Efficacy of Influenza Vaccination and Tamiflu® Treatment – Comparative Studies with Eurasian Swine Influenza Viruses in Pigs

Recent epidemiological developments demonstrated that gene segments of swine influenza A viruses can account for antigenic changes as well as reduced drug susceptibility of pandemic influenza A viruses. This raises questions about the efficacy of preventive measures against swine influenza A viruses. Here, the protective effect of vaccination was compared with that of prophylactic Tamiflu® treatment against two Eurasian swine influenza A viruses. 11-week-old pigs were infected by aerosol nebulisation with high doses of influenza virus A/swine/Potsdam/15/1981 (H1N1/1981, heterologous challenge to H1N1 vaccine strain) and A/swine/Bakum/1832/2000 (H1N2/2000, homologous challenge to H1N2 vaccine strain) in two independent trials. In each trial (i) 10 pigs were vaccinated twice with a trivalent vaccine (RESPIPORC® FLU3; 28 and 7 days before infection), (ii) another 10 pigs received 150 mg/day of Tamiflu® for 5 days starting 12 h before infection, and (iii) 12 virus-infected pigs were left unvaccinated and untreated and served as controls. Both viruses replicated efficiently in porcine respiratory organs causing influenza with fever, dyspnoea, and pneumonia. Tamiflu® treatment as well as vaccination prevented clinical signs and significantly reduced virus shedding. Whereas after homologous challenge with H1N2/2000 no infectious virus in lung and hardly any lung inflammation were detected, the virus titre was not and the lung pathology was only partially reduced in H1N1/1981, heterologous challenged pigs. Tamiflu® application did not affect these study parameters. In conclusion, all tested preventive measures provided protection against disease. Vaccination additionally prevented virus replication and histopathological changes in the lung of homologous challenged pigs.


Introduction
Vaccines and antiviral drugs are essential means for control of influenza [1]. The fast spread and frequent mutation rate of influenza viruses contribute to high incidence and variability of these viruses in seasonal, epidemic, and pandemic influenza [2,3]. The area-wide and permanent circulation of swine influenza A viruses together with the possibility of interspecies transmission and replication of avian and human influenza A viruses enables reassortment of new viruses in pigs [4][5][6][7][8][9]. As shown by the emergence of pandemic influenza A H1N1(2009) virus (pH1N1/ 2009) such reassorted viruses can represent a worldwide threat [10][11][12]. The antigenic properties as well as drug susceptibility of pH1N1/2009 are determined by gene segments of swine influenza A viruses. In particular, pH1N1/2009 became resistant to M2 channel inhibitors [13,14] by accepting the matrix protein-coding gene of European swine influenza A viruses which confers the drug resistance [15,16]. Since H3N2 viruses circulating in humans are also resistant to this drug class [17,18] a situation of nearly 100% prevalence of ion channel inhibitor resistance was caused worldwide and neuraminidase inhibitors (NAI) like TamifluH and RelenzaH are the only drugs considered for additional prophylactic use at the moment.
The current knowledge about the efficacy of existing NAI against Eurasian swine influenza A viruses is based only on in vitro data [19,20]. To extend this knowledge, in the present study the efficacy of vaccination as well as the application of TamifluH against two Eurasian swine influenza A viruses was compared under experimental conditions in their natural host. The protective effect of vaccination was comparatively studied in a vaccine-heterologous as well a vaccine-homologous challenge.

Results
Comparison of efficacy of vaccination and TamifluH treatment against H1N1/1981 (vaccine-heterologous challenge) H1N1/1981 had been isolated within the first period after introduction of avian-like viruses into the European pig population [21,22]. Because the vaccine strain H1N1/2003 was isolated after 22 years of evolution of these viruses in pigs and vaccinated pigs do not cross-react in HI with H1N1/1981, challenge with H1N1/ 1981 allows studying the efficacy of vaccination against heterologous challenge with a not cross-reactive strain of the same influenza A virus subtype in comparison to the prophylactic effect of TamifluH.
Just 24 hours after infection with H1N1/1981 unvaccinated untreated pigs developed influenza with dyspnoea diagnosed until day 3 p.i. (Fig. 1A). Coughing was observed rarely in individual animals only (data not shown). Furthermore, a significant rise in body temperature was observed on day 1 p.i. (Fig. 1B). Vaccination and TamifluH treatment significantly reduced clinical signs ( Fig. 1A and 1B). Reduction of body weight was not observed (data not shown).
Macroscopic lung lesions of control pigs mainly affected the margins of the cardiac lobes, followed by lesions on the margins of the apical lobes. The diaphragmatic lobe was only rarely affected near to the cardiac lobe. In general the extent of lung consolidation did not exceed 5-10% of the lung surface. On day 2 p.i., a protective effect was neither observed in TamifluH-treated nor in FLU3-vaccinated animals. Two vaccinated pigs had larger lung lesions than any other pig. On day 10 p.i., lesions were significantly lower in vaccinated pigs and TamifluH-treated pigs ( Table 1). A histopathological score of about 3 and 2 was detected in untreated, infected as well as TamifluH-treated pigs on day 2 and 10 p.i. (Fig. 1D). Despite similar virus replication in the lung, a significantly lower histopathological score was observed in vaccinated pigs at both time points (Fig. 1D).
After first vaccination with FLU3 marginal antibody titres to the vaccine strains H1N1/2003 and H1N2/2000 were detected ( Fig. 2A). These antibodies had risen to highly significant titres after second vaccine administration and did not differ significantly between H1N1/2003 and H1N2/2000. The challenge induced a strong H1N1/1981-specific antibody response on 6 days p.i. in vaccinated pigs whereas it was observed in pigs of the TamifluHtreated as well as the untreated group two days later ( Fig. 2A, B, C). Vaccinated pigs developed significantly higher H1N1/1981specific antibody titres in comparison to the TamifluH-treated and the untreated group on day 6, 8, and 10 p.i. (vaccinated versus TamifluH group: p = 0.008; vaccinated versus untreated group: p = 0.003). Starting on day 6 p.i., antibodies generated in vaccinated, H1N1/1981-infected pigs did cross-react with pH1N1/2009 ( Fig. 2A). The vaccine-induced antibodies against H1N1/2003 were boosted after H1N1/1981 infection and rose significantly from day 4 p.i. to day 8 p.i. (Fig. 2A).
The vaccine induced highly significant NI antibody titres to all tested viruses (''before'' versus ''vaccination'': p,0.001; for antibody titres compare with

Comparison of efficacy of influenza vaccination and TamifluH treatment against H1N2/2000 (vaccinehomologous challenge)
The FLU3 vaccine contains a high passage of strain H1N2/ 2000. Therefore, challenge with the same H1N2 enables studying the effect of vaccination against homologous virus infection.
Influenza induced by H1N2/2000 in untreated, infected control animals was characterized by two dyspnoea peaks on day 1 and 4 p.i (Fig. 4A) and temperature .41uC on day 1 p.i. (Fig. 4B). All pigs recovered from clinical signs within 5 days. Clinical signs were neither observed in vaccinated nor in TamifluH-treated pigs. None of the H1N2/2000-infected animals lost body weight (results not shown).
Vaccination as well as TamifluH-treatment caused a significant virus titre reduction in nasal swabs (Fig. 4C). All unvaccinated, untreated pigs shed virus from day 1 to 5 p.i. Virus titres ranged from 1.3 to 2.7 log 10 EID 50 /ml. Mean virus titres of TamifluHtreated pigs were significantly reduced from 1 to 4 days p.i. (day 1 p.i. 6 of 10 pigs -6/10 pigs -shed virus at titres ranging from 0.7-0.9 log 10 EID 50 /ml, day 2 p.i. 5/10 pigs 0.7-1.3 log 10 EID 50 /ml, day 3 p.i. 2/5 pigs 0.7-2.1 log 10 EID 50 /ml, day 4 p.i. 3/5 pigs 0.9-2.3 log 10 EID 50 /ml). After stopping TamifluH-treatment H1N2/ 2000 shedding increased in four of five pigs on day 5 (1.3-2.3 log 10 EID 50 /ml) but vanished already 6 days p.i. due to the appearing antibodies (Fig. 4C, 5B). In contrast, five of 10 pigs of the vaccinated group did not shed the virus at all. Low virus titres of 0.7 log 10 EID 50 /ml were detected in the nasal swabs of three vaccinated pigs 24 h p.i., from a fourth pig from day 2 to 5 p.i. Table 1. Summary of the virus titres determined in left and right lung lobes and macroscopic lung lesions at ventral and dorsal view (mean 6 standard deviation; n = 5; control group day 10 p.i. n = 7); on day 10 p.i. there was no virus in the lungs anymore (data not shown). While vaccination completely prevented H1N2/2000 replication in lungs of vaccinated pigs, the viral titres between the TamifluH-treated and untreated animals did not differ ( Table 1).
The mean extent of lung consolidation of control animals challenged with H1N2/2000 ranged from 1.9 to 3.0% (Table 1). Vaccinated pigs had few or no lesions at all on day 2 and 10 p.i. A significantly reduced lung histopathology was also observed in vaccinated pigs in comparison to untreated, unvaccinated pigs on days 2 and 10 p.i. (Fig. 4D). A high mean histopathological score of about 3 was characteristic for lung tissue samples of untreated as well as TamifluH-treated pigs on day 2 p.i. (Fig. 4D). Whereas in control animals lung histology worsened till day 10 p.i., an improvement was observed in TamifluH-treated pigs (3.2 versus 2.3; not significant, Fig. 4D).

Discussion
High-dose aerosol infection of pigs with H1N1/1981 and H1N2/2000 caused sudden onset of high fever and dyspnoea like influenza in humans [2,3]. Compared with pig infection trials reported so far the observed clinical symptoms were stronger [23][24][25][26][27][28]. These similarities between influenza in aerosol-infection pig models and influenza in humans and pigs in addition to the similar disease course induced by H1N1/1981 and H1N2/2000 provided a good basis for comparatively evaluating the efficacy of vaccination against heterologous and homologous challenge as well as NAI treatment in the present study. The results reveal different degrees of protection.
Like in humans [29,30], (i) vaccination as well as TamifluHtreatment significantly reduced clinical symptoms and virus shedding whereas (ii) vaccination was less effective when the challenge occurred with heterologous H1N1/1981 than with homologous H1N2/2000. The faster and stronger antibody response against the heterologous challenge strain H1N1/1981 may explain the efficacy of vaccination in the absence of virusspecific HI antibodies against the challenge virus. It suggests a certain degree of reactivity between older and more recent H1N1 strains. The latter could also account for detection of crossprotecting HI antibodies against pH1N1/2009 that concurs strongly with recently published studies [31][32][33][34]. Moreover, a higher antibody response against the vaccine strain H1N1/2003 was detected after H1N1/1981 challenge reflecting the ''antigenic sin'' [35,36]. Additionally, NI can contribute to the protective effect seen after vaccination and heterologous challenge. N1 crossreacting NI antibodies were detected indicating that neuraminidases of H1N1 strains are still antigenetically related to each other. Moreover, major histocompatibility complex restricted epitopes conserved in nucleoprotein and matrix protein could be involved in protection as discussed for human seasonal influenza A viruses and pH1N1/2009 virus [37]. European swine influenza A viruses share similar nucleoprotein, matrix, and polymerase genes.
Marked differences were found comparing the efficacy of the studied preventive measures regarding lung viral load, macro-scopic lesions, and inflammation. The lack of virus inhibition in the lung after vaccination and heterologous challenge reflects the pathogenic role of antigenetic drift in European swine Influenza A (H1N1) viruses between 1981 and 2003. It also demonstrates a low efficacy of TamifluH regarding this study parameter. Macroscopic lung lesions were almost absent after vaccination and homologous challenge. They were also reduced by TamifluH treatment as well as in vaccinated, heterologous challenged pigs on day 10 p.i. after primary enhancement on day 2 p.i. Based on similar observations until day 5 p.i., Gauger et al. postulated that vaccination may potentiate influenza following challenge with divergent homosubtypic viruses that do not share cross-reacting hemagglutinin or serum neutralizing antibodies [32]. But, the significant reduced lung consolidation on day 10 p.i. reported here suggests that this effect is transient and reversed by antibodies specific to the challenge virus. With regard to inflammation, a significant score reduction was found after vaccination but not after drug treatment. Taken together, these results suggest that the pathogenetic processes which lead to induction of disease are blocked at different stages by vaccination and TamifluH. The latter prevented disease despite high viral lung load and interstitial lymphoid tissue hyperplasia. Two further aspects should be mentioned concerning TamifluH treatment. First, the increased virus shedding one day after drug cessation suggest a need for prolonged treatment of pigs until the appearance of protective antibodies in serum. Detection of virusspecific protective antibodies in the blood correlating with virus clearance in the nose underlines this conclusion. Secondly, H1N2/ HI assay was carried out with serum from pigs vaccinated with RESPIPORCH FLU3 (A), pigs treated with TamifluH (B), and untreated pigs (C). Geometric mean and standard deviation of antibody titres determined in serum samples of 10 vaccinated and TamifluH-treated or 12 control animals/day are shown until day 2 p.i. and 5 or 7 from day 3-10 p.i., respectively. Mann-Whitney-U-test was used to calculate p statistical probability: *p,0.05, **p,0.01, ***p,0.001. The detection limit of HI antibody titre determination is shown as dotted line. doi:10.1371/journal.pone.0061597.g005 2000 but not H1N1/1981 is additionally glycosylated at Asn163 in the HA and NAI-resistant in cell culture [19]. Glycosylation in position 163 of HA hampers the HA-NA balance and reduces NAI efficacy in vitro [38]. In contrast to in vitro results however, the prevention of influenza in pigs indicates that HA glycosylation at Asn163 does not necessarily affect the efficacy of TamifluH in the natural host.
In summary, due to the high similarity in the course of influenza A virus infection in pigs and seasonal influenza in humans, the pig infection model described here provides a valuable tool for antiviral investigations. In comparison of the tested preventive measures (vaccination and treatment with TamifluH), all protected against disease. Vaccination provided the most optimal protection at homologous challenge. Here, virus replication and histopathological changes in the lung were prevented.
Influenza  Pigs had been proved to be free of influenza during their life span as well as free of maternally-derived antibodies against pH1N1/ 2009, avian H1N1 and human H1N2 influenza A viruses. They were housed in identical isolation rooms based on their challenge status and were provided with feed and water ad libitum.

Compounds
Commercially available TamifluH capsules (F. Hoffmann-La Roche AG, Basel, CH, batch B113313, 75 mg oseltamivir per capsule) were used for in vivo antiviral studies according to summary of product characteristics.

Experimental Design
Group classification and experimental conditions. Two independent trials, one with H1N1/1981 (heterologous challenge with homosubtypic virus not cross-reactive to sera of vaccinated pigs) and another with H1N2/2000 (homologous challenge with the same strain as in the vaccine highly cross-reactive to sera of vaccinated pigs) were performed. The experimental design is summarized in Table 2. In each trial 32 pigs were allotted randomly into 3 groups. One group of 10 pigs was vaccinated i.m. with 2.0 ml of FLU3 21 and 7 days before challenge. Another group of 10 pigs was treated orally with TamifluH starting with 2 capsules the evening before challenge. Then, 2 TamifluH capsules were administered twice daily for 4 days. The third group included 12 unvaccinated untreated pigs as control.
At an age of 11 weeks, pigs of all 3 groups were simultaneously challenged by one-hour-aerosol exposure. Aerosols of H1N1/1981 and H1N2/2000 were dispersed through a flow aerosol generator which produces droplets of 0.5 to 20 mm under atmospheric pressure. H1N1/1981 was nebulised at a dose of 10 7.85 TCID 50 / m 3 and H1N2/2000 at a dose of 10 7.33 TCID 50 /m 3 .
Experimental infections were done in BSL-2 infection units with High Efficiency Particulate Airfilter H13 filters.
In each trial 5 animals of each group were stunned by electrical stunning tongs 2 days p.i. and bled to death. On 10 days p.i. the remaining animals were slaughtered in the same way.
Lung tissue samples were taken from each lobe for virus detection. Samples of the right and left halves of the lungs were pooled, ground with sterile sea sand, and diluted 1:10 in dilution medium (1.0 ml Amphotericin B and 0.1 ml Gentamycin, made up to 100 ml with phosphate buffered saline solution). Additionally, lung tissue was collected and fixed in 10% neutral buffered formalin for histopathological evaluation.
Blood samples for immunological analysis were taken immediately before the first and second vaccinations, 7 days after the second vaccination (before challenge), and 2, 4, 6, 8, and 10 days p.i.
8 hemagglutinating units (HU) of the 3 vaccine strains, H1N1/ 1981, and pH1N1/2009 were used as antigens and incubated with 1:10 prediluted sera in microtitre plates for 30 min at room temperature. Then a 0.5% chicken erythrocyte suspension was added and incubated for 30 min at room temperature.

Determination of 50% Egg Infectious Dose (EID 50 )
Dilution series (log 10 ) from both lung and nasal swab samples were injected into the allantois cavity of 11-day-old chicken embryos (0.1 ml; 5 eggs per dilution). After sealing the perforation point eggs were incubated at 37uC and checked daily for vitality using an egg candler. On day 4 p.i., the allantois fluid was collected and tested in the hemagglutination test. The Spearman and Kaerber method was used to calculate the EID 50 from the hemagglutinating activity [39,40]. The detection limit was 0.7 log 10 EID 50 /ml.

NA Inhibition (NI)
NI was analyzed using modified protocol of Sandbulte et al. [41]. Briefly, OD was measured at wavelength 550 nm. For data analysis, the absorbance of the fetuin control wells was subtracted from the OD values and the dilution of sera that resulted in a reading equal to 50% of the positive control (virus, no serum) was determined. The inverse of this dilution was the NI titre. Assay validity was supported by positive control samples (virus+fetuin) with mean absorbance of 0.7-1.3, negative control samples (fetuin only) with mean absorbance ,0.08, and control serum which did not significantly inhibit NA activity.

Lung Pathology and Histopathology
The pathology of the lungs was evaluated macroscopically, photographs were taken, and observed lesions were recorded onto a lung diagram. Percentage of affected lung surface area was assessed for each lobe at dorsal and ventral view.
Formalin-fixed lung tissue samples were embedded in paraffin. 5 mm-thick sections were stained with haematoxylin and eosin for light microscopy. Inflammation was scored on a semi quantitative scale from 0-4: 0, no inflammation; 1, discreet interstitial alveolar macrophages; 2, slight interstitial bronchial associated lymphoid tissue hyperplasia; 3, distinct interstitial alveolar macrophages; 4, distinct interstitial and massive broncholuminal alveolar macrophages.

Statistical Analysis
Mann-Whitney-U-test was performed to evaluate statistical significances.