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    • Veterinary Immunology and Immunopathology 112 (2006) 117–128 The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination 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,* a Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA b Department of Veterinary Diagnostics and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA c 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 Abstract 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: (E.L. Thacker). 1 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. doi:10.1016/j.vetimm.2006.02.008
    • 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. Use. 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 TM 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. Table 1 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 necropsied at 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 a Days post infection. b Pigs had HI titers between 1:40 and 1:80 at the time of the first vaccination. c Pigs were vaccinated at 3 and 5 weeks of age. d 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  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 1 Present 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  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  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  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  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  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  107 PBMCs 2.8. Statistical analysis were centrifuged (400  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. Results 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 observed. 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 Table 3 Percentage of lung with visible macroscopic lesions and microscopic lesion scores Æ S.E.M. from pigs infected with H1N1 SIV at both necropsies Group Percentage of macroscopic lesions a Microscopic lesion scoresb c 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). a As determined by lesion sketches and image analysis. b SIV microscopic lesion scores are based on the severity of bronchiolar epithelial damage (necrotic bronchiolitis). c 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  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 Table 4 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). a 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. b 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. c 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.
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