Research Article: Development of Recombinant Vaccine against A(H1N1) 2009 Influenza Based on Virus-like Nanoparticles Carrying the Extracellular Domain of M2 Protein

Date Published: July , 2010

Publisher: A.I. Gordeyev

Author(s): R.Y. Kotlyarov, V.V. Kuprianov, A.I. Migunov, L.A. Stepanova, L.M. Tsybalova, O.I. Kiselev, N.V. Ravin, K.G. Skryabin.

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Abstract

The conventional vaccines currently being used to deal with influenza are based on a virus
obtained in chicken embryos or its components. The high variability of the major immunogenic
surface proteins – hemagglutinin and neuraminidase–require the development of
strain–specific vaccines that match the antigenic specificity of a newly emerging virus.
Recombinant vaccines based on single viral proteins that could be easily produced in standard
expression systems are attractive alternatives to traditional influenza vaccines. We
constructed recombinant nanosized virus–like particles based on a nuclear antigen of the
hepatitis B virus. These particles expose on the surface the extracellular domain of the M2
protein of the highly pathogenic A(H1N1) 2009 influenza virus. The methods of production of
these virus–like particles in Escherichia coli and their purification
were developed. Experiments on animals show that M2sHBc particles are highly
immunogenic in mice and provide complete protection against the lethal influenza challenge.

Partial Text

Influenza is the most common viral disease in humans and animals. Type A influenza viruses
vary in their degrees of pathogenicity. In recent years, the H5N1strain has caused local
outbreaks of the disease with a high morbidity rate in Southeast Asia. The H1N1 virus
originating in swine was behind the flu pandemic that lasted from 2009 to 2010, with an
unexpectedly high morbidity rate among middle–aged and high–risk individuals. Given
its large amount of phenotypic attributes and its phylogenic origin, the H1N1 virus is akin to
the virus that was deemed responsible for the Spanish Flu epidemic that lasted between 1918 and
1920. These characteristics provide evidence of a possible return of a highly pathogenic virus
into circulation throughout the human population. The current influenza vaccines are based on a
virus obtained from chicken embryos, or from its components [1]. The high variability of the viral surface proteins, hemagglutinin and
neuraminidase, leads to the appearance of an epidemic strain every 1–2 years [2], which requires the development of a “standard”
strain–specific vaccine at the same rate.

Construction of expression vector pQE–M2sHBc andE. coliproducer strain. The gene that codes for the hybrid
protein M2sHBc was synthesized using a three–step PCR.
During the first step, the portion of the M2HBc sequence was obtained as a result of
PCR with the primers M2F3 (C GAA TGG GAA TGC CGT TGC AGC GAT AGC AGC GAT GAC
CCT) and HBC–R2 (A GGA TCC TCA GCA AAC AAC AGT AGT CTC CGG AAG) and DNA copy of the
hepatitis B virus genome as a template. During the second step, the obtained fragment was used
as a template for the PCR with the primers M2sF1 (GAA ACC CCG ACC CGT AGC GAA
TGG GAA TGC CGT TGC AGC) and HBC–R2. During the third step, a full–sized gene,
M2sHBc, was obtained as a result of the PCR amplification
with the primers M2sF2 (CTC ATC AGC CTG CTG ACC GAA GTG GAA ACC CCG ACC CGT AGC) and
HBC–R2. The fragment obtained, 525 bp, was digested with the restriction enzymes PagI and
BamHI, whose recognition sites were entered into the sequence of primers M2sF2 and
HBC–R2, respectively, and cloned into the expression vector pQE60 (Qiagen) using sites
for NcoI and BamHI. The expression vector pQE–M2sHBc was used in further
work. Sequencing verified the absence of the PCR–specified mutations in
the synthesized gene.

Design and production of M2sHBc nanoparticles . The Hepatitis B
nuclear antigen is one of the most effective carriers of antigen determinants. Monomers of this
protein, consisting of 183 amino acid residues, self–assemble into icosahedral particles
with a 34 nm diameter, made of 240 subparticles organized in dimeric blocks [19]. Two HBc antigen regions can be used for
the presentation of foreign peptides on the surface of the HBc particles
– the protein N–terminus and the immunodominant loop located between the
75th and 85th amino acid residues of the protein [20–22]. Based on our experience,
the introduction of the foreign sequence into the immunodominant loop results, in most cases,
in the disturbance of the assembly and/or the solubility of the particles. Therefore, as a site
for the introduction of the M2e peptide for the construction of the hybrid
protein M2sHBc, the N–terminus of HBc was used. The
HBc sequence contains an arginine–rich C–terminal domain, which
binds viral DNA during the viron assembly. When expressed in E. coli ,
this domain binds bacterial RNA [23],
whose presence in the preparation is undesired. Since the C–terminal domain (150 –
183 amino acid residues) is not necessary for the assembly of the particles [24], it was removed and replaced by a cysteine residue, whose
introduction increases the stability of HBc particles [16]. Therefore, our hybrid protein M2sHBc comprises, starting
from the N–terminus, the sequence of M2e peptide of the swine flu virus
A/California/04/2009 (H1N1), the sequence of HBc antigen from the
4th to 149th amino acid residues, and the C–terminal cysteine.

The purpose of this study was to develop a recombinant candidate vaccine against a new, highly
pathogenic strain of the influenza A virus: the swine flu H1N1. We used an approach intended to
design a nanovaccine in which an extracellular domain of the M2 protein of the influenza virus
was introduced on the surface of the virus–like particles formed by a nuclear antigen of
hepatitis B. Our data show that the hybrid protein M2sHBc was efficiently
expressed in E. coli and self–assembled in nanosized virus–like
particles. Mice immunization with M2sHBc particles generates an effective
immune response against M2e, and it ensures immunity against an influenza
virus strain that has an identical M2e peptide sequence. Thus,
M2sHBc particles can be used as a basis for the development of a recombinant
vaccine against the modern pandemic swine flu H1N1 and other viruses whose appearance is
expected in the coming years.

 

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