The immune response and maternal antibody interference to a ...Document Transcript
Veterinary Immunology and Immunopathology 112 (2006) 117–128
The immune response and maternal antibody interference
to a heterologous H1N1 swine inﬂuenza virus infection
Pravina Kitikoon a, Dachrit Nilubol a,1, Barbara J. Erickson a,
Bruce H. Janke b, Thayer C. Hoover c, Steve A. Sornsen c, Eileen L. Thacker a,*
Department of Veterinary Microbiology and Preventive Medicine,
College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
Department of Veterinary Diagnostics and Production Animal Medicine,
College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
Pﬁzer Animal Health, 16996-255th AVE, Francis Sites, Spirit Lake, IA 51360, USA
Received 21 October 2005; received in revised form 30 January 2006; accepted 13 February 2006
This study investigated the efﬁcacy of a bivalent swine inﬂuenza virus (SIV) vaccine in piglets challenged with a
heterologous H1N1 SIV isolate. The ability of maternally derived antibodies (MDA) to provide protection against a
heterologous challenge and the impact MDA have on vaccine efﬁcacy were also evaluated. Forty-eight MDA+ pigs and 48
MDAÀ pigs were assigned to 8 different groups. Vaccinated pigs received two doses of a bivalent SIV vaccine at 3 and 5 weeks of
age. The infected pigs were challenged at 7 weeks of age with an H1N1 SIV strain heterologous to the H1N1 vaccine strain.
Clinical signs, rectal temperature, macroscopic and microscopic lesions, virus excretion, serum and local antibody responses,
and inﬂuenza-speciﬁc T-cell responses were measured. The bivalent SIV vaccine induced a high serum hemagglutination-
inhibition (HI) antibody titer against the vaccine virus, but antibodies cross-reacted at a lower level to the challenge virus. This
study determined that low serum HI antibodies to a challenge virus induced by vaccination with a heterologous virus provided
protection demonstrated by clinical protection and reduced pneumonia and viral excretion. The vaccine was able to prime the
local SIV-speciﬁc antibody response in the lower respiratory tract as well as inducing a systemic SIV-speciﬁc memory T-cell
response. MDA alone were capable of suppressing fever subsequent to infection, but other parameters showed reduced
Abbreviations: BAL, bronchoalveolar lavage; CMI, cellular mediated immune; DPI, days post infection; HA, hemagglutination; HI,
hemagglutination-inhibition; HMI, humoral mediated immune; IHC, immunohistochemistry; MDA, maternally derived antibodies; MDCK,
Madin-Darby canine kidney; NW, nasal washes; OD, optical density; PI, post infection; SIV, swine inﬂuenza virus; SN, serum neutralization;
TCID, tissue culture infective dose
* Corresponding author at: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, P.O. Box 3020,
Iowa State University, Ames, IA 50010-3020, USA. Tel.: +1 515 294 5097; fax: +1 515 294 8500.
E-mail address: email@example.com (E.L. Thacker).
Present address: Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd.,
Pathumwan, Bangkok 10330, Thailand.
0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
118 P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128
protection against infection compared to vaccination. The presence of MDA at vaccination negatively impacted vaccine efﬁcacy
as fever and clinical signs were prolonged, and unexpectedly, SIV-induced pneumonia was increased compared to pigs
vaccinated in the absence of MDA. MDA also suppressed the serum antibody response and the induction of SIV-speciﬁc memory
T-cells following vaccination. The results of this study question the effectiveness of the current practice of generating increased
MDA levels through sow vaccination in protecting piglets against disease.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Inﬂuenza; Heterologous H1N1; Maternal antibodies; Vaccine; Immune response
1. Introduction updated annually as occurs with human inﬂuenza
vaccines. The level of cross protection between
The emergence of an H3N2 virus in the late 1990s genetically heterologous isolates of the same subtype
has altered the impact of swine inﬂuenza virus (SIV) is unpredictable. Thus, it is important to investigate the
infection on the US swine industry (Karasin et al., cross protection of genetically heterologous strains of
2000b; Olsen, 2002). The epidemiology of SIV- the same subtype.
induced disease has evolved from a seasonal epidemic Studies investigating the role of SIV-MDAs have
disease pattern to more of an endemic proﬁle (Choi produced differing results. One study found that
et al., 2003). Many production systems currently face MDA provided complete protection against homo-
increased respiratory disease due to SIV infection in logous SIV infection (Blaskovic et al., 1970).
pigs of all ages including nursery and ﬁnishing pigs. However, only virus isolation from lung tissue was
Vaccination against SIV is a tool used to help prevent assayed and nasal swabs and viral antigen load in the
and control disease in pigs and is most commonly used upper or lower respiratory tract was not assessed.
in sow herds. Sow herd immunization typically Another study concluded that MDA provided no
provides protection against clinical disease associated protection against SIV-induced disease based on the
with SIV infection in the adult sows and enhances percentage of lung lesions and viral levels in the
passive immunity to the piglets as well. The role of lungs (Mensik et al., 1971), while Renshaw (1975)
maternally derived antibodies (MDAs) in protecting found that the level of protection in piglets correlated
the young offspring from clinical disease has been to the MDA level at the time of infection. A more
recognized in many viral diseases including SIV recent study by Loeffen et al. (2003) demonstrated
(Blaskovic et al., 1970; Renshaw, 1975; Puck et al., incomplete protection against disease by MDA and
1980; Englund et al., 1998; Choi et al., 2004). The use piglets with MDA shed more virus following
of vaccine-enhanced MDA to control SIV-induced infection with the homologous virus than piglets
clinical disease in nursery pigs was successful in the without MDA. These experiments differed in their
years prior to 1997 when a single H1N1 SIV subtype experimental design including the level of MDA, but
predominated in the US swine herds. The emergence all studies were based on homologous virus infection
of a new H3N2 subtype in 1997 resulted in the US in piglets. In addition, all studies demonstrated that
swine population facing new genetically diverse SIV MDA, depending on the level present at the time
subtypes with genetic materials from multiple origins, suppress the hemagglutinin-inhibition (HI) antibody
including avian and human viruses (Karasin et al., response by the piglets to SIV infection (Blaskovic
2000b; Webby et al., 2000; Zhou et al., 2000). et al., 1970; Mensik and Pokorny, 1971; Renshaw,
Recently, the H1 viruses in the US herds have evolved 1975; Loeffen et al., 2003).
to include H1N1 and H1N2 subtypes with genomes In the present study, we were interested in
consisting of avian, swine and human genes that had simulating ﬁeld conditions where sows and piglets
been acquired from the H3N2 subtype (Karasin et al., are exposed to multiple SIV strains and receive
2000a; Choi et al., 2002). Current commercial SIV vaccines that may differ genetically. The research
vaccines contain one, two or three different SIV objectives were three-fold; the ﬁrst objective was to
isolates. However, swine vaccines are not presently investigate protection by MDA against experimental
P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128 119
challenge with a heterologous SIV isolate; the second respiratory syndrome virus and Mycoplasma hyop-
objective assessed vaccine efﬁcacy against a hetero- neumoniae. The pigs were identiﬁed by numbered ear
logous SIV isolate in the absence of MDA; and the tags and assigned to 8 groups of 12 pigs each divided
third objective examined the effect of MDA on vaccine into 2 replicates with stratiﬁcation by arrival weight.
efﬁcacy in the piglets when experimentally challenged The experimental design is summarized in Table 1.
with a heterologous SIV. Cross-protection, vaccine Pigs were housed in two identical rooms in the
efﬁcacy and the immune response induced by Livestock Infectious Disease Facility at ISU based on
vaccination and infection were determined using their challenge status. Pigs in the vaccinated groups V,
clinical signs, lung lesions, HI antibody titers, virus MV, VS and MVS were inoculated with a bivalent SIV
isolation from nasal swabs, and SIV-speciﬁc anti- vaccine containing the H1N1 and H3N2 subtype
bodies in the upper and lower airways. (FluSure1, Pﬁzer Animal Health, New York, NY,
USA) according to label directions at 3 and 5 weeks of
age. Pigs’ positive for MDA had HI titers to both the
2. Material and methods vaccine and challenge H1N1 antigens between 1:40
and 1:80 at the time of the ﬁrst vaccination. Pigs in the
2.1. Pigs and experimental design infected groups S, VS, MS and MVS were challenged
intratracheally at 7 weeks of age (0 days post
All study procedures and animal care activities infection; DPI) with 10 ml of 105.5TCID50/ml of
were conducted in accordance with the guidelines H1N1 strain A/Swine/Iowa/40776/92 virus, which
and under the approval of the Iowa State University demonstrated minimal serum antibody cross-reactiv-
(ISU) Institutional Committee on Animal Care and ity to the vaccine H1N1 strain.
Ninety-six 8- to 12-day-old crossbred pigs 2.2. Clinical evaluation
were obtained from two commercial herds: 48 pigs
with MDA (MDA+) were obtained from a herd with Pigs were evaluated for 7 days to assess respiratory
stable SIV status where the sows were routinely disease after SIV infection. The pigs were observed
vaccinated (MaxiVac1 Excell , Schering-Plough and scored (Table 2) at rest followed by rectal
Animal Health, Union, New Jersey, USA) and 48 temperature measurement. Pigs were weighed upon
pigs without MDA (MDAÀ) were procured from a arrival and at À1, 5 and 21 DPI to evaluate production
herd seronegative for SIV, porcine reproductive and performance.
Experimental design-group description, maternal derived antibody (MDA) status, vaccination status, SIV infection status and numbers of pigs
necropsied on each necropsy days
Group MDA status Vaccination status Infection status Number of pigs Total number of pigs
5 DPI a 21 DPI
NEG No No No 6 6 12
V No Yes No 6 6 12
M Yesb No No 6 6 12
MV Yes Yesc No 6 6 12
S No No Yes d 6 6 12
VS No Yes Yes 6 6 12
MS Yes No Yes 6 6 12
MVS Yes Yes Yes 6 6 12
Days post infection.
Pigs had HI titers between 1:40 and 1:80 at the time of the ﬁrst vaccination.
Pigs were vaccinated at 3 and 5 weeks of age.
Pigs were infected with H1N1 SIV that was heterologous to the vaccine isolate at 7 weeks of age.
120 P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128
Table 2 method (Vincent et al., 1997). IHC was performed
Clinical sign scores
on sections cut from one parafﬁn-embedded lung
Parameters Scores Description tissue block and included three pieces (1 cm Â 2 cm)
Respiratory rate 0 Normal of lung collected at 5 DPI.
1 Slightly elevated
2 Moderately elevated,
2.4. Virus isolation
slight abdominal breathing
3 Clearly elevated,
distinct abdominal breathing Following collection at À1, 3, 5, and 7 DPI, nasal
Coughing 0 Absent swabs were immediately placed in infecting medium
1 Present (MEM with 7% BSA, 300 U/ml penicillin, 300 mg/ml
Sneezing 0 Absent
streptomycin and 1 mg/ml trypsin). Ten-fold serial
dilutions of the viral solution were prepared in the
All scores per topic are accumulated for a total clinical score. infecting medium and inoculated onto Madin-Darby
canine kidney (MDCK) cells prior to incubation at
2.3. Necropsy 37 8C with 5% CO2. All of the following steps that
required incubation were carried out at room
Pigs were euthanized with a pentobarbital-based temperature. Prior to staining, the cells were ﬁxed
euthanasia solution (Beuthanasia1, Schering-Plough, with 4% phosphate-buffered formalin and washed
Kenilworth, NJ, USA) followed by exsanguination. with 0.5% Tween-20 in PBS (washing solution).
Nasal washes (NW) were collected using a previously Subsequently, the cells were incubated for 1 h with
published methodology (Larsen et al., 2000). Brieﬂy, anti-inﬂuenza A nucleoprotein monoclonal antibo-
10 ml of sterile phosphate buffer saline (PBS) with 1% dies (clone HB-65, ATCC, Rockville, Maryland)
bovine serum albumin (BSA), penicillin (300 U/ml) diluted 1:650 in the washing solution containing 1%
and streptomycin (300 mg/ml) were infused into the BSA (diluting solution). After washing, the cells were
nasal passages. The head was moved gently and the incubated 1 h with the rabbit anti-mouse IgG
ﬂuid was allowed to drain into a collection cup. The conjugated horseradish peroxidase (Dako Cytoma-
lungs were removed and evaluated for pneumonia. tion, Carpinteria, California) diluted 1:250 with the
Macroscopic lesions associated with SIV pneumonia, diluting solution. The color was developed using a
consisting of well demarcated dark-purplish areas of chromogen aminoethyl carbazole substrate (Sigma,
lung consolidation, were sketched onto a standard lung St. Louis, Missouri). Each procedure contained
diagram. The proportion of lung surface with lesions mock-infected negative control cells and positive
was determined from the diagram using a Zeiss SEM- control cells infected with a virus with a known titer.
IPS image analyzing system as previously described The titer of the virus in each nasal swab was expressed
(Thacker et al., 2001). Bronchial swabs were obtained as log 10 TCID50 per milliliter and calculated by the
from each pig and cultured for swine respiratory method of Reed and Muench (Reed and Muench,
bacteria using standard procedures. Bronchoalveolar 1938).
lavage (BAL) was performed as previously described
using the same PBS solution as used for the nasal 2.5. Hemagglutination-inhibition (HI) assay
washes (Mengeling et al., 1995). A portion of lung
tissue was collected from all lung lobes, ﬁxed in 10% Blood was collected at À28, À14, À1, 4 and 20
neutral buffered formalin, processed and embedded in DPI. Sera was stored at À20 8C and assayed
parafﬁn using an automated tissue processor. Lung simultaneously following both trials. The HI assays
sections were scored for microscopic lung lesions were tested according to the standard protocol
consistent with SIV (necrotic bronchiolitis) as pre- routinely performed at ISU-Veterinary Diagnostic
viously described (Thacker et al., 2001). Laboratory (Yoon et al., 2004) using 0.5% rooster
The presence of SIV-speciﬁc antigen was assessed erythrocytes for hemagglutination. Virus antigens
in the formalin-ﬁxed lung tissues using a previously utilized in the HI assays included the challenge virus
described immunohistochemistry (IHC) staining (strain A/Swine/Iowa/40776/92 H1N1) and the H1N1
P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128 121
vaccine antigen (provided by Pﬁzer Animal Health, 1 ml of PKH67 (2 Â 10À6 M) and incubated for 5 min,
New York, NY, USA). followed by 2 min incubation with fetal bovine serum
(FBS) to adsorb the dye and stop the dye uptake. Cells
2.6. ELISA for local SIV-speciﬁc antibody were then washed three times with RPMI 1640
production (Mediatech, Huntingford, VA). Once stained, cells
were recounted and added to 96-well U-bottomed
The NW and BAL ﬂuids were incubated at 37 8C microtiter plates (Costar, Corning, NY) at a density of
for 1 h with an equal amount of 10 mM dithiothreitol 5 Â 105 cells per well in 100 ml medium (RPMI
(DTT; Sigma-Aldrich, St. Louis, MO) to disrupt containing 10% fetal calf serum, 2 mM L-glutamine,
mucus present in the ﬂuids. ELISA assays for SIV 100 U/ml penicillin and 100 mg/ml streptomycin).
antibodies in the respiratory tract were performed as PBMCs were cultured with inactivated challenge and
previously described (Larsen et al., 2000). Brieﬂy, vaccine antigen, 100 HA units/100 ml in duplicate.
inactivated challenge virus and vaccine antigen were Positive control samples were cultured with 5 mg/ml
diluted to a hemagglutination (HA) concentration of PHA in duplicate and the culture media was used as
100 HA units/50 ml. Immulon-2HB 96-well plates negative control.
(Dynex, Chantilly, VA) were coated with 100 ml of
SIV antigen and incubated at room temperature 2.7.2. Cell surface marker staining
overnight. Plates were blocked for 1 h with 100 ml Cells were centrifuged (300 Â g) for 10 min and
of 10% BSA in PBS and washed three times with the supernatant was discarded. Primary antibodies to
0.05% Tween-20 in PBS (PBS-T). The assay was swine leukocyte surface antigens in PBS containing
performed on each NW and BAL sample in triplicate. 1% BSA and 0.1% sodium azide (FACS buffer) was
Negative controls (DTT with equal amount of PBS added to wells containing cells. Primary antibodies,
solution) were included on each plate. Plates were including phycoerythrin (PE)-conjugated anti-CD4
incubated at room temperature for 1 h, washed three and biotinylated anti-CD8a were added to the
times with PBS-T, then incubated with peroxidase- appropriate wells. After incubating for 20 min, the
labeled goat anti-swine IgG (Kirkegaard and Perry, cells were washed with FACS buffer and resuspended
Gaithersburg, MD) or peroxidase-labeled goat anti- in 50 ml of secondary antibody streptavidin-conju-
swine IgA (Bethyl, TX) at 37 8C for 1 h. The ABTS/ gated cychrome dye secondary antibody (Pharmingen,
peroxidase was added as the substrate (Kirkegaard BD Bioscience, CA). Cells were incubated, washed,
and Perry, Gaithersburg, MD). Antibody levels were resuspended and ﬁxed with 2% formalin in PBS before
reported as the mean optical density (OD) and the ﬂow cytometric analysis.
mean OD of each treatment group was compared. The program Modﬁt Proliferation Wizard (Verity
Software House Inc., Topsham, Maine) was used to
2.7. Flow cytometry analysis analyze cell proliferation. The results are presented as
the mean number of proliferating cells Æ standard
2.7.1. Culture procedures error mean per 10,000 PBMCs. The number of cells
Peripheral blood mononuclear cells (PBMC) were proliferating was calculated by the following formula:
collected in heparinized blood collection tubes and (% proliferation to mitogen Â number of cells in the
isolated by differential centrifugation. PBMCs were R1 gate) À (% proliferation with no stimula-
collected 1 day prior to the second vaccination, prior tion Â number of cells in the R1 gate) (Waters
to challenge and prior to each necropsy at 4 and 20 et al., 2002). R1 is the region containing live
DPI. The PBMCs were counted prior to staining with lymphocytes based on forward and side light scatter
PKH67 green ﬂuorescent dye (Sigma–Aldrich, St. properties of porcine lymphocytes (Dorn et al., 2002).
Louis, MO) following a procedure previously
described (Dorn et al., 2002). Brieﬂy, 2 Â 107 PBMCs 2.8. Statistical analysis
were centrifuged (400 Â g) for 10 min, supernatants
were aspirated, and cells were resuspended in 1 ml of Comparisons of results between experimental
diluent C (Sigma). Cells in diluent C were added to groups were performed using a non-parametric
122 P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128
Wilcoxon/Kruskal–Wallis test (Rank sum test) from
JMP 5.1 Software (SAS Institute, Cary, NC). For all
analyses, statistically signiﬁcant difference between
groups were considered when P 0.05.
3.1. Clinical evaluation
All pigs inoculated with SIV developed a fever
(!104 8F) by 24 h post infection (PI) with the exception
of pigs in group MS (nonvaccinated MDA+) which
remained normal throughout the trial (Fig. 1a). The
fever resolved by 2 DPI in all SIV infected groups
except the MDA+-vaccinated, challenged group (group
MVS) which remained febrile for 4 additional days. No
fever was detected in the nonchallenged control pigs at
any time (data not shown).
The summary of total accumulated clinical scores Fig. 1. Mean rectal temperatures (a) and clinical scores (b) of
is illustrated in Fig. 1b. At 24 h PI, all pigs inoculated nonvaccinated, challenged pigs (^), MDAÀ-vaccinated, challenged
with SIV had increased clinical scores. Clinical signs pigs (&), MDA+-nonvaccinated, challenged pigs (*) and MDA+-
vaccinated, challenged pigs (Â) before (À1 days post infection;
consisted of increased respiratory rates which DPI) and after (1–7 DPI) infection with H1N1 SIVat 7 weeks of age.
decreased over the next few days with the exception Results of nonchallenged pigs are not included. Different letters in
of group MVS. In all other SIV infected groups, the ﬁgure are signiﬁcant difference between values (P 0.05).
coughing was rare and was detected in only one pig
from group MS at 24 h PI. Seven pigs in group MVS
continued to cough until 5 DPI after which coughing than pigs in group VS. The percentage of macroscopic
was no longer detected. No coughing was present in lung lesions in group MVS was signiﬁcantly
any of the nonchallenged control pigs (group NEG). increased in severity compared to all other challenged
Overall, pigs in group MVS showed the most clinical groups. No signiﬁcant lesions consistent with SIV
disease while pigs that were MDAÀ, vaccinated and were present in any nonchallenged pigs. By 21 DPI,
challenged (group VS) demonstrated the least disease the percentage of pneumonia was minimal in all SIV
following infection. infected groups and no statistical differences were
3.2. Necropsy In contrast to the SIV-associated macroscopic lung
lesions, the microscopic difference between the
Half of the pigs in each group were necropsied at 5 groups was less obvious. However, the trend of the
DPI with the remaining pigs necropsied at 21 DPI. microscopic ﬁndings supported the macroscopic
Macroscopic lung lesions and histopathological results. Bronchiolar epithelial damage (necrotic
ﬁndings from both necropsies are summarized in bronchiolitis) was considered speciﬁc for SIV infec-
Table 3. Pigs in group VS had signiﬁcantly lower tion. Scoring focused on airway damage and the
percentages of macroscopic lung lesions consistent degree of inﬂammation surrounding the airways and
with SIV (lung consolidation with dark-purplish well alveoli. The microscopic score was based on the
demarcated areas) than the MDAÀ-nonvaccinated number of airways in the section involved. Pigs in
challenged control group (group S). Pigs in group MS group VS, demonstrated less microscopic damage
had signiﬁcantly less pneumonia than pigs in group S than pigs in any of the other SIV challenged groups.
but the lesion levels were still signiﬁcantly higher The necrotizing bronchiolitis lesion scores decreased
P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128 123
Percentage of lung with visible macroscopic lesions and microscopic lesion scores Æ S.E.M. from pigs infected with H1N1 SIV at both
Group Percentage of macroscopic lesions a Microscopic lesion scoresb
5 DPI 21 DPI 5 DPI 21 DPI
NEG 0.23 Æ 0.05 a 0.24 Æ 0.11 a 0Æ0 a 0
V 0.09 Æ 0.06 a 0.11 Æ 0.04 a 0Æ0 a 0
M 0.12 Æ 0.15 a 0.44 Æ 0.19 a,b 0Æ0 a 0
MV 0.22 Æ 0.09 a 0.17 Æ 0.07 a 0Æ0 a 0
S 10.03 Æ 1.71 c 1.25 Æ 0.09 b 2.17 Æ 0.31 b 0
VS 1.59 Æ 0.51 a 0.21 Æ 0.12 a 0.71 Æ 0.42 a 0
MS 4.46 Æ 1.10 b 0.48 Æ 0.24 a 2.14 Æ 0.40 b 0
MVS 18.33 Æ 1.88 d 0.85 Æ 0.26 a,b 2.60 Æ 0.40 b 0
Means with different letters within a column are statistically different (P 0.05).
As determined by lesion sketches and image analysis.
SIV microscopic lesion scores are based on the severity of bronchiolar epithelial damage (necrotic bronchiolitis).
Days post infection.
at 21 DPI in all SIV infected groups and no statistical 3.4. Hemagglutination-inhibition (HI) test
differences remained. Microscopic lesions consistent
with SIV were not detected in any nonchallenged pigs. Antibodies were measured by HI assays using both
Detection of SIV antigen by IHC was performed on the vaccine antigen (Fig. 3a) and the challenge antigen
all lungs. SIV antigen was detected only in the lungs (Fig. 3b). Prior to the ﬁrst vaccination (À28 DPI)
collected at necropsy on 5 DPI (data not shown). Only MDAÀ pigs had no HI titers, while pigs that were
one pig in group VS was positive for SIV antigen by MDA+ had HI titers that averaged 1:80 (HI score = 4)
IHC at that time, while SIV antigens were detected in to both antigens. Pigs in nonvaccinated, nonchal-
all pigs in groups MS, S and MVS. No SIVantigen was lenged group M had average HI titers (to both
detected in the lungs of any of the nonchallenged antigens) that gradually declined to a titer of less then
groups. 1:10 at 21 DPI ($10 weeks of age). Group NEG
3.3. Virus isolation
Fig. 2 shows the level of virus detected from the
nasal swabs from groups that were inoculated with
SIV. Virus was not detected in the nasal swabs
collected from the non-infected groups or any pigs
prior to challenge (data not shown). At 3 DPI, pigs in
group VS had signiﬁcantly lower amounts of virus in
the nasal swabs compared to all other SIV- challenged
pigs (groups S, MS and MVS). At 5 DPI, groups S and
MS had increased levels of virus compared to group
VS. Although the virus in group MVS was not Fig. 2. Virus titers in nasal swabs from nonvaccinated, challenged
signiﬁcantly different from group VS, the level were pigs (S), MDAÀ-vaccinated, challenged pigs (VS), MDA+-nonvac-
also similar to groups S and MS. By 7 DPI, virus was cinated, challenged pigs (MS) and MDA+-vaccinated, challenged
no longer detected from pigs in groups VS and MS, pigs (MVS) following H1N1 SIV infection at 7 weeks of age.
Results are represented as mean log10 TCID50/ml Æ S.E.M. Differ-
whereas one pig in group S and one pig in group MVS ent superscription letters within the ﬁgure are signiﬁcant difference
were still shedding low levels of virus (data not between the values (P 0.05). The results of nonchallenged pigs are
shown). not included.
124 P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128
À1 DPI) indicates some cross reactivity between HI
antibodies induced by vaccination to the challenge
antigen (groups V and VS); and the HI MDA induced
antibodies (groups M, MV and MVS). Two weeks
following the ﬁrst vaccination (À14 DPI), MDAÀ pigs
in groups V and VS had no HI antibodies against the
challenge antigen. After the second vaccination, pigs
in groups V and VS developed low levels of HI
antibodies to the challenge virus (1:29 Æ 1:15 and
1:20 Æ 1:11). MDA+ (groups M, MV and MVS) had
HI antibodies to the challenge virus. Vaccination of
pigs in the presence of MDA+ did not increase the HI
antibody levels to the challenge virus. The HI antibody
levels to the challenge virus decreased a minimum of
one-fold every 2 weeks until levels were less than 1:20
on the day prior to challenge in groups MV and MVS.
However, following challenge all vaccinated pigs
independent of MDA status had increased HI antibody
responses to the challenge antigen.
3.5. SIV-isotype speciﬁc ELISAs
Fig. 3. Mean hemagglutinin-inhibition (HI) antibody titers against
the vaccine antigen (a) and challenge antigen (b) from pigs prior to An ELISA to measure the local immune antibody
the ﬁrst vaccination and second vaccination (À28 and À14 days post
infection; DPI), prior to SIV infection (À1 DPI) and prior to both
response to SIV vaccine antigen and challenge antigen
necropsy dates (4 and 20 DPI). The NEG group is not shown. The HI was performed on both BAL and NW ﬂuids. Little
score (n): n = 2n Â 5 serum HI antibody titer. antibody response to either SIV antigen was observed
in the NW ﬂuid (data not shown). In BAL ﬂuid, IgA
was the dominant SIV-speciﬁc antibody at both 5 and
remained HI-antibody negative throughout the trial 21 DPI. The levels of IgA antibodies in the BAL
(data not shown). speciﬁc to the vaccine antigen (Table 4) were
As shown in Fig. 3a, pigs that were MDA+ had no signiﬁcantly higher in pigs in groups VS and MVS
increase in HI antibody titers to the vaccine antigen at 1 compared to the pigs in groups S and MS at both
day prior to the 2nd vaccination (À14 DPI). In contrast, necropsy dates.
an increase in HI titers occurred in groups V and VS
indicating an active antibody response to vaccination in 3.6. Flow cytometry analysis
the absence of MDA. Two weeks after the second
vaccination (À1 DPI) vaccinated pigs that were MDAÀ Table 4 demonstrates that at 21 DPI MDAÀ pigs in
(groups V and VS) demonstrated signiﬁcantly higher group VS, CD4+/8+ T-cells showed signiﬁcantly
(P < 0.0317) HI antibody titers than all other vacci- increased proliferation when stimulated with either
nated groups. Pigs in group MV with MDA+ demon- the challenge or vaccine antigen. No signiﬁcant
strated a slight increase in the HI titer following the differences were observed between groups in the other
second vaccination. However, group MVS which also populations of lymphocytes.
was MDA+ and vaccinated showed no rise in the HI titer
and had levels that did not differ from the MDA+-
nonvaccinated pigs (groups M and MS). 4. Discussion
The HI titers to the challenge antigen are shown in
Fig. 3b. The presence of low levels of HI antibodies to The study reported here had three objectives which
the challenge antigen prior to infection (À28, À14 and included; evaluating the protection provided by MDA,
P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128 125
Lower airway SIV-speciﬁc IgA antibody and T-cell proliferation analysis from pigs following H1N1 SIV infection at 7 weeks of age
Group SIV-speciﬁc IgA antibodies in BALa Number of CD4+/8+ cellsb at 21 DPI c
5 DPI 21 DPI Vaccine Ag Challenge Ag
S 0.00 Æ 0.00 a 0.30 Æ 0.07 a 2298.31 Æ 293.1 a,b 1211.97 Æ 272.5 a
VS 0.28 Æ 0.11 b 0.73 Æ 0.15 b 5426.13 Æ 1026.8 b 4520.70 Æ 676.0 b
MS 0.00 Æ 0.01 a 0.18 Æ 0.06 a 1469.60 Æ 740.1 a 805.70 Æ 429.9 a
MVS 0.45 Æ 0.17 b 1.35 Æ 0.19 b 707.25 Æ 354.9 a 282.70 Æ 180.0 a
Means with different letters within a column are statistically different (P 0.05).
Mean OD of SIV-speciﬁc IgA antibodies Æ S.E.M. against the vaccine antigen from bronchoalveolar lavage ﬂuid (BAL) measured by
ELISA. Results of nonchallenged pigs are not shown.
Mean numbers of CD4+/8+ cells Æ S.E.M. that proliferated to the vaccine and the challenge antigen as determined by ﬂow cytometry and
cell surface marker staining. Results of nonchallenged pigs are not shown.
Days post infection.
evaluating vaccination efﬁcacy against a heterologous intratracheally and as a result the immune response in
SIV isolate, and the effect of MDA on vaccine efﬁcacy. the upper airways was reduced. The presence of MDA
Similar to previous ﬁndings (Loeffen et al., 2003; Choi at the time of vaccination did not appear to reduce the
et al., 2004), MDA were found to be partially local IgA response to the vaccine antigen and the
protective as at 1 DPI, MDA+-nonvaccinated, chal- presence of vaccine-speciﬁc IgA antibodies did not
lenged pigs had no fever. However, other clinical provide protection against the heterologous infection.
symptoms such as increased respiratory rates and These results bring into question the signiﬁcance of
coughing occurred in the presence of MDA. The MDA IgA antibodies in providing lower respiratory tract
alone did not protect against SIV infection as virus protection against inﬂuenza induced disease.
antigen was detected in the lungs and no reduction in Previous studies investigated the signiﬁcance of
virus shedding from the nasal cavity was observed. MDA to homologous SIV infection (Blaskovic et al.,
However, the prolonged viral shedding in MDA+ pigs 1970; Renshaw, 1975; Loeffen et al., 2003) and no
as described in earlier studies was not observed in this enhancement of SIV-induced disease was observed.
study (Renshaw, 1975; Loeffen et al., 2003). MDAÀ- No studies have been conducted to study the response
vaccinated pigs were protected against the hetero- to heterologous SIV infection which is more likely to
logous H1 virus used in this trial. In addition, occur under ﬁeld conditions. This study suggests that
vaccination signiﬁcantly reduced the level of virus MDA at the time of vaccination may possibly enhance
in the lungs and in the upper airways and nasal SIV-induced pneumonia resulting from heterologous
cavities. The ﬁndings in this study matched the results H1 infection. The exact mechanism for this enhance-
from a previous study in Europe that reported cross- ment is unknown. It is possible that MDA+ interfered
protection was elicited with H1 SIV vaccination with the cell mediated immune (CMI) response to
followed by a heterologous H1 virus infection (Van infection by skewing the T-helper 1 (Th1) type of
Reeth et al., 2001). response to a more Th2-like response through the
Induction of a local antibody response following formation of antibody–antigen (Ab–Ag) complexes
SIV infection has been determined in previous studies (Casadevall and Pirofski, 2003). Inﬂuenza virus
(Larsen et al., 2000; Heinen et al., 2000, 2001). This infection normally induces an effective innate immune
study conﬁrmed this response as vaccination primed response and the production of proinﬂammatory
the immune system for a local response as vaccine- cytokines (Van Reeth, 2000; Van Reeth et al., 2002)
speciﬁc IgA antibody levels were increased in the which is responsible for effective adaptive humoral
BAL ﬂuid after infection. A minimal antibody mediated immune (HMI) and CMI responses. In mice,
response to either SIV antigen was observed in the clearance of the virus is mediated by T-cells following
NW ﬂuid. The lack of a nasal mucosal response may primary infection and memory T-cells following
be attributed to the fact that the pigs were challenged secondary exposure (Flynn et al., 1998; Woodland
126 P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128
et al., 2001) while protection against infection and antibody levels to the vaccine antigen at the time of
clinical disease is mediated by neutralizing antibodies infection were protected against disease. This is an
directed against the major envelope glycoproteins of important ﬁnding, since HI antibody levels are
the virus (Jakeman et al., 1989). A recent study by commonly thought to correlate with protection against
Anderson and Mosser (2002) demonstrated that the clinical disease (de Jong et al., 1999, 2001; Hannoun
innate immune system could divert a Th1 to a Th2 et al., 2004). The results of this study reﬂect the limited
adaptive immune response by binding the antigen to ability of HI antibody levels to be used as tools to predict
the IgG Fc portion of the macrophage (FcgR). protection; however they can be useful for herd health
Typically, activated macrophages acting as antigen monitoring or antibody surveillance for vaccination.
presenting cells, induce a Th1-like response. However, We also demonstrated that vaccination induced a
when the antigen is bound to an antibody, the FcgR on memory T-cell response that appears to be important in
the macrophage can be targeted resulting in a more clearing infection. While the presence of MDA
Th2-like phenotype. MDA+ at the time of vaccination decreased clinical disease, they did not reduce the
in the pigs in this study may have resulted in an amount of virus present in the respiratory tract of
increased Th2-type response due to the presence of infected pigs and their presence suppressed the HI
Ab–Ag complexes forming from the vaccine antigen. antibody response to vaccination. In addition, MDA at
Thus following the heterologous H1 infection, MDA+- the time of vaccination reduced vaccine efﬁcacy and
vaccinated pigs were unable to rapidly mount an possibly enhanced the SIV-induced pneumonia. In this
effective CMI response to clear the virus from the study, it appears that MDA inhibited the production of
lungs. memory T-cells by the vaccine. However, more
Our theories are supported by the observed pro- investigation is required to determine the exact
liferation of CD4+/8+ T-cells (memory T-cell) in mechanism of the MDA-induced disease observed
response to both the vaccine and challenge antigens at here. The ﬁndings in our study clearly demonstrated
21 DPI in MDAÀ-vaccinated pigs. In contrast, MDA+- that vaccination provides better protection than MDA
vaccinated pigs had no memory T-cells although high against inﬂuenza and brings into question the common
levels of HI antibodies to the challenge antigen were practice of immunizing sows to increase MDA levels
present at 21 DPI. While the MDAÀ-vaccinated pigs for piglet protection.
had lower HI antibody levels, signiﬁcantly higher
levels of memory T-cells were present which may
explain the rapid recovery of MDAÀ-vaccinated pigs Acknowledgements
following infection. In addition, these results conﬁrm
previous studies in mice (Flynn et al., 1998; Woodland The authors would like to thank Pﬁzer Animal
et al., 2001) that demonstrated the importance of Health for support of this project. We thank Dr. Van De
memory T-cells in clearing inﬂuenza virus and con- Woestyne for assistance with statistical analysis. In
trolling clinical disease. addition, we would like to thank Nancy Upchurch and
This study investigated the efﬁcacy of SIV vacci- the students in the Thacker Lab for their assistance in
nation against a heterologous challenge and the role this project.
MDA play in vaccination efﬁcacy for protection against
clinical disease and pneumonia. We demonstrated that a
complete match between the vaccine strains to the ﬁeld References
strains detected by HI test may not always be required
for a successful vaccination strategy. Interestingly, at Anderson, C.F., Mosser, D.M., 2002. Cutting edge: biasing immune
the time of infection no signiﬁcant differences in the HI responses by directing antigen to macrophage Fc gamma recep-
antibody levels to the challenge antigen were present in tors. J. Immunol. 168, 3697–3701.
vaccinated pigs independent of MDA status, yet the Blaskovic, D., Rathova, V., Kociskova, D., Kaplan, M.M., Jamri-
chova, O., Sadecky, E., 1970. Experimental infection of weanl-
outcome following experimental infection differed ing pigs with A-swine inﬂuenza virus. 3. Immunity in piglets
signiﬁcantly between the groups. Pigs with low levels of farrowed by antibody-bearing dams experimentally infected a
HI antibodies to the challenge antigen and high HI year earlier. Bull. World Health Organ. 42, 771–777.
P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128 127
Casadevall, A., Pirofski, L.A., 2003. Antibody-mediated regulation Larsen, D.L., Karasin, A., Zuckermann, F., Olsen, C.W., 2000.
of cellular immunity and the inﬂammatory response. Trends Systemic and mucosal immune responses to H1N1 inﬂuenza
Immunol. 24, 474–478. virus infection in pigs. Vet. Microbiol. 74, 117–131.
Choi, Y.K., Goyal, S.M., Farnham, M.W., Joo, H.S., 2002. Phylo- Loeffen, W.L., Heinen, P.P., Bianchi, A.T., Hunneman, W.A., Ver-
genetic analysis of H1N2 isolates of inﬂuenza A virus from pigs heijden, J.H., 2003. Effect of maternally derived antibodies on
in the United States. Virus Res. 87, 173–179. the clinical signs and immune response in pigs after primary and
Choi, Y.K., Goyal, S.M., Joo, H.S., 2003. Retrospective analysis of secondary infection with an inﬂuenza H1N1 virus. Vet. Immu-
etiologic agents associated with respiratory diseases in pigs. nol. Immunopathol. 92, 23–35.
Can. Vet. J. 44, 735–737. Mengeling, W.L., Lager, K.M., Vorwald, A.C., 1995. Diagnosis of
Choi, Y.K., Goyal, S.M., Joo, H.S., 2004. Evaluation of transmission porcine reproductive and respiratory syndrome. J. Vet. Diagn.
of swine inﬂuenza type A subtype H1N2 virus in seropositive Invest. 7, 3–16.
pigs. Am. J. Vet. Res. 65, 303–306. Mensik, J., Pokorny, J., 1971. Development of antibody response to
de Jong, J.C., Heinen, P.P., Loeffen, W.L., van Nieuwstadt, A.P., swine inﬂuenza virus in pigs. I. The inﬂuence of experimental
Claas, E.C., Bestebroer, T.M., Bijlsma, K., Verweij, C., Oster- infection of pregnant sows on serum antibody production by
haus, A.D., Rimmelzwaan, G.F., Fouchier, R.A., Kimman, T.G., their progeny during postnatal development. Zentralbl. Veter-
2001. Antigenic and molecular heterogeneity in recent swine inarmed. B 18, 177–189.
inﬂuenza A(H1N1) virus isolates with possible implications for Mensik, J., Valicek, L., Pospisil, Z., 1971. Pathogenesis of swine
vaccination policy. Vaccine 19, 4452–4464. inﬂuenza infection produced experimentally in suckling pig-
de Jong, J.C., van Nieuwstadt, A.P., Kimman, T.G., Loeffen, W.L., lets. 3. Multiplication of virus in the respiratory tract of suck-
Bestebroer, T.M., Bijlsma, K., Verweij, C., Osterhaus, A.D., ling piglets in the presence of colostrum-derived speciﬁc
Class, E.C., 1999. Antigenic drift in swine inﬂuenza H3 hae- antibody in their blood stream. Zentralbl. Veterinarmed. B
magglutinins with implications for vaccination policy. Vaccine 18, 665–678.
17, 1321–1328. Olsen, C.W., 2002. The emergence of novel swine inﬂuenza viruses
Dorn, A.D., Waters, W.R., Byers, V.M., Pesch, B.A., Wannemuehler, in North America. Virus Res. 85, 199–210.
M.J., 2002. Characterization of mitogen-stimulated porcine Puck, J.M., Glezen, W.P., Frank, A.L., Six, H.R., 1980. Protection of
lymphocytes using a stable ﬂuorescent dye (PKH2) and multi- infants from infection with inﬂuenza A virus by transplacentally
color ﬂow cytometry. Vet. Immunol. Immunopathol. 87, 1–10. acquired antibody. J. Infect. Dis. 142, 844–849.
Englund, J., Glezen, W.P., Piedra, P.A., 1998. Maternal immuniza- Reed, L.J., Muench, H., 1938. A simple method of estimating ﬁfty
tion against viral disease. Vaccine 16, 1456–1463. percent endpoints. Am. J. Hyg. 27, 493–497.
Flynn, K.J., Belz, G.T., Altman, J.D., Ahmed, R., Woodland, D.L., Renshaw, H.W., 1975. Inﬂuence of antibody-mediated immune
Doherty, P.C., 1998. Virus-speciﬁc CD8+ T cells in primary and suppression on clinical, viral, and immune responses to swine
secondary inﬂuenza pneumonia. Immunity 8, 683–691. inﬂuenza infection. Am. J. Vet. Res. 36, 5–13.
Hannoun, C., Megas, F., Piercy, J., 2004. Immunogenicity and Thacker, E.L., Thacker, B.J., Janke, B.H., 2001. Interaction between
protective efﬁcacy of inﬂuenza vaccination. Virus Res. 103, Mycoplasma hyopneumoniae and swine inﬂuenza virus. J. Clin.
133–138. Microbiol. 39, 2525–2530.
Heinen, P.P., van Nieuwstadt, A.P., de Boer-Luijtze, E.A., Bianchi, Van Reeth, K., 2000. Cytokines in the pathogenesis of inﬂuenza. Vet.
A.T., 2001. Analysis of the quality of protection induced by a Microbiol. 74, 109–116.
porcine inﬂuenza A vaccine to challenge with an H3N2 virus. Van Reeth, K., Labarque, G., De Clercq, S., Pensaert, M.,
Vet. Immunol. Immunopathol. 82, 39–56. 2001. Efﬁcacy of vaccination of pigs with different H1N1
Heinen, P.P., van Nieuwstadt, A.P., Pol, J.M., de Boer-Luijtze, E.A., swine inﬂuenza viruses using a recent challenge strain
van Oirschot, J.T., Bianchi, A.T., 2000. Systemic and mucosal and different parameters of protection. Vaccine 19, 4479–
isotype-speciﬁc antibody responses in pigs to experimental 4486.
inﬂuenza virus infection. Viral Immunol. 13, 237–247. Van Reeth, K., Van Gucht, S., Pensaert, M., 2002. In vivo studies on
Jakeman, K.J., Smith, H., Sweet, C., 1989. Mechanism of immunity cytokine involvement during acute viral respiratory disease of
to inﬂuenza: maternal and passive neonatal protection following swine: troublesome but rewarding. Vet. Immunol. Immuno-
immunization of adult ferrets with a live vaccinia-inﬂuenza virus pathol. 87, 161–168.
haemagglutinin recombinant but not with recombinants contain- Vincent, L.L., Janke, B.H., Paul, P.S., Halbur, P.G., 1997. A mono-
ing other inﬂuenza virus proteins. J. Gen. Virol. 70 (Pt. 6), 1523– clonal-antibody-based immunohistochemical method for the
1531. detection of swine inﬂuenza virus in formalin-ﬁxed, parafﬁn-
Karasin, A.I., Olsen, C.W., Anderson, G.A., 2000a. Genetic char- embedded tissues. J. Vet. Diagn. Invest. 9, 191–195.
acterization of an H1N2 inﬂuenza virus isolated from a pig in Waters, W.R., Harkins, K.R., Wannemuehler, M.J., 2002. Five-color
Indiana. J. Clin. Microbiol. 38, 2453–2456. ﬂow cytometric analysis of swine lymphocytes for detection of
Karasin, A.I., Schutten, M.M., Cooper, L.A., Smith, C.B., Subbarao, proliferation, apoptosis, viability, and phenotype. Cytometry 48,
K., Anderson, G.A., Carman, S., Olsen, C.W., 2000b. Genetic 146–152.
characterization of H3N2 inﬂuenza viruses isolated from pigs in Webby, R.J., Swenson, S.L., Krauss, S.L., Gerrish, P.J., Goyal, S.M.,
North America, 1977–1999: evidence for wholly human and Webster, R.G., 2000. Evolution of swine H3N2 inﬂuenza viruses
reassortant virus genotypes. Virus Res. 68, 71–85. in the United States. J. Virol. 74, 8243–8251.
128 P. Kitikoon et al. / Veterinary Immunology and Immunopathology 112 (2006) 117–128
Woodland, D.L., Hogan, R.J., Zhong, W., 2001. Cellular immunity antibody against swine inﬂuenza viruses. J. Vet. Diagn. Invest.
and memory to respiratory virus infections. Immunol. Res. 24, 16, 197–201.
53–67. Zhou, N.N., Senne, D.A., Landgraf, J.S., Swenson, S.L., Erickson,
Yoon, K.J., Janke, B.H., Swalla, R.W., Erickson, G., 2004. Compar- G., Rossow, K., Liu, L., Yoon, K.J., Krauss, S., Webster, R.G.,
ison of a commercial H1N1 enzyme-linked immunosorbent 2000. Emergence of H3N2 reassortant inﬂuenza A viruses in
assay and hemagglutination inhibition test in detecting serum North American pigs. Vet. Microbiol. 74, 47–58.