncated NS1 LAIV with H3 surface genes was further shown to elicit protection against homologous and heter-ologous H3 challenge viruses, as well as partial heterosubtypic protection when vaccinated pigs were challenged with H1 virus (35).
Human replication-defective adenovirus serotype 5 encoding HA (Ad5-HA) has been shown experimentally to provided ho-mologous protection (24,31,36) when administered by either the intramuscular 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 provide partial heterologous protection when administered intranasally (24).
Despite advantages of LAIV- and Ad5-based vaccines, these platforms have yet to come to market. A live-animal sample and assay to predict vaccine efficacy and cross-protection, as well as additional cross-protection studies using contemporary viruses, will aid in the continued development of these platforms. Serum HI titer is the established gold-standard method to predict IAV cross-reactivity and, subsequently, the cross-protective efficacy of a particular vaccine. A reciprocal serum HI titer of⬎40 is usually considered protective (correlated with a 50% reduction in the risk of IAV infection [37]), and a greater than 4-fold reduction in HI titer between viruses is considered to represent significant anti-genic drift with a predicted loss in cross-protection (37). LAIV vaccination elicits modest serum HI antibody responses to ho-mologous antigens, but LAIV also provides protection against heterologous IAV in which LAIV antibodies did not cross-react (25,38). Thus, HI titers following LAIV vaccination may be useful for evaluating vaccine immunogenicity and predicting
homolo-gous protection, but predictions of cross-protection using this gold standard method are unreliable.
Since intranasal LAIV vaccination elicits IAV-specific antibody at mucosal surfaces (29,39), it is possible that a live-animal mu-cosal sample would be better for evaluating intranasal IAV vaccine immunogenicity and cross-protective efficacy. To test this hy-pothesis, a study was completed in which groups of pigs were exposed to wild-type (WT) IAV, LAIV, or Ad5-HA, all with a pandemic lineage surface gene(s), for collection of mucosal and serum samples postexposure. Vaccinated pigs were then chal-lenged with either a heterologous-cluster or␥-cluster IAV to evaluate 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, WT IAV inoculation or LAIV or Ad5-HA vaccination provided some level of cross-protection (depending on the challenge strain), and immunogenicity likely impacted the extent of cross-protective ef-ficacy. IAV-specific IgA in oral fluids was found to be associated with cross-protective efficacy; the data may serve as predictive indices, and additional work is warranted to further validate these findings.
MATERIALS AND METHODS
Ethics statement.Animal experiments were approved by the Institutional Animal Care and Use Committee of the National Animal Disease Center in 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 pandemic H1N1 (H1N1pdm09) (pdm) surface genes from A/New York/18/2009 (NY/09) with the A/turkey/Ohio/313053/2004 (H3N2) internal genes, in which 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 attenuation mutations introduced) as previously described (40,41) and is referred to as RG NY/09. The replication-defective adenovirus type 5 vector HA (Ad5-HA) vaccine was derived using an AdEasy system (Agilent, Santa Clara, CA) by cloning the A/California/04/2009 HA gene into the pShuttle-cytomegalovirus (CMV) vector, and adenovirus rescue was per-formed as previously described (42). Heterologous challenge viruses were H1N1 -cluster A/swine/Minnesota/03012/2010 virus (MN/10) and H1N2 ␥-cluster A/swine/Illinois/3134/2010 IAV (IL/10) obtained through submission of clinical samples to the University of Minnesota Veterinary 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 cells with high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum. Ad5-HA was purified using a double discontinuous cesium chloride gradient per the 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 a high-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 the manufacturers. IAV antibody status was confirmed to be negative using the IDEXX Multi-Screen Ab test (IDEXX, Westbrook, ME). In part I of the experiment (attenuation), piglets were randomly assigned to groups Hughes et al.
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of eight and immunized i.n. at 4 weeks of age. Groups received LAIV, WT NY/09, or RG NY/09 parent virus or were not vaccinated (NV). Pigs were humanely euthanized at 3 days postvaccination (dpv) to assess the atten-uation and pathogenicity of the LAIV compared to the wild-type parent virus. 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 the same dose (Table 1). LAIV vaccine and WT NY/09 virus were targeted at 10650% tissue culture infective doses (TCID
50)/ml and at 2 ml per dose.
The back titer of the inoculum after administration indicated that the WT NY/09 dose was 105.5TCID
50per pig, whereas the LAIV dose was 104.2
TCID50per pig. Pigs in the Ad5-HA group received 1010TCID50in 2 ml
i.n. Nonvaccinated (NV) controls received 2 ml PBS i.n. Three weeks following the boost, all pigs except for the nonvaccinated/nonchallenged (NV/NC) controls were challenged i.n. with 105.8TCID
50MN/10() or
IL/10(␥). Pigs were humanely euthanized at 5 days postinfection (dpi) for collection 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 3 postpriming. 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 was collected by centrifugation at 800⫻gfor 20 min, divided into aliquots, and frozen at⫺80°C. NW were collected by instilling 5 ml PBS into a naris and collecting effluent as previously described (43), divided into aliquots, and frozen at⫺80°C. Typically, 0.5 to 2 ml of NW was collected. OF were obtained following previously described methods (44) with few modifications. Briefly, cotton ropes were hung in each room for approx-imately 30 min for the pigs to chew. Ropes from each group were placed into separate ziplock bags. The rope was manually squeezed inside the bag and liquid sample decanted into a 50-ml conical tube. The tubes were centrifuged at 800⫻g for 20 min, and the supernatant was filtered through 0.45-m-pore-size syringe filters and immediately frozen at ⫺80°C. For postchallenge sample collection, nasal swabs were collected daily beginning on the day of challenge (0 dpi) through necropsy (5 dpi) using polyester-tipped swabs (Puritan, Guilford, ME) prewetted in 2 ml minimal essential media (MEM) and stored frozen. At necropsy, trachea wash was obtained by removing the trachea prior to lung lavage. The trachea, from 1 cm below the larynx to 2 cm above the bifurcation, was removed and submerged in 3 ml MEM and vigorously agitated for 15 s. The trachea was then removed, and the medium was aliquoted and frozen at⫺80°C. Lung lavage samples were obtained by lavage performed with 50 ml MEM as previously described (38). An aliquot of lung lavage fluid was 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 out bacterial infection. All postchallenge samples were stored on ice until they were 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 was evaluated (45). The total percentage of pneumonia was calculated on the basis of the weighted proportions of each lobe with respect to the total lung 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 were evaluated and scored by a veterinary pathologist blinded to the treatment groups using parameters previously described (47).
Antibody evaluation.The HI assay was performed as recommended in the WHO animal influenza training manual using turkey red blood cells and WT NY/09 virus or H1 challenge virus as the target antigen as previously described (41). The HI assay using OF and NW was performed following the same protocol as the serum HI assay, beginning with an initial dilution of 1:10. Data are expressed as the reciprocal titer. The serum neutralization assay was performed by generating serial 2-fold di-lutions of heat-inactivated sera in MEM supplemented with 5% bovine serum albumin and antibiotics and by incubating with 100 TCID50before
inoculation of confluent MDCK monolayers in 96-well plates as previ-ously described (48). Cells were incubated for 48 h, fixed, and stained for viral nucleoprotein (NP) antigen by immunocytochemistry (49). OF and NW neutralization assays were performed similarly except that the NW samples were not heat inactivated prior to use in the neutralization assay. Reciprocal titers were Log2transformed and used for statistical analysis of
HI and neutralization titer data. IAV-specific IgA and IgG levels in the OF and the NW were recorded as the optical density (OD) value using an indirect whole-virus ELISA and the viruses listed inTable 2as previously described (38), with the exception that the NW and OF samples were diluted 1:2 in PBS for use in the assay. Antibody levels were reported as the mean OD at 405 nm for each vaccine group. Endpoint antibody titers were obtained by initially diluting OF and NW samples (pooled by treatment group) 1:2 in PBS and titrating 2-fold in duplicate before performing the ELISA. The resulting OD data were modeled as a nonlinear function of the Log10dilution using the GraphPad Prism (GraphPad software Inc., La
Jolla, CA) Log (agonist) versus response-variable slope four-parameter logistic model. Endpoints were interpolated by using 2⫻the average OD of the nonvaccinated control as the cutoff. Endpoints for each virus were organized by cluster, and average cluster endpoint dilutions are expressed for both OF and NW. Total IgA and IgG ELISAs were performed using a pig 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 the TCID50/ml as previously described (38). Briefly, frozen samples were
thawed 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) (1g/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 1Experimental design for evaluation of IAV vaccine immunogenicity and cross-protective efficacya
Group (n⫽8) Vaccine Challenge virus
NV/NC None None
NV/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 2IAV isolates used to evaluate antibody responses following intranasal immunization
Cluster Virus name Abbreviation Subtype
Pandemic A/NY/18/2009 NY/09 H1N1
Pandemic A/CA/04/2009 CA/09 H1N1
Pandemic 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 H1N2 H3 A/turkey/OH/313053/2004 OH/04 H3N2
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tion, the Log10transformed TCID50/ml was calculated for each sample using the method of Reed and Muench (50).
Statistical analysis.Reciprocal HI and neutralization titers were Log2 transformed and viral titers were Log10transformed for analysis and ex-pressed as reciprocal titer values. ELISA data are presented as the average optical density at 405 nm. Statistical analysis was completed with Graph Pad prism version 6 using the Kruskal-Wallis test followed by Dunn’s multiple comparison or using two-way analysis of variance (ANOVA) and Tukey’s multiple comparison where appropriate.
RESULTS
Attenuation 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 lung lesions (pneumonia) at 3 dpv were significantly reduced in pigs inoculated with LAIV compared to those in pigs inoculated with RG NY/09 (Fig. 1A). Amounts of WT NY/09 virus were detected in nasal swabs by 1 dpv, peaked at 2 dpv, and began to decline by 3 dpv. The amount of virus detected in nasal swabs from pigs inoculated with LAIV was significantly reduced at each time point tested compared to the amount of virus detected in nasal swabs from pigs exposed to WT NY/09 and was significantly reduced at 3 dpv compared to the amount detected in pigs exposed to RG NY/09 (average, 0.03 versus 1.8 Log10TCID50/ml) (Fig. 1B). Pigs
inoculated with LAIV had significantly reduced mean viral titers in the trachea (0.2 Log10TCID50/ml) compared to animals
ex-posed to WT NY/09 and RG NY/09 (4.5 and 3.9 Log10TCID50/ml,
respectively) (Fig. 1C). Additionally, LAIV pigs had reduced mean viral titers in the lung lavage fluid compared to animals exposed to
WT NY/09 (P⫽0.01) (Fig. 1D). The back titer of the challenge indicated that each pig in the RG NY/09 groups received 105.9
TCID50, each pig in the WT NY/09 group received 105.5TCID50,
and each pig in the LAIV group received 104.2TCID50.
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 evaluation of IAV-specific antibodies. At 42 dpv, serum HI antibodies against WT 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/09 virus were 180, 55, and 31 in animals immunized with WT NY/09, LAIV, and Ad5-HA, respectively. Average serum HI titers against the challenge viruses were below the limit of detection (⬍10), with the 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 evaluated as a sample source in the HI assay, but there was no measureable HI activity using these samples, even at a dilution of 1:10. All samples from nonvaccinated animals (NV) had no detectable HI titers (⬍10).
Functional IAV-specific antibody levels were also measured using a virus neutralization assay (Table 3). WT NY/09 and LAIV exposure induced production of serum neutralizing antibody against NY/09 virus (titers, 168⫾27 and 74⫾27, respectively), and titers against IL/10(␥) virus were similar. However, serum neutralization 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 1Attenuation 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) and lung lavage (D) fluids. Each dot represents a single animal in that respective treatment group, with the means⫾standard errors of the means (SEM) forn⫽8 shown for the group indicated. Data were analyzed with the Kruskal-Wallis test and Dunn’s posttest.Pvalues of⬍0.05 were considered significant. *,P⬍0.05; **,P⬍0.01.
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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 WT NY/09 were able to neutralize WT NY/09 virus (titer 8⫾1) but not the heterologous challenge viruses, while NW samples were unable to neutralize any virus used in the assay.
NW and OF samples were used in an indirect whole-virus ELISA to detect antibody specific to H1N1pdm09 lineage virus throughout 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 and OF,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 samples beginning at 28 dpv (1 week postboost) and was maintained above background levels (NV group) only in samples from pigs in the WT NY/09 group (Fig. 2C). The total IgA and IgG antibody levels in each sample were determined, and amounts never varied more than 2-fold between time points (data not shown), suggesting that sample collection was consistent throughout the experiment. The Ad5-HA vaccine did not elicit detectable levels of H1N1pdm09-specific IgA or IgG in NW or OF samples (see Fig. S2A in the supplemental material). Due to an inability to measure H1N1pdm09-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.Macroscopic and microscopic lung lesions were evaluated at 5 dpi following challenge with heterologous H1 -cluster MN/10 or ␥-cluster IL/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 minimal macroscopic pneumonia (3.5%⫾1.1 and 2.1% ⫾0.3, respec-tively). Pigs with prior exposure to WT NY/09 virus, challenged with MN/10(), did not have reduced pneumonia compared to NV/CH pigs due to overall low scores at 5 dpi (Fig. 3A); however, these animals did not have significantly increased pneumonia scores compared to the NV/NC controls (P⫽0.63). Prior WT NY/09 inoculation did reduce macroscopic lung lesions following IL/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 reduced macroscopic pneumonia following challenge and the percentage of pneumonia for the LAIV groups was not significantly increased over that seen with NV/NC pigs (P⫽0.41). While LAIV-vacci-nated pigs challenged with MN/10() had microscopic pathology scores greater than those seen with NV/NC controls (1.1 and 1.3 versus 0.3, respectively), these scores were not significantly in-creased over NV/CH pig scores (0.79) (Fig. 3C). Pigs challenged with IL/10(␥) demonstrated mild microscopic lesions and did not have 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, viral titers in nasal swabs (NS), trachea washes, and lung lavage fluid were measured to evaluate protection against virus replication. NS were collected daily from 0 dpi through necropsy at 5 dpi (Fig. 4A
andD). Pigs exposed to WT NY/09 virus and subsequently chal-lenged with MN/10() had reduced average viral titers in NS on dpi 1 through 4 (P⬍0.0001), but the average viral titers in NS
TABLE 3Serum 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 ⬍2
NV ⬍10 ⬍10 ⬍10 ⬍2 ⬍2 ⬍2
a*,P⫽0.03; **,P⫽0.005 (compared to NY/09 neutralization). Statistical significance was measured by the Kruskal-Wallis test (n⫽8).
FIG 2Kinetics of IAV-specific mucosal IgA and IgG levels following immunization. Groups of pigs were intranasally inoculated with WT NY/09 or LAIV on day 0 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 ELISA compared 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. Data are expressed as means⫾SEM of OD determined forn⫽8 for the indicated group.
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collected on dpi 5 were similar to the titers measured in NS col-lected from NV/CH animals (Fig. 4A). Virus was not recovered from any NS collected from pigs exposed to WT NY/09 and chal-lenged with IL/10(␥) (Fig. 4D). LAIV vaccination limited the amount 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, LAIV vaccination significantly limited the amount of IL/10(␥) challenge virus shed and the length of time during which the virus was shed in nasal passages (P⫽ 0.005) (Fig. 4D). Pigs vaccinated with Ad5-HA also had reduced amounts of challenge virus in NS on dpi 1 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 supplemental material).
Mean titers of the MN/10() challenge virus were significantly reduced in trachea washes from pigs previously exposed to WT NY/09 virus compared to NV/CH pigs (1.5 Log10TCID50/ml and
4.6 Log10TCID50/ml, respectively) (Fig. 4B). LAIV-vaccinated
an-imals had mean titers of MN/10() virus in the trachea on dpi 5 similar to those measured from NV/CH pigs (Fig. 4B). Addition-ally, mean viral titers in the lung lavage fluid of all immunization groups challenged with MN/10() did not differ from the levels seen with NV/CH pigs (Fig. 4C). Pigs exposed to WT NY/09 or LAIV and challenged with IL/10(␥) did not have any virus recov-ered from trachea wash or lung lavage fluid (Fig. 4EandF) on dpi 5. 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. Since both live-virus exposure and LAIV vaccination provided in-creased cross-protection against the␥-cluster H1 variant and less cross-protection against the-cluster H1 variant, we evaluated whether levels of IAV-specific IgA in a live-animal sample source were predictive of cross-protection, given that the standard HI assay was unable to measure cross-reactivity to either challenge virus (Table 3). Using NW and OF samples collected from all pigs at 42 dpv, the cross-reactivity of IgA to eight H1 viruses from different phylogenetic clusters (listed inTable 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 representacross-reac-tive viruses from each H1 cluster and an H3 virus was 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 NV controls (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 cluster viruses (P⫽0.0006), and H3 viruses (P⫽0.01) compared to the OD values for homologous NY/09 antigen. However, IgA in OF from LAIV-vaccinated animals had lower OD values across all
FIG 3Pneumonia following heterologous IAV-challenged, IAV-vaccinated pigs. Groups of pigs were inoculated with the indicated vaccine and subsequently challenged intranasally with heterologous virus, and lung pathology was evaluated at 5 dpi. (A and B) Macroscopic lesion scores of animals following challenge with 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 that respective treatment group, with the mean⫾SEM forn⫽8 shown for the group indicated. Data were analyzed with the Kruskal-Wallis test and Dunn’s posttest.
Pof⬍0.05 was considered significant. *,P⬍0.05. Hughes et al.
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heterologous viruses compared to those seen with viruses of the pandemic cluster (P⬍0.001).
Since reductions in OD values compared to those of homolo-gous IAV antigen were similar across tested heterolohomolo-gous H1 vari-ants, average endpoint titers of IgA specific to each H1 cluster were determined 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. 5CandD). Differences between the OF IgA endpoint titer against the NY/09 vaccine virus and mean IgA endpoint titers in OF from WT NY/09-exposed pigs were an average of 1.8-fold for similar H1N1pdm09 lineage viruses, 4-fold for␥-cluster viruses, 4.8-fold for-cluster viruses, 6.7-fold for
␦-cluster viruses, and 8.4-fold for H3 virus (Fig. 5D). IgA endpoint titers 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 seen with␥-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 H1 LAIV and a replication-defective viral vector encoding IAV H1N1pdm09 HA to provide cross-protection against representa-tive heterologous␥- and-cluster H1 IAV infections compared to live virus infections and investigated predictive immune measures of cross-protection in samples collected from a live animal. In evaluations 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(␥) compared to MN/10() was somewhat anticipated. Together, these data sug-gest that a multivalent next-generation swine IAV vaccine may be necessary to protect against the diverse H1 viruses cocirculating in U.S. swine.
Although cold-adapted, temperature-sensitive LAIV has been approved for use in humans (51) and in horses (52), LAIV vac-cines have not been approved for use in pigs. Original work on the LAIV vaccine focused on H3N2 surface genes (25,34,35). Here we describe the first use of an H1N1 NS1-truncated virus as an LAIV vaccine and compared the levels of immunogenicity and efficacy following exposure to WT NY/09 virus, which shares the same HA and NA as the LAIV. Similarly to previous reports of H3 NS1-truncated LAIV vaccination in pigs (35) and H1 NS1-truncated LAIV vaccination in mice (33), the H1 NS1-truncated LAIV in pigs was attenuated, with significantly less virus detected in the nose, trachea, and lungs as well as reduced levels of macroscopic pneumonia (Fig. 1). A previous study used a lower dose of chal-lenge WT virus (3 Log10TCID50less than was used in the current
study) (27), and it was as pathogenic as the typical dose of WT virus used in the current study. This suggests that, regardless of dose, WT IAV induces significant pathology, and although LAIV was administered at a level 1.8 Log10TCID50lower than that of the
WT virus, the LAIV was attenuated. In addition, there are
signif-FIG 4IAV vaccination impacted IAV burden in the respiratory tract following heterologous challenge. Groups of pigs were inoculated with the indicated vaccine and 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) of pigs 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 the mean⫾SEM forn⫽8 shown for the group indicated. The Kruskal-Wallis test and Dunn’s multiple-comparison test were used for analysis.Pof⬍0.05 was considered significant. ***,P⬍0.001; ****,P⬍0.0001.
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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 replicationin vivo, the LAIV elicited a measureable IAV-specific antibody response, sim-ilarly to previous studies (33,35). Administering a higher dose of LAIV may increase the degree of cross-protection, similarly to the cross-protection observed with live virus; however, this may not translate into a substantial increase in immune responses. In a follow-up study using a 1.5-Log10-higher dose of LAIV
adminis-tered i.n. to 21-day-old pigs, minimal LAIV was recovered from NS and IAV-specific mucosal IgA endpoint titers were not in-creased over the titers observed in the current study (data not shown).
Direct and indirect transmission of IAV is a major animal health as well as public health concern. Direct pig-to-pig transmis-sion has been shown with the 2009 pandemic H1N1 virus (55), as well as indirect aerosol transmission with emerging H3N2 variant viruses (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 not always limit virus shedding (21,38). An IAV vaccine that limits nasal shedding of virus would likely have the ability to limit fur-ther transmission; fur-therefore, we evaluated nasal shedding as a measure 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 (5 dpi). In contrast, LAIV-vaccinated pigs challenged with IL/10(␥) demonstrated reduced virus replication in the nose that was cleared by 4 dpi. These reductions in nasal shedding in vaccinated, challenged animals suggests that LAIV vaccination could limit the transmission of heterologous virus to naive or vaccinated contact animals (38,56,57), thus slowing or breaking the transmission cycle. Studies are under way to investigate this hypothesis.
A common parameter used to evaluate IAV vaccine efficacy is the prevention or significant reduction of lung lesions following challenge compared to nonvaccinated challenged animal results (45). LAIV-vaccinated animals challenged with IL/10(␥) had pneumonia scores that were not significantly different from the scores calculated for NV/CH controls. However, pneumonia scores following challenge were relatively low across all treatment groups, with NV/CH pigs averaging 3.5%, making significant re-ductions in pneumonia difficult to assess. Our observation of low pneumonia scores following challenge is consistent with previous studies of H1 IAV intranasal challenge in pigs and is not uncom-mon in 10-week-old pigs (24,58). Despite the low pneumonia scores, virus was recovered from the lungs of all NV/CH pigs, and IL/10(␥) was not recovered from lung lavage fluid or trachea washes of LAIV-vaccinated pigs, indicating protection against vi-rus replication. The observation of lung pathology in the absence of virus in the respiratory tract is consistent with previous work evaluating LAIV or Ad5-HA efficacy in pigs (24,29,53). Whereas Ad5-HA-vaccinated animals had increased macroscopic lung
le-FIG 5LAIV 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 to measure 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 and B) 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 NV control. (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). Each dot represents a viral antigen in the indicated cluster with the mean endpoint dilution for the cluster indicated (see Materials and Methods).
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sions following MN/10() challenge compared to the NV/CH controls (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 enhanced respiratory disease (47). Collectively, these observations suggest that intranasal LAIV and/or Ad5-HA vaccination primes a muco-sal cell-mediated immune response that is rapidly activated upon heterologous challenge and that this activation may control viral burden but result in immunopathology (59). Future work evalu-ating the mucosal IAV-specific cell-mediated immune response following intranasal IAV vaccination may provide insight into the mechanism associated with pneumonia following heterologous challenge; however, such an evaluation is unlikely to provide a useful live-animal sample that could be readily used for evaluating LAIV immunogenicity and cross-protection. While lung lesions are often used to evaluate IAV vaccine efficacy, together, these data 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 (generated through WIV vaccination) are often used to evaluate antigenic relatedness between swine IAV strains (17,60). Previous studies have shown that LAIV or Ad5-HA vaccination results in lower serum 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 robust peripheral HI antibody response and that the commonly used serum HI assay may not be suitable for predicting LAIV or Ad5-HA vaccine efficacy. In contrast, serum virus neutralization antibody was detected following LAIV vaccination and neutraliza-tion titers may be a useful measure of LAIV immunogenicity and efficacy. The greater sensitivity of the neutralization assay than the HI assay has made it an important tool in the development of IAV vaccines that are poorly immunogenic (37,61,62). Serum from LAIV vaccinates neutralized homologous H1N1pdm09 virus and IL/10(␥) virus equally. Pigs exposed to WT NY/09 or LAIV had significant reductions in serum neutralization against MN/10() compared to homologous NY/09 virus neutralization. Higher se-rum neutralization titers measured against IL/10(␥) virus than against MN/10() virus were associated with control of virus rep-lication in the lungs and trachea. These data suggest that the serum neutralization assay may be an effective tool to predict cross-pro-tection of LAIV vaccines in pigs, though further work is warranted to 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 antibody assays to predict immunogenicity and efficacy associated with in-tranasally delivered vaccines. Previous studies have shown that IAV antigen-specific antibody can be detected by ELISA in NW and OF samples collected following vaccination or infection (24,
38, 39, 63). Experimental IAV infection elicits an IAV-specific antibody response detectable in OF (63), and OF have become a rapidly evolving diagnostic sample source for several human and animal pathogens (63,64). We detected IAV-specific antibody in both 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 to NW samples as determined by a standard OD reading performed with 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 and that the response was associated with protection from homolo-gous challenge in humans (65) as well as in pigs (21,38). Although we did not evaluate protection from homologous challenge, the higher antibody response observed in OF than in NW following vaccination would suggest that OF could serve as a sample source to 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 heterologous cross-protection in humans and in animal models of human IAV infection (26,65,66). Given the reductions in heterologous virus nasal shedding and in measureable IAV-specific IgA levels in mu-cosal samples prechallenge (42 dpv), we evaluated the association of cross-reactive mucosal IgA in immunized pigs with the level of cross-protection observed. Both NW and OF had measurable cross-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 nearly the same across heterologous phylogenetic clusters. Using this as-sociation alone, we would have predicted complete protection against both challenge viruses, since mucosal IAV-specific IgA was cross-reactive with both MN/10() and IL/10(␥) antigens at sim-ilar OD values; however, this was not the case. Endpoint titrations of IAV-specific IgA in NW and OF from WT NY/09-exposed pigs separated 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 levels were associated with cross-protection. Due to the reduced immu-nogenicity of the LAIV vaccine, there was no clear association between IgA endpoint titers and cross-protection. Performing this analysis with OF samples from WT NY/09-inoculated animals, which had higher IAV-specific IgA titers, did show an association with antigenic relatedness, reactive IgA titers, and cross-protection (Fig. 4and5B). Collectively, these data suggest that IAV-specific IgA in OF could be used to assess antigenic related-ness and cross-protection of circulating and emerging IAVs but that it may require that pigs be inoculated with wild-type viruses to elicit a response such that sufficient levels of mucosal IAV-specific IgA are induced. Vaccine or surveillance researchers would be able to curate a collection of OF samples from IAV-infected pigs and measure the antigenic relatedness of the IAV immunogen to currently circulating IAV strains and update and modify vaccine strains as necessary. This approach is somewhat similar to that of human seasonal IAV vaccine selection, where ferrets are infected with live IAV to elicit a high serum HI titer in order to assess the antigenic relatedness of circulating IAV isolates (67,68).
ACKNOWLEDGMENTS
We thank Zahra Olson, Lilia Walther, and Steven Kellner for technical assistance 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
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viding specific information and does not imply endorsement by the USDA. USDA is an equal opportunity provider and employer.
The findings and conclusions in the manuscript are ours and do not necessarily 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.
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