Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein

Abstract

Several types of influenza vaccines are available, but due to the highly unpredictable variability of influenza virus surface antigens (hemagglutinin (HA) and neuraminidase) current vaccines are not sufficiently effective against broad spectrum of the influenza viruses. An innovative approach to extend the vaccine efficacy is based on the selection of conserved influenza proteins with a potential to induce inter-subtype protection against the influenza A viruses. A promising new candidate for the preparation of broadly protective vaccine may be a highly conserved N-terminal part of HA2 glycopolypeptide (HA2 gp) called fusion peptide. To study its capacity to induce a protective immune response, we immunized mice with the fusion peptide (aa 1-38 of HA2 gp). The protective ability of fusion peptide was compared with the ectodomain aa 2-23 of M2 protein (eM2) that is antigenically conserved and its immunogenic properties have already been well documented. Corresponding peptides (both derived from A/Mississippi/1/85 (H3N2) virus) were synthesized and conjugated to the keyhole limpet hemocyanin (KLH) and used for the immunization of mice. Both antigens induced a significant level of specific antibodies. Immunized mice were challenged with the lethal dose of homologous (H3N2) or heterologous A/PR/8/34 (H1N1) influenza A viruses. Immunization with the fusion peptide led to the 100% survival of mice infected with 1 LD50 of homologous as well as heterologous virus. Survival rate decreased when infectious dose was raised to 2 LD50. The immunization with eM2 induced effective cross-protection of mice infected even with 3 LD50 of both challenge viruses. The lower, but still effective protection induced by the fusion peptide of HA2 gp suggested that besides ectodomain of M2, fusion peptide could also be considered as a part of cross-protective influenza vaccine. To our knowledge, this is the first report demonstrating that active immunization with the conjugated fusion peptide of HA2 gp provided the effective production of antibodies, what contributed to the cross-protection against influenza infection.

Full text

Acta virologica 55: 261 – 265, 2011 doi:10.4149/av_2011_03_261
Evaluation of anti-inuenza eciency of polyclonal IgG antibodies specic to
the ectodomain of M2 protein of inuenza A virus by passive
immunizationof mice
J. KIRÁLY, E. VAREČKOVÁ, V. MUCHA, F. KOSTOLANSKÝ*
Department of Orthomyxoviruses, Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava,
Slovak Republic
Received May 24, 2011; accepted June 27, 2011
Summary. – We attempted to quantify the protective potential of polyclonal IgG antibodies specic to the
ectodomain of M2 protein (eM2) of inuenza A virus (IAV) against lethal inuenza infection of mice. For
this purpose, eM2 conjugated with keyhole limpet hemocyanin (KLH) or KLH alone were administered with
Freund’s adjuvant intraperitoneally (i.p.) to BALB/c mice. IgG antibodies specic to the KLH-eM2 conjugate
(anti-KLH-eM2 IgGs) and KLH (anti-KLH IgGs), respectively, were puried from ascitic uids. Analysis of the
preparation of anti-KLH-eM2 IgGs by ELISA revealed that it contained about 25% of anti-eM2 IgGs and 75%
of anti-KLH IgGs. Taking into account this nding mice were passively immunized by intravenous route with
320, 160, 80, and 40 µg of anti-eM2 IgGs per mouse, respectively, while 320 µg of anti-KLH IgGs were used
in control. Following subsequent infection with 3 LD50 IAV the survival of mice was determined. An absolute
protection (100% survival) was obtained with 320 µg of anti-eM2 IgGs, and a relatively strong signicant
protection (~80% survival, p = 0.024) with 160 µg. e amount 160 µg of IgGs represents approx. 100 µg IgGs
per 1 ml of blood.
Keywords: inuenza; M2 protein; eM2-specic IgG concentration; protection
*Corresponding author. E-mail: viruos@savba.sk; fax: +421-2-
54114284.
Abbreviations: IAV = inuenza A virus; eM2 = ectodomain of M2
protein; HA = hemagglutinin; anti-KLH-eM2 IgGs = IgG antibodies
to KLH-eM2; anti-KLH IgGs = IgG antibodies to KLH; i.n. = in-
tranasally; i.p. = intraperitoneally; i.v. = intravenously; KLH = key-
hole limpet hemocyanin; MAb(s) = monoclonal antibody(ies);
NA = neuraminidase
Introduction
ere are current eorts to avoid every year vaccination
against inuenza with a vaccine containing seasonally actual-
ized hemaglutinin (HA) and neuraminidase (NA) antigens
by use of a vaccine based on a conserved antigen(s). Such
a vaccine should evoke a long-lasting immune response
eciently suppressing the infection with various antigenic
variants or even subtypes. e main candidate molecule is
the M2 protein of IAV, particularly its 23 aa-long ectodomain
(eM2) that is characteristic by an outstanding antigenic con-
servativity and is abundantly expressed in the membrane of
infected cells (Neirynck et al., 1999; Palese and Garcia-Sastre,
2002; Lamb, 1985; Zebedee and Lamb, 1988). Nevertheless,
its immunogenicity during the natural inuenza infection is
very weak and short-term (Feng et al., 2006). ese incon-
venient properties of eM2 represent the main issues to be
solved. A number of various approaches for preparation of
eM2-based vaccine focused on increasing its immunogenicity
have been published (Slepuskin et al., 1995; Neirynck et al.,
1999; Wynne et al., 1999; Okuda et al., 2001; Liu et al., 2004;
Ben-Yedidia and Arnon, 2005; Huleatt et al., 2008; DeFilette
et al., 2008). e mechanism of the eM2-associated biological
action was described as the antibody-dependent cell-mediated
cytotoxicity (ADCC), in which NK cells with their low-anity
Fc gamma receptors bind IgG antibodies already bound to
the M2 protein expressed on the surface of infected cells and
mediate their lysis (Jagerlehner et al., 2004). is hypothesis
explains why anti-eM2 IgGs, when present in suciently high

262 SHORT COMMUNICATIONS
concentration, only attenuate the infection while neutralizing
antibodies to HA that directly inhibit the binding of virus to
the cell receptor provide a very eective instant protection.
Despite the lower eciency of eM2-specic antibodies, the
idea of utilization of eM2 in future inuenza vaccine remains
actual and promising as this molecule has a potential to elicit
a a broad cross-protective response (Sui et al., 2010; Stanekova
et al., 2011). In addition to the eM2 immunogenicity, the con-
centration of eM2-specic antibodies remains an important
characteristic to be followed that can also serve as an indicator
of eectivity of particular eM2-based vaccine preparation.
In this study, we atttempted to quantify the protective po-
tential of polyclonal IgG antibodies specic to eM2 IAV against
lethal inuenza infection of mice by determining the depend-
ence of survival of infected mice on actual concentration of these
antbodies in their blood following passive immunization.
Materials and Methods
Virus. A/Mississippi/1/85 (H3N2) was propagated in allantoic
uid of 10-day chicken embryos and stored at -70°C.
Mice. Six-week-old female BALB/c mice purchased from the
Faculty of Medicine, Masaryk University, Brno, Czech Republic,
were used. e animals were treated according to the European
Union standards and fundamental ethical principles including
animal welfare requirements.
eM2 peptide and KLH-eM2 conjugate. A 23-aa-long synthetic
eM2 peptide of IAV (H3 subtype) of the sequence SLLTEVET-
PIRNEWGSRSNDSSD, Mr of 2,592.74 and 93.94% purity was
purchased from ProImmune (USA). e peptide contained substi-
tutions C17S and C19S to avoid formation of disulphide bonds in
the peptide. e conjugation of eM2 with KLH (Sigma) was done
using glutaraldehyde as described by Staneková et al. (2011).
Immunization of mice. BALB/c mice were immunized i.p. with
three doses of KLH-eM2 (30 µg of eM2 per mouse) or KLH (30 µg
pre mouse), respectively, supplemented with Freund’s adjuvant, in
14 day intervals Staneková et al. (2011).
Polyclonal antibodies were puried from ascitic uids by anity
chromatography on Protein A-Sepharose columns (Ey et al., 1978).
Passive immunization of mice. BALB/c mice – 5 animals per
group – were administered i.v. anti-eM2 IgGs in 200 µl doses of 320,
160, 80, and 40 µg per mouse, respectively, while control mice ob-
tained 320 µg of anti-KLH IgGs and 200 µl of PBS, respectively.
Infection of mice. Two hrs aer passive immunization the mice
were intranasally (i.n.) inoculated with 3 LD50 of A/Mississippi/1/85
(H3N2) in 40 μl. Survival of mice was recorded daily for 14 days.
Statistical signicance of survival was evaluated by Fisher exact
test.
ELISA. eM2- or KLH-specic antibodies were assayed by a bind-
ing test according to Varečková et al. (2003a). Wells of 96-well plates
were coated overnight with 100 ng of eM2 or KLH as antigens in
100 μl at 4°C. e antibody titer was calculated as the reciprocal
of sample dilution at the point where the regression line drawn
through the titration curve crossed the cut-o line. e latter value
was the mean from 5 negative control samples plus 3 SD.
Results and Discussion
Antibody response in mice to immunization with eM2
In this work, we used a simple model of the eM2-KLH
conjugate as immunogen supplemented with the Fre-
Fig. 1
Antibody response in mice to immunization with KLH-eM2
Mice were i.p. immunized with 3 doses of KLH-eM2 and their sera were assayed for eM2-specic IgG antibodies by ELISA.

SHORT COMMUNICATIONS 263
und’s adjuvant to induce an antibody response in mice.
Control mice were given KLH alone. Following three im-
munizations the majority of mice developed ascites due to
administration of the adjuvant. e antibody response to
eM2 gradually increased, corresponding to serum titers of
528, 10,800, and 28,800, respectively (Fig. 1). Ascitic uids
obtained aer the last immunization served for purication
of polyclonal IgG antibodies.
Proportions of eM2- and KLH-specic antibodies in IgGs
puried from ascitic uid from mice immunized with KLH-
eM2
Proportions of IgG antibodies specic to eM2 and KLH,
present in IgGs puried from mice immunized with KLH-
eM2, were assayed by ELISA (Fig. 2). e distance of titra-
tion curves at A492 of 1.5 was estimated at 1.49 log2 units and
corresponding titers of anti-eM2 and anti-KLH IgGs were
1120 and 3149, respectively. is means that provided equal
numbers of accessible eM2 epitopes specic to anti-eM2 IgGs
and KLH epitopes specic to anti-KLH IgGs adsorbed onto
respective wells, the ratio of anti-eM2 and anti-KLH IgGs
was approximately 1:3.
Eective anti-eM2 IgG concentration required for the
protection to inuenza infection
Taking into account the anti-eM2 IgGs content of the
anti-KLH-eM2 IgGs puricate, groups of mice were given
i.v. 320, 160, 80, and 40 µg of anti-eM2 IgGs per animal.
Control mice were administered 320 µg of anti-KLH IgGs
and 200 µl of PBS, respectively. Two hrs later the mice were
infected with 3 LD50 IAV and observed for survival. An ab-
solute protection (100% survival) was obtained with 320 µg,
a relatively strong signicant protection (~80% survival) with
160 µg (p = 0.024), and a weak protection (~20% survival)
with 80 and 40 µg of anti-eM2 IgGs (Fig. 3). Control mice
scored a 100% mortality.
Estimating total blood volume in mouse at 1.5 ml, the
applied doses 320 µg and 160 µg of anti-eM2 IgGs resulting
in signicant protection corresponded to the concentra-
tions of 213 and 107 µg/ml anti-eM2 IgGs, respectively, in
the blood.
ese results roughly agree with those of Fu et al. (2009),
who also evaluated the anti-eM2 protective response, how-
ever, by use of MAbs. ey found that two of four tested
MAbs at doses of 0.2–2.0 mg per mouse ensured a high sur-
vival, while 20 µg resulted in a low survival. Another group
of authors (Beerli et al., 2009) found that a most eective
anti-eM2 MAb exhibited a fair protection against infection
with 4 LD50 of virus at a dose of 20 µg per mouse, but only
a weak one at a dose of 6 µg. Such a high eciency of this
MAb as compared with our observations as well as those
of Fu et al. (2009) can be most probably ascribed to a high
anity (Kd = 4 nmol/l) of that particular MAb.
In conclusion, we assume that results of this study con-
tribute to a recently accepted presumption that the eM2
molecule can be an eective and cross-protective immuno-
gen. Its immunogenicity can be enhanced by applying it in
appropriate form and/or with an optimally selected adjuvant.
Its cross-reactivity can be ensured mainly by polyclonal
character of the resulting antibody response. Moreover, the
Fig. 2
Proportions of eM2- and KLH-specic antibodies in IgGs puried from ascitic uid of mice immunized with KLH-eM2
Titration curves of anti-eM2 and anti-KLH IgGs (a) and titers of anti-eM2 and anti-KLH IgGs at A492 of 1.5 (b) in ELISA.

264 SHORT COMMUNICATIONS
latter may contribute to the prevention of the appearance
of antibody-escape mutants during natural infection (Zha-
rikova et al., 2005).
Acknowledgements. e authors thank Mmes M. Némethová and
M. Mišovičová for excellent technical assistance. is work was
supported by the VEGA grants Nos. 2/0101/10 and 2/0154/09 from
the Scientic Grant Agency of Ministry of Education of Slovak Re-
public and Slovak Academy of Sciences and the Slovak Research and
Development Agency under the contract No. APVV-0250-10.
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