Download Title: Investigation of the ability of the M2 protein to induce cross

Title: Investigation of the ability of the M2 protein to induce cross protection
against different H1 SIV isolates
Principal Investigator: Eileen Thacker, DVM, PhD, DACVM
Professor
Veterinary Microbiology and Preventative Medicine
College of Veterinary Medicine
Iowa State University, 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 types that 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 these newly 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 vaccines are needed to provide costeffective 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
1
No
2
Yes
3
Yes
4
No
5
Yes
6
Yes
7
No
rM2 protein
No
Yes
No
No
Yes
No
No
SIV infection # pigs necropsied
No
5
cH1N1
5
cH1N1
5
cH1N1
5
H1N2
5
H1N2
5
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/40776/92 classic
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 febrile at 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 differences
in the percentage of pneumonia were detected between any of the vaccinated and challenged
groups. The pigs vaccinated 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).
Vaccinated pigs, 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 IgA were 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 groups that 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 HIantibodies and 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 of the 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 rM2specific 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 earlier in vitro study demonstrated
reduced influenza virus spread in cell cultures with 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 as potentially 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 DPI
1
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 DPI
a
a
a
1
0.0±0.0
0.0±0.0
0.0±0.0
0.0±0.0
0.0±0.0a
b
b
b
2
0.0±0.0
2.0±0.0
1.8±0.3
1.5±0.3
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
b
b
b
4
0.0±0.0
2.0±0.0
2.0±0.0
1.8±0.2
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
b
b
b
6
0.0±0.0
2.0±0.0
1.5±0.3
1.0±0.0
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 positive
to SIV-antigen
a
a
1
0.93 ± 0.3
0.0±0.0
0a
a,b
a
2
3.60 ± 1.6
0.6±0.2
0a
3
1.50 ± 0.7a
1.9±0.5b
40a
b
b
4
17.86 ± 2.8
2.7±0.4
40a
5
0.79 ± 0.5a
0.2±0.2a
20a,b
a,b
a
6
1.86 ± 1.0
0.2±0.2
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
a
a
a
1
0±0
0±0
0±0
0±0a
b
c
b,c
2
4.6±0.5
6.3±0.5
3.0±0.0
4.5±0.6c,d
3
4.0±0.4b
6.0±0.3c
3.0±0.3b,c
4.8±0.7c,d
a
b
a
4
0±0
2.6±0.2
0±0
2.0±0.3a,b
5
3.4±0.6b
4.8±0.6c
2.4±0.2b
5.0±0.5c,d
b
c
c
6
3.6±0.4
5.3±0.3
3.2±0.2
5.8±0.3d
a
a
a
7
0±0
0.6±0.4
0±0
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
a
a
a,b
3
0.0±0.0
0.2±0.2
0.148±0.035
0.410±0.078a,b
4
0.0±0.0a
0.2±0.2a
0.086±0.011a
0.335±0.021a,b
b
b
b
5
2.0±0.0
2.0±0.0
0.357±0.132
0.716±0.135b
a
a
a.b
6
0.0±0.0
0.0±0.0
0.156±0.049
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)
CD4+ T cells
proliferated/10,000 PBMCs
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) ± SEM
IgA
IgG
IgA
IgG
a
a
a
1
0.099±.030
0.048±0.035
0.094±0.018
0.021±0.018a
a,b,c
b
a,b
2
0.380±0.083
0.479±0.147
0.506±0.145
0.567±0.196b
3
0.739±0.190c
0.580±.076b
0.861±0.257b
0.915±0.059b,c
a,b
a
a,b
4
0.237±0.029
0.034±0.011
0.369±0.034
0.008±0.004a
5
0.521±0.134a,b,c
0.616±0.096b
0.850±0.182b
1.067±0.163c
b,c
b
b
6
0.623±0.171
0.608±0.053
0.902±0.236
1.140±0.156c
a,b
a
a,b
7
0.171±0.039
0.038±0.012
0.312±0.042
0.023±0.009a
a-c
Values with superscripts within each column are significantly different (p<0.001)
1800
1600
1400
b
1200
1000
800
600
400
200
0
NEG
BiVac
BiVac+M2
b
b
a
a
a
cH1N1
H1N2
Antigen used in test
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) being
statistically different compared within the box (p ≤ 0.004).
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Frace, A.M., Klimov, A.I., Rowe, T., Black, R.A. & Katz, J.M. Modified M2 proteins
produce heterotypic immunity against influenza A virus. Vaccine 1999, 17(18), 22372244.
De Filette, M., Min Jou, W., Birkett, A. et al. Universal influenza A vaccine:
optimization of M2-based constructs. Virology 2005, 337(1), 149-161.
Liu, W., Zou, P., Ding, J., Lu, Y. & Chen, Y.H. Sequence comparison between the
extracellular domain of M2 protein human and avian influenza A virus provides new
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Presentations:
Thacker, E.l., Kitikoon, P., Yager, G., Strait, E., Yu, S., Janke, B. 2006. Investigation of the
ability of the M2 protein to enhance cross protection against different SV isolates. In: Proc. 19th
IPVS, Copenhagen, DK. July 16-19, Vol. 1. p. 268.
The effect of the matrix 2 (M2) protein on the efficacy of a commercial bivalent swine influenza
vaccine.P. Kitikoon, E. L. Strait, S. Yu, B. H. Janke, B. Z. Erickson, E. L.Thacker (submitted
Vet Record)
Matrix 2 protein contributes to protection against different H1 swine influenza isolates.
P. Kitikoon, A. Vincent, B. Janke, B. Erickson, E. Strait, S. Yu, M. Gramer and E. Thacker. To
be presented at the Emerging Disease Conference, June 24-27,2007, Krakow, Poland.