Title: Investigation of the ability of the M2 protein to induce cross protection
againstdifferent H1 SIV isolates
Principal Investigator: Eileen Thacker, DVM, PhD, DACVM
Professor
Veterinary Microbiology and Preventative Medicine
College of Veterinary Medicine
IowaStateUniversity, Ames, IA 50010
Co-Investigator:Pravina Kitikoon, DVM, MS
Graduate Student
Industry Summary
Swine influenza virus (SIV) is currently the cause of significant respiratory diseases in the swine industry. Prior to 1998, SIV in the U.S. swine population consisted of viruses of the H1N1 subtype comprised of swine genetics (cH1N1). In 1998, a H3N2 virus emerged that consisted of genetics from avian and human influenza lineage in addition to the swine lineage of the original virus. The emergence of this virus has resulted in the continuous evolution to new antigenic typesthat differ in the hemagglutinin (HA) and neuraminidase (NA) proteins both genetically and antigenically from the original virus. Currently, disease control in nursery and finishing pigs has become challenging in some production systems due to the emergence of these antigenically different influenza viruses. Historically SIV-induced disease in young pigs was controlled primarily through the production of increased levels of maternally derived antibodies in sows through vaccination. Sow vaccination has become less successful in controlling disease in young pigs in recent years due to reduced cross reaction of the maternal antibodies in protecting against disease induced by thesenewly diverse viruses. While optimally SIV vaccines would be updated on a regular basis with new predominant viruses, similar to what is done with human influenza vaccines,unfortunately, government regulations, financial concerns and diagnostic differences in the swine industry make this strategy difficult to implement. As a result, intervention strategies that utilize current commercial vaccinesare needed to provide cost-effective SIV control for swine producers and veterinarians.
The goal of the research reported here was to evaluate the ability of the matrix 2 (M2) protein to induce cross protection against genetically different SIV isolates. The M2 protein is a minor transmembrane protein which unlike the HA and NA protein, is highly conserved within influenza A viruses [8,25]. Antibodies to M2 protein have been shown to reduce viral spread in cell culture [13] and when passively transferred was capable of inhibiting influenza A replication in mice [15]. In addition, M2-based vaccines were shown to provide protection in studies conducted in experimental animals such as mice and ferrets [11,23,24]. In contrast, a single study conducted in pigs demonstrated enhanced clinical disease when pigs were immunized with a M2 DNA-based vaccine indicating that an immune response against the M2 protein alone was insufficient to provide protection against a SIV challenge. In this study, we hypothesized that incorporating a recombinant M2 (rM2) protein into a commercial SIV H1N1 vaccine will result in pigs mounting an immune response to the major HA and NA membrane proteins and the conserved M2 protein resulting in a broader immune response against the swine H1 viruses and increased protection against clinical disease. The efficacy of the administration of rM2 protein with the conventional vaccine was difficult to determine from this study as the vaccine alone without rM2 protein provided protection against lung pneumonia and reduced viral shedding following infection with both challenge viruses. However, including rM2 protein reduced the microscopic lesion scores and viral levels in the lungs at 5 days post infection in pigs that were challenged with the virus that differed from that included in the commercial vaccine. In addition, the rM2 protein appeared to enhance the proliferative response of CD4+ T cells compared to cells from pigs vaccinated without rM2. The findings in the study support the positive role of rM2 protein in enhancing a broad immune response against SIV infection. Further studies are needed using vaccines that have no cross-protection with the challenge viruses to confirm the possible role of M2 protein addition to vaccines for the control of SIV-induced disease.
Scientific Report
Materials and Methods:
Experimental design
Thirty-five pigs were procured from a herd free of porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, and SIV. Upon arrival, pigs were assigned to one of 7 groups. The experimental design is summarized in Table 1. All animal procedures were conducted under the supervision and approval of the Iowa State University Institutional Animal Care and Use Committee (IACUC).
Table 1: Experimental design
Group / SIV Vaccination / rM2 protein / SIV infection / # pigs necropsied1 / No / No / No / 5
2 / Yes / Yes / cH1N1 / 5
3 / Yes / No / cH1N1 / 5
4 / No / No / cH1N1 / 5
5 / Yes / Yes / H1N2 / 5
6 / Yes / No / H1N2 / 5
7 / No / No / H1N2 / 5
Challenge and vaccines
Pigs were vaccinated with 2 doses of a commercial bivalent vaccine (EndFluence 2™, Intervet Animal Health),consisting of the classic H1N1 and H3N2 viruses, at 3 and 5 weeks of age. Simultaneously the pigs will be vaccinated with rM2 protein in adjuvant at a different site. SIV was challenged via intratracheal inoculation with either the A/Swine/IA/40776/92classic H1N1 (cH1N1) strain or A/Swine/Indiana/9K035/99 (H1N2) strain. We have previously demonstrated minimal cross reactivity serologically between these two viruses.
Clinical evaluation
All pigs were evaluated for both clinical respiratory disease and fever based on rectal temperature daily after challenge. Clinical observations included coughing and respiratory rate.
Pathological examination
All pigs were necropsied at 5 days post infection (DPI). The vaccine efficacy was determined by measuring the presence and level of viral antigen in the lungs by immunohistochemistry test (IHC) [16], viral load in nasal swabs at -1, 2, 4, and 5 DPI by virus isolation on MDCK cells, and the percentage of pneumonia at necropsy as previously described [17].
Antibody evaluation
The humoral immune response was assessed by hemagglutinin-inhibition (HI) assay utilizing both viruses. Anti-M2 antibodies were measured using an indirect ELISA recently developed in our laboratory [18]. The presence of anti-M2 antibodies prior to challenge confirmed that the pig’s immune system recognized the rM2 protein and was used to assess the correlation between anti-M2 antibody levels and protection against disease. Bronchoalveolar lavage fluid (BAL) was collected at 5 dpi to measure SIV-specific IgG and IgA using an ELISA assay performed in our laboratory [19].
Identification of populations of lymphocytes responding to SIV
Cell mediated immune responses were assessed using a proliferation assay coupled to a phenotypic evaluation of the lymphocyte subsets. Heparinized peripheral blood mononuclear cells (PBMCs) were collected to assess systemic lymphocyte proliferation. Lymphocyte proliferation was measured using a membrane dye and lymphocyte population identification by flow cytometry [20].
Statistical analysis
Data were analyzed by analysis of variance to ascertain group differences for each parameter. Pairwise comparisons was performed using least significant differences if the differences are p<0.05.
Results:
Mean rectal temperatures at 1DPI were significantly higher in all SIV challenged pigs compared to pigs in the negative control (group 1) and group 3 (Table 2). Fever was detected only one day following challenge in all groups except for the nonvaccinated, H1N2-challenged group which were also febrileat 3 DPI. Respiratory rates were elevated in all SIV challenged groups and were significantly higher compared to group 1. However, no significant differences were detected between treatment groups (Table 3).
Non-vaccinated, SIV-challenged pigs had significantly more SIV-induced pneumonia compared to vaccinated and negative control pigs (Table 4). However, no significant differencesin the percentage of pneumonia were detected between any of the vaccinated and challenged groups. The pigsvaccinated with rM2 followed by challenge with cH1N1 (group 2) exhibited significantly lower microscopic lesion scores compared to groups vaccinated without rM2 and nonvaccinated, cH1N1-challenged (groups 3 and 4). Although the amount of SIV-antigen in the lungs did not differ statistically between the vaccinated groups that were challenged with H1N2 (groups 5 and 6) the pattern of virus infected cells differed (data not shown). Pigs that received the rM2 protein in addition to the commercial vaccine (groups 5) had SIV antigen confined to a few airways while pigs that received commercial vaccine only (group 6) had SIV-antigen scattered throughout the lung tissues. Non-vaccinated challenged pigs shed significantly more virus compared to all other groups. No significant difference in viral shedding was detected between the vaccinated-challenged groups. At 5 DPI, pigs in groups 4, 6 and 7 shed significantly more virus (p < 0.006)compared to the negative control group (data not shown).
Results from the HI assays demonstrated that the bivalent vaccine induced cross-reactive antibodies to both challenge antigens as all vaccinated pigs (groups 2, 3, 5 and 6) had HI titers to both the cH1N1 and H1N2 antigens prior to SIV-infection (Table 5). Pigs infected with the H1N2 virus (group 7) produced low HI cross-reactive antibodies to the cH1N1 antigen while pigs infected with cH1N1 virus (group 4) produced higher levels of HI cross-reactive antibodies to the H1N2 antigen. The addition of rM2 protein in the vaccine did not appear to reduce the ability of the vaccine to induce a HI-antibody response. No HI-antibodies were detected in any of the negative control pigs throughout the trial. Prior to SIV-infection, rM2-specific antibody secreting cells (ASC) and rM2-antibodies in serum were present only in pigs that received rM2 with vaccination (Table 6). At 5 DPI, low positive rM2-specific ASCs were detected in pigs from groups 3, 4 and 7 in addition to groups 2 and 5. However, the rM2-antibody levels in the sera from those groups were not statistically different from the negative control groups.
Antibodies specific to SIV were detected in the lower airways at 5 DPI (Table 7). Vaccinatedpigs, with or without rM2, had significantly higher levels of SIV-specific IgG to both challenge antigens compared to the nonvaccinated, SIV-infected pigs and negative control pigs. The levels of SIV-specific IgAwere not significantly different between vaccinated pigs, with or without rM2,and nonvaccinated, SIV-challenged pigs except for the level of IgA against cH1N1 antigen detected in group 3. Pigs vaccinated and challenged with the cH1N1 (groups 2 and 3) tended to have increased levels of IgA and IgG in BAL responding to the H1N2 antigen compared to antibody levels to the cH1N1 antigen,. Interestingly, vaccinated pigs challenged with H1N2, independent of rM2 vaccination, (groups 5 and 6) exhibited significantly higher IgG levels to H1N2 antigen than to the cH1N1 antigen (p<0.0001) while no such differences were detected in the nonvaccinated, H1N2-challenged pigs (group 7).
Data from flow cytometry analysis at -1 DPI differed according to the antigen used in the test. When PBMCs were stimulated with the cH1N1 antigen, only groups that were vaccinated with rM2 had significantly increased proliferation of CD4+ T cells. However, when stimulated with the H1N2 antigen CD4+ T cells from both vaccinated groups, independent of rM2 vaccine, showed increased proliferation compared to the nonvaccinated group (Figure 1). At 5 DPI, no statistical differences in T cell proliferation were observed between any of the groups of pigs. The -T cell population from rM2 vaccinated groups challenged with either virus tended to show increased proliferation with the H1N2 antigen compared to the groupsthat did not receive rM2 or the nonvaccinated, SIV-infected group. Stimulation of -T cells with cH1N1 antigen resulted in proliferation of cells only in the group vaccinated with rM2 that was infected with cH1N1 (data not shown).
Discussion:
This study demonstrated that a commercial bivalent SIV vaccine provided protection against SIV-associated pneumonia and reduced viral shedding from two genetically different challenge isolates. Vaccination primed the local airway antibody response as vaccinated pigs, independent of rM2, had increased SIV-specific IgA and IgG levels compared to the nonvaccinated, SIV-challenged pigs. In contrast to an earlier study [12]; there was no disease enhancement in SIV challenged pigs that received the rM2 protein. However, it could not be determined if the addition of rM2 protein to the commercial vaccine enhanced vaccine efficiency since the commercial vaccination alone reduced clinical disease, induced cross-reactive HI-antibodiesand primed the CD4+ T-cells to both challenge viruses.
It is possible that the presence of the H3N2 vaccine antigen in the commercial bivalent vaccine contributed to the protection against the disease induced by the H1N2 isolate used in this study. The suggestion that the commercial vaccine cross-reacted with the H1N2 virus more than the cH1N1 virus was due to significantly higher levels of IgA and IgG against the H1N2 antigen compared to the cH1N1 antigen in the lower airways at 5 DPI in vaccinated groups (groups 2, 3, 5 and 6) especially groups 5 and 6. Flow cytometry analysis at -1 DPI also demonstrated that the vaccine induced increased proliferation of CD4+ T cells in response to the H1N2 virus compared to the cH1N1 virus in all vaccinated groups, independent ofthe addition of rM2. In contrast, only lymphocytes from the groups vaccinated with rM2 proliferated significantly to cH1N1 antigen stimulation. Since the rM2 protein was constructed from the M2 gene of the cH1N1 virus used in the study, it is likely that the M2 protein in the cH1N1 antigen induced that proliferative response.
Although findings from this study could not provide a definite answer of whether the addition of rM2 protein in the vaccine increased the protective efficacy of SIV vaccines, the reduction in microscopic lesions and reduced IHC positive results in the cH1N1-challenged pigs (groups 2-4) are promising. Previous studies showed that M2-antibodies are capable of reducing influenza virus replication both in vitro[13] and in vivo[21]. Significantly higher levels of rM2-specific antibody secreting cells were detected only in the pigs that received the rM2 with vaccine; however, the level of rM2-antibodies in the serum was not statistically different from the group vaccinated without rM2. It is possible that the rM2-antibody produced prior to challenge was not sufficient to reduce virus levels.An earlierin vitro study demonstrated reduced influenza virus spread in cell cultureswith rM2-antibodies was dose-dependent [13]. In addition, earlier M2-vaccine based strategies used a 3 dose vaccination protocol [11,22-24]. Therefore, future studies could investigate enhancing the rM2-antibody response through the administration of more vaccine boosters as well aspotentially different doses and/or adjuvants. Further studies utilizing a vaccine that does not cross-protect with the challenge virus is required to further identify the role that a M2-immune response plays in protecting against SIV-induced clinical disease.
In conclusion, this study demonstrated that the addition of a rM2 protein reduced viral antigen and microscopic lesions in the lungs following infection with a SIV strain that differed from the virus in a commercial SIV inactivated vaccine. These findings provide encouragement for further studies investigating the ability of the addition of the rM2 protein to conventional SIV vaccines to improve SIV control that will benefit the swine industry.
Table 2: Average group rectal temperatures(mean ± SEM)
Group / -1 DPI / 1 DPI / 2 DPI / 3 DPI / 1-2 DPI1 / 103.3±0.17 / 102.8±0.24 / 103.1±0.24 / 103.7±0.18 / 102.7±0.19a
2 / 103.6±0.14 / 104.1±0.38 / 102.6±0.25 / 103.2±0.37 / 103.3±0.31a,b
3 / 103.4±0.10 / 103.8±0.34 / 102.8±0.20 / 103.3±0.51 / 103.3±0.22a,b
4 / 103.1±0.16 / 104.5±0.16 / 103.6±0.37 / 103.3±0.25 / 104.1±0.22a,b
5 / 103.4±0.16 / 104.0±0.24 / 103.2±0.20 / 103.5±0.24 / 103.6±0.21a,b
6 / 103.5±0.18 / 104.2±0.18 / 103.5±0.19 / 103.4±0.08 / 103.8±0.10b
7 / 103.5±0.14 / 104.7±0.45 / 103.4±0.23 / 104.3±0.28 / 104.1±0.29b
a,b Values with superscripts within each column are significantly different (p<0.007)
Table 3: Average group respiratory scores (mean ± SEM)
Group / -1 DPI / 1 DPI / 2 DPI / 3 DPI / 1-2 DPI1 / 0.0±0.0 / 0.0±0.0a / 0.0±0.0a / 0.0±0.0a / 0.0±0.0a
2 / 0.0±0.0 / 2.0±0.0b / 1.8±0.3b / 1.5±0.3b / 1.9±0.1b
3 / 0.0±0.0 / 2.0±0.0b / 1.8±0.2b / 1.6±0.2b / 1.9±0.1b
4 / 0.0±0.0 / 2.0±0.0b / 2.0±0.0b / 1.8±0.2b / 2.0±0.0b
5 / 0.0±0.0 / 2.0±0.0b / 1.4±0.2b / 1.0±0.3b / 1.7±0.1b
6 / 0.0±0.0 / 2.0±0.0b / 1.5±0.3b / 1.0±0.0b / 1.8±0.1b
7 / 0.0±0.0 / 2.0±0.0b / 1.6±0.2b / 1.6±0.2b / 1.8±0.1b
a,b Values with superscripts within each column are significantly different (p<0.0001)
Table 4: Average group percentage of pneumonia, microscopic lesions and SIV antigen detection (IHC)
Group / % lung lesion ± SEM / Microscopic lesion ± SEM / % of pigs positiveto SIV-antigen
1 / 0.93 ± 0.3a / 0.0±0.0a / 0a
2 / 3.60 ± 1.6a,b / 0.6±0.2a / 0a
3 / 1.50 ± 0.7a / 1.9±0.5b / 40a
4 / 17.86 ± 2.8b / 2.7±0.4b / 40a
5 / 0.79 ± 0.5a / 0.2±0.2a / 20a,b
6 / 1.86 ± 1.0a,b / 0.2±0.2a / 25a,b
7 / 15.04 ± 3.9b / 2.8±0.2b / 100b
a,b Values with superscripts within each column are significantly different (p<0.001)
Table 5: Group average hemagglutinin-inhibition (HI) antibody†to SIV-challenge antigen
Group / HI antibody to cH1N1 antigen ± SEM / HI antibody to H1N2 antigen ± SEM-1 DPI / 5 DPI / -1 DPI / 5 DPI
1 / 0±0a / 0±0a / 0±0a / 0±0a
2 / 4.6±0.5b / 6.3±0.5c / 3.0±0.0b,c / 4.5±0.6c,d
3 / 4.0±0.4b / 6.0±0.3c / 3.0±0.3b,c / 4.8±0.7c,d
4 / 0±0a / 2.6±0.2b / 0±0a / 2.0±0.3a,b
5 / 3.4±0.6b / 4.8±0.6c / 2.4±0.2b / 5.0±0.5c,d
6 / 3.6±0.4b / 5.3±0.3c / 3.2±0.2c / 5.8±0.3d
7 / 0±0a / 0.6±0.4a / 0±0a / 3.6±0.4b,c
† HI antibody (n): n = 2n × 5 serum HI antibody titer
a-d Values with superscripts within each column are significantly different (p<0.05)
Table 6: Average score of rM2-specific antibody secreting cells (ASC)†and rM2-specific antibody by ELISA
Group / Mean rM2-ASC score ± SEM / rM2 ELISA mean OD ± SEM-1 DPI / 5 DPI / -1 DPI / 5 DPI
1 / 0.0±0.0a / 0.0±0.0a / 0.083±0.007a / 0.294±.039a
2 / 2.0±0.0b / 2.0±0.0b / 0.180±0.015b / 0.602±0.180b
3 / 0.0±0.0a / 0.2±0.2a / 0.148±0.035a,b / 0.410±0.078a,b
4 / 0.0±0.0a / 0.2±0.2a / 0.086±0.011a / 0.335±0.021a,b
5 / 2.0±0.0b / 2.0±0.0b / 0.357±0.132b / 0.716±0.135b
6 / 0.0±0.0a / 0.0±0.0a / 0.156±0.049a.b / 0.264±0.054a
7 / 0.0±0.0a / 0.4±0.2a / 0.080±0.013a / 0.390±0.053a,b
†Score 0 = No signal, 1 = low positive signal < 50% cells/well (duplicated), 2 = high
positive signal 50% cells/well (duplicated)
a,b Values with superscripts within each column are significantly different (p<0.01)
Table 7: Group average optical density (OD) to SIV-specific antibodies in lower airways (BAL fluid) at 5 DPI
Group / Mean OD (to cH1N1) ± SEM / Mean OD (to H1N2) ± SEMIgA / IgG / IgA / IgG
1 / 0.099±.030a / 0.048±0.035a / 0.094±0.018a / 0.021±0.018a
2 / 0.380±0.083a,b,c / 0.479±0.147b / 0.506±0.145a,b / 0.567±0.196b
3 / 0.739±0.190c / 0.580±.076b / 0.861±0.257b / 0.915±0.059b,c
4 / 0.237±0.029a,b / 0.034±0.011a / 0.369±0.034a,b / 0.008±0.004a
5 / 0.521±0.134a,b,c / 0.616±0.096b / 0.850±0.182b / 1.067±0.163c
6 / 0.623±0.171b,c / 0.608±0.053b / 0.902±0.236b / 1.140±0.156c
7 / 0.171±0.039a,b / 0.038±0.012a / 0.312±0.042a,b / 0.023±0.009a
a-c Values with superscripts within each column are significantly different (p<0.001)
Figure 1: Average number of proliferating CD4+ T cells from 10,000 peripheral blood mononuclear cells (PBMCs) collected at -1 DPI from nonvaccinated (NEG), vaccinated without the addition of rM2 protein (BiVac) and vaccinated with the addition of rM2 protein (BiVac+M2). Data is expressed as group average ± SEM with different letters (a and b) beingstatistically different compared within the box (p ≤ 0.004).
References:
1Olsen, C.W. The emergence of novel swine influenza viruses in North America. Virus Res 2002, 85(2), 199-210.
2Karasin, A.I., Landgraf, J., Swenson, S. et al.Genetic characterization of H1N2 influenza A viruses isolated from pigs throughout the United States. J Clin Microbiol 2002, 40(3), 1073-1079.
3Hierholzer, J.C., Suggs, M.T. & Hall, E.C. Standardized viral hemagglutination and hemagglutination-inhibition tests. II. Description and statistical evaluation. Appl Microbiol 1969, 18(5), 824-833.
4Richt, J.A., Lager, K.M., Janke, B.H., Woods, R.D., Webster, R.G. & Webby, R.J. Pathogenic and antigenic properties of phylogenetically distinct reassortant H3N2 swine influenza viruses cocirculating in the United States. J Clin Microbiol 2003, 41(7), 3198-3205.
5Webby, R.J., Rossow, K., Erickson, G., Sims, Y. & Webster, R. Multiple lineages of antigenically and genetically diverse influenza A virus co-circulate in the United States swine population. Virus Res 2004, 103(1-2), 67-73.
6Rimmelzwaan, G.F., Baars, M., van Beek, R. et al.Influenza virus subtype cross-reactivities of haemagglutination inhibiting and virus neutralising serum antibodies induced by infection or vaccination with an ISCOM-based vaccine. Vaccine 1999, 17(20-21), 2512-2516.