Development of single-chain variable fragments (scFv) against
inﬂuenza virus targeting hemagglutinin subunit 2 (HA2)
Received: 24 June 2015 / Accepted: 23 September 2015 / Published online: 8 October 2015
Ó Springer-Verlag Wien 2015
Abstract Inﬂuenza A viruses (IAV) are widespread in
birds and domestic poultry, occasionally causing severe
epidemics in humans and posing health threats. Hence, the
need to develop a strategy for prophylaxis or therapy, such
as a broadly neutralizing antibody against IAV, is urgent.
In this study, single-chain variable fragment (scFv) phage
display technology was used to select scFv fragments
recognizing inﬂuenza envelope proteins. The Tomlinson I
and J scFv phage display libraries were screened against
the recombinant HA2 protein (rHA2) for three rounds.
Only the third-round elution sample of the Tomlinson J
library showed high binding afﬁnity to rHA2, from which
three clones (3JA18, 3JA62, and 3JA78) were chosen for
preparative-scale production as soluble antibody by E. coli.
The clone 3JA18 was selected for further tests due to its
broad afﬁnity for inﬂuenza H1N1, H3N2 and H5N1.
Simulations of the scFv 3JA18-HA trimer complex
revealed that the complementarity-determining region of
the variable heavy chain (V
-CDR2) bound the stem
region of HA. Neutralization assays using a peptide derived
-CDR2 also supported the simulation model. Both
the selected antibody and its derived peptide were shown to
suppress infection with H5N1 and H1N1 viruses, but not
H3N2 viruses. The results also suggested that the scFvs
selected from rHA2 could have neutralizing activity by
interfering with the function of the HA stem region during
virus entry into target cells.
Inﬂuenza A viruses (IAVs) are negative single-stranded
RNA viruses that cause signiﬁcant morbidity and mortality
in the human population because they are occasionally
transmitted from a natural reservoir to humans . IAVs
are classiﬁed according to the antigenic properties of the
envelope surface proteins hemagglutinin (HA) and neu-
raminidase (NA) . In recent years, various IAV strains of
H1N1, H3N2, H5N1 and H7N9 have taken an enormous
economic toll and raised public-health concerns [3, 4].
Because the virus undergoes continuous antigenic drift and
shift, development of an effective strategy for prophylaxis
or therapeutics against IAV infections has been slow [5, 6].
HA is the major surface glycoprotein of the virion
envelope, plays a crucial role during virus entry into the
cell to initiate infection and is thus a determinant of host
range . In the ﬁrst step of infection, the HA1 subunit of
HA binds to the sialic acid moiety of a glycan on the cell
membrane to induce endocytosis . In the low-pH envi-
ronment of the late endosome, the HA2 subunit undergoes
a conformational change , enabling the N-terminal
fusion peptide of HA2 to project toward the target to
induce fusion between the viral envelope and the endoso-
mal membrane . Antibodies targeting the HA globular
head have been developed, such as CH65, 5J8, CR8033
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-015-2625-6) contains supplementary
material, which is available to authorized users.
& Ding-Kwo Chang
Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Agricultural Biotech Research Center, Academia Sinica,
Institute of Biotechnology, National Tsing Hua University,
Institute of Epidemiology and Preventive Medicine, National
Taiwan University, Taipei, Taiwan
Arch Virol (2016) 161:19–31