Abstract
Influenza viruses can cause respiratory illnesses and are very contagious, leading to either
pandemics or seasonal epidemics and resulting in significant mortality and morbidity rates.
The main symptoms of the influenza virus infection are high fever, cough, sore throat, and
rhinitis. In most cases, the illness resolves after two weeks. However, in a person with a
weak immune system, it might be fatal.
Based on the differences in the matrix protein processing, influenza viruses are classified
into four types: A, B, C, and D. Influenza A has a high rate of evolution and causes
pandemic outbreaks, which have resulted from viral jumps from the aquatic birds or other
animal reservoirs to humans. Influenza B virus has a lower rate of evolution then influenza
A, exclusively infects humans, and causes annual epidemics. Influenza C causes only mild
infection in humans, and Influenza D is not known to infect humans.
Influenza A is further divided into 18 Hemagglutinin (HA) and 9 Neuraminidase (NA)
subtypes (Tong et al., 2013). Based on phylogenetic analysis of HA, the Influenza A virus
is clustered into two groups: Group 1 and Group 2. Based on the difference in
hemagglutinin (HA) surface glycoproteins, Yamagata-like (B/Yamagata/16/88) and
Victoria-like (B/Victoria/02/87) are the two lineages of the influenza B virus, co-circulating
in the human population since the 1980’s, although B/Yamagata appears to have recently
become extinct. H1N1 (Influenza A: Group 1), H3N2(Influenza A: Group 2), and Influenza
B (Victoria Lineage) are the currently circulating Influenza virus in humans, while a few
incidents of H5N1, H7N2, and H9 infections have also been reported in the past.
Previously, in our lab, we designed a headless stem domain immunogens containing the
entire stem pf HA from the H3 (H3HA6 (Bommakanti et al, 2010)) and H1 (H1HA6
(Bommakanti et al, 2012) forming inclusion bodies and were aggregation-prone. Later, a
smaller soluble stem domain immunogen containing a conserved region of HA2 (H1HA10)
was designed (Mallajosyula et al, 2014). This conferred complete homologous protection
with partial heterologous protection. The above-designed immunogens did not include all
epitope residues of conformation-specific broadly neutralizing antibodies CR9114 and
CR6261. To address the shortcomings of our previous constructs, we re-analyzed the HA
interaction network and engineered novel immunogens. Here, we report the design of
bacterially expressed stem domain immunogens containing all the epitope residues of
bnAbs CR9114 and CR6261 and the entire HA2 chain of the Influenza virus.
The immunogens were subjected to random mutagenesis library display. Yeast surface
display was used in which individual designs (or immunogen libraries) were expressed on
the yeast surface and were screened for surface expression and binding to conformationspecific
bnAbs. A few clones with improved expression and binding were isolated. These
mutants differ from wild-type and have mutations outside of the antibody binding epitopes
of both bnAbs CR9114 and CR6261, but showed improved binding to both bnAbs.
