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Isolation of an in vitro affinity-matured, thermostable "Headless" HA stem fragment that binds broadly neutralizing antibodies with high affinity Tariq Ahmad Najar, Uddipan Kar, Jessica A Flynn, and Raghavan Varadarajan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00267 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Biochemistry

Defining the strategy used for the design and construction of mutagenesis library, display of library on the yeast cell surface followed by the sort and enrichment process to isolate well folded, native-like HA-stem immunogen using FACS. 327x222mm (72 x 72 DPI)

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Title:

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Isolation of an in vitro affinity-matured, thermostable “Headless” HA stem fragment that

3

binds broadly neutralizing antibodies with high affinity

Authors: Tariq Ahmad Najara, Uddipan Kara, Jessica A. Flynnb,1, Raghavan Varadarajana,1

Affiliations: a

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.

b

MRL, West Point, PA 19486 USA.

1

To whom correspondence should be addressed

Jessica Flynn, email: [email protected] Raghavan Varadarajan, email: [email protected]

Key words: Vaccines; Protein design and stabilization; yeast surface display; Humoral immunity; Heterologous protection.

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Biochemistry

Abstract: 4

The surface glycoprotein hemagglutinin (HA) of influenza virus is the primary target for design of

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an effective universal influenza vaccine as it is capable of eliciting broadly cross-reactive

6

antibodies against different HA subtypes. Several monoclonal antibodies targeting the stem region

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of HA that are able to neutralize various subtypes of influenza virus have been isolated in the

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recent past. Designing a stable, HA stem immunogen that attains a native-like conformation and

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can elicit such antibodies has been a challenge. We describe the affinity maturation of a previously

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designed stem immunogen (H1HA6) by random mutagenesis, followed by selection using yeast

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surface display. The affinity-matured mutant protein (H1HA6P2), upon bacterial expression,

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attained a stable, native-like, trimeric conformation without any heterologous trimerization motif

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and showed a significant improvement in thermal stability and binding to several stem-specific,

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conformation-sensitive, broadly neutralizing antibodies (bnAbs) relative to H1HA6. These results

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point to an effective strategy for design of stabilized HA stem immunogens that can be tested for

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their protective ability.


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Introduction

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The influenza virus is responsible for frequent epidemics and sometimes pandemics resulting

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in significant morbidity and mortality worldwide. Vaccination is the primary method of controlling

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the infection 1. Current seasonal vaccines, both inactivated as well as live attenuated influenza

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virus based vaccines, typically generate a strain-specific immune response in vaccinees and have

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to be reformulated every year to match the circulating virus strains

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considerable interest in developing a universal influenza vaccine that can focus the host immune

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response toward conserved regions of the viral surface glycoproteins and provide broader

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protection without the need for frequent vaccination 4.

2, 3

. Therefore, there is

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The envelope of influenza virus has two major glycoprotein., Hemagglutinin (HA), is

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responsible for viral entry into the host cell by binding to host sialic acid receptors and

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neuraminidase (NA) is responsible for facilitating the exit of newly synthesized virions from the

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infected cell 5. The HA glycoprotein, which is highly immunogenic, is a trimer of HA1 and HA2

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dimers that are produced by cleavage of the precursor HA0 by host proteases. The membrane distal

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globular head domain of the protein contains the receptor binding site and is comprised exclusively

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of the HA1 subunit. The membrane proximal stem region harboring the fusion peptide is

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predominantly composed of the HA2 subunit 6, 7.

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Influenza HA sequences from various strains and subtypes reveal that the HA stem domain is

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more conserved than the globular head domain 8, 9. The strain specific immune response induced

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by seasonal vaccines specifically targets defined antigenic regions surrounding the receptor

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binding site on the HA head domain. Antibodies generated against these immunodominant sites

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are very potent but mostly strain specific, and neutralize virus by blocking its access to the host 3 ACS Paragon Plus Environment

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Biochemistry

10

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receptor

. These immunodominant sites are sufficiently flexible to accommodate mutations in

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response to host immune pressure, resulting in escape variants and limiting the breadth of

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neutralizing antibodies against divergent strains

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the sub-dominant stem of HA block viral infection by locking HA in its prefusion conformation,

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inhibiting membrane fusion. Extensive efforts in the recent past from several groups have resulted

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in the isolation of anti-stem antibodies 13-21. These anti-stem antibodies specifically target highly

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conserved regions on the HA stem and thus show cross-reactivity between HAs of distinct

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subtypes

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domain of HA and is capable of eliciting or boosting antibodies against the conserved stem

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epitopes in order to confer protection against multiple strains of the virus is highly desirable 24.

22, 23

11, 12

. Less abundant antibodies directed against

. Therefore, an immunogen that focuses the immune response toward the stem

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Several groups, including our own, have designed constructs consisting of either the whole

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stem domain or portions of the stem region in an effort to elicit or boost bnAb responses in animal

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models 8, 25-32. The major challenge in this approach is that of designing a stem construct which is

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capable not only of folding in the proper prefusion, native-like conformation but also trimerizing

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as an independent unit. We had earlier designed stem fragment immunogens from both group 1

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(strain A/Puerto Rico/8/1934 H1N1 hereafter referred to as PR8)

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Kong/1/1968 hereafter referred to as HK68 8 virus strains and evaluated their protective efficacy

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in vivo. These stem immunogens conferred robust subtype specific protection but failed to show

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heterosubtypic protection in vivo against lethal virus challenge. One of the immunogens, H1HA6,

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a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. In an

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attempt to further improve the biophysical properties and immunogenicity of this stem-derived

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immunogen, we constructed a random mutagenesis library of H1HA6. The library was displayed

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on the yeast cell surface to isolate mutants that are highly expressed and show improved binding

25

and group 2 (A/Hong


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to the bnAb CR6261 compared to the parent H1HA6 protein. We expect that this should result in

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the isolation of better folded and more stable variants for the protein. After five rounds of sorting

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and enrichment, we isolated a few clones of which one, H1HA6P2, dominated the enriched

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population. The other isolated mutants differed in having only one of the two mutations present in

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H1HA6P2, and in addition contained other diverse mutations; hence we did not characterize them

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further. The mutant H1HA6P2 differs from the parent H1HA6 by two mutations, namely K314E

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and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody

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footprint. This mutant showed significant improvement in binding to the conformation sensitive

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bnAb CR6261 and retained this binding after heat stress. Improvements in binding to other bnAbs,

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like F10-ScFv and FI6v3-ScFv, were also observed. Biophysical and biochemical studies showed

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that H1HA6P2 is well-folded, independently trimerizes in the absence of a trimerization motif,

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and attains a native, neutral pH- like conformation that appropriately presents the natural epitopes

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of multiple bnAbs.

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Materials and Methods

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Strains, antibodies and reagents

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S. cerevisiae EBY100 strain (yeast surface display strain) was provided by Prof. K. Dane

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Wittrup (MIT, USA). Yeast surface display vector pPNLS was provided by Prof. Dennis R. Burton

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(TSRI, USA). Monoclonal antibody (MAb) IgG-CR6261 was provided by Merck (USA). F10-

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ScFv and FI6v3-ScFv having a 3×FLAG tag at the C-terminus for detection in FACS experiments

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was designed based on the published sequence

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Alexa-Fluor 633, anti-mouse Alexa-Fluor 633 and anti c-myc antibodies were purchased from

13

. Anti-chicken Alexa-Fluor 488, anti-human


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Biochemistry

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Invitrogen (Life Technologies). Mouse anti-FLAG antibody was purchased from Sigma (Sigma

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Aldrich).

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Cloning of H1HA6 into yeast surface display vector (pPNLS)

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The yeast codon optimized gene for H1HA6 (PR8) was synthesized at Genscript (Piscataway,

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NJ, USA) 33. The gene product was PCR amplified with primers having Sfi1 overhang sequence

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and cloned into SfiI digested linear pPNLS vector by ligation using T4 DNA ligase. The final yeast

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display construct includes, the N-terminal Aga2p gene and a C-terminal c-myc epitope tag for

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displayed protein detection and quantification.

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Display of H1HA6 on the yeast cell surface

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The yeast surface display vector pPNLS contains an AGA2p fusion at the N-terminus of the

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surface displayed protein along with two epitope tags, N-terminal HA (YPYDVPDYA) and C-

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terminal c-myc (EQKLISEEDL) for detection (Figure 2)

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pPNLS as an N-terminal Aga2p fusion protein under the control of an inducible GAL promoter,

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was displayed on the cell surface of S. cerevisiae strain EBY100 using a standard protocol

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Briefly, EBY100 cells transformed with plasmids were plated on SDCAA (selection media) agar

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plates and single colonies were picked up and grown in glucose containing liquid SDCAA media

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(pH 4.0) till mid-log phase at 30°C, followed by induction in galactose containing liquid SGCAA

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(pH 4.0) media at 20°C for 24 hours.

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Flow Cytometry analysis

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. The H1HA6 construct cloned into

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.

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The surface expression of displayed H1HA6 protein was monitored by incubating the cells

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(~106cells) expressing the protein with chicken anti-c-myc antibody (1:250 dilution) followed by

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anti-chicken Alexa-Fluor 488 (1:300 dilution) after washing with ice cold labeling buffer (0.25% 6 ACS Paragon Plus Environment

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BSA in PBS, pH 7.4). Conformational integrity of displayed protein was detected by monitoring

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the binding to broadly neutralizing antibody CR6261. Briefly, ~106 cells were stained with human

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CR6261-IgG (1 µM) as a primary antibody followed by goat anti-human Alexa-Fluor 633 (1:700

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dilution) as the secondary antibody after washing with labeling buffer. All the antibody labeling

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was done in the labeling buffer for 30 minutes at 4°C. After each step, cells were washed with

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labeling buffer followed by centrifugation at 10,000 rpm for 2 minutes at 4 oC. Cells were

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resuspended in 500µL of labeling buffer (0.25% BSA in PBS, pH 7.4) and analyzed on a BD-

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Canto II cytometer (BD Biosciences NJ, USA).

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Generation of Random mutagenesis library

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The H1HA6 gene in pPNLS was subjected to random mutagenesis by error prone PCR (ep-

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PCR) using either modified nucleotide analogues 8-oxo-dGTP and dPTP as described in Table S1

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35

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ensure a low mutagenesis rate, we amplified 20pg of target DNA, using pPNLS-H1HA6 as

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template, for 15 cycles with flanking primers (vector specific primers). The PCR products were

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subjected to agarose gel electrophoresis (1%) and purified using a gel extraction kit (Thermo

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Scientific). These were further re-amplified in the presence of regular dNTP’s and absence of

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nucleotide analogues for 25 cycles. 5µg of re-amplified PCR product, was combined with 1µg SfiI

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digested, gel band purified pPNLS gapped vector and transformed into EBY100 cells by

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electroporation, where the full plasmid was reassembled by homologous recombination as

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described 34. Briefly, ~109 yeast cells in mid log-growth phase were resuspended in filter sterilized

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(using 0.2 µm filter) E-buffer (100 mM Tris-HCl, 270 mM Sucrose and 1 mM MgCl2, pH 7.5) and

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then mixed with 1µg of gapped vector and 5µg of mutated H1HA6 PCR product. The cells were

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then distributed into five 0.2 cm cuvette (100µL each) and pulsed at 2.5 kV using Biorad Gene-

or MnCl2 as described in Table S2

36

. The ep-PCR conditions are described in Table S3. To


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Biochemistry

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pulser. Transformation mixtures were then passaged twice at 30 oC for 20 hours in SDCAA to

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minimize the growth of untransformed cells. Library size was determined by plating serial

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dilutions of the transformation mixture on SDCAA plates. The final diversity of the library was

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estimated to be 5× 106.

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Sequencing of the library

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To get an approximate idea of the average number of mutations per residue in the gene,

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plasmid was extracted from the pooled transformation mix after two rounds of passage, by the

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Hoffman method 37, transformed into E.coli DH5α cells and plated on 150 mm diameter LB agar

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plates. Single clones (30 clones from both libraries) were picked up and subjected to Sanger

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sequencing using vector specific primers. The mutagenesis rate was calculated to be 0.25-5% at

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the amino acid level.

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Library expression and equilibrium sort

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After transformation into EBY100, the library was grown at 30 oC overnight in SDCAA

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medium. Subsequently the library was passaged twice in SDCAA at an OD600 of 1 before induction

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to reduce the number of untransformed cells. To induce the culture, ~108 cells were transferred to

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SGCAA liquid media (~200mL) in such a way that the final OD600 of the culture is ~0.5. Cells

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were allowed to grow at 20 oC for 24 hours under shaking conditions. After induction, ~108 cells

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displaying the mutant library were aliquoted into microcentrifuge tubes and washed thrice with

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labeling buffer before incubating with primary antibodies (CR6261 and anti-c-myc) for 1 hour at

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4 oC with mild shaking to facilitate the binding. Finally, cells were stained with secondary

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antibodies, anti-human Alexa-Fluor 633 (1:700 dilution) and anti-chicken Alexa-Fluor 488 (1:300


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dilution). Secondary antibody binding was allowed to take place for 30 minutes at 4 oC, in the

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dark.

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For sorting, a total of ~108 cells were resuspended in 1 mL of ice cold labeling buffer and

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sorted on a BD-FACS Aria-III cell sorter with a sort window as shown in Figure 2 (a) with an

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event rate of ~3,000 cells/second. During round 1 of sort, 5 ×107 cells were analyzed and the

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sort window was set to collect ≤0.2% of the total population. The collected cells were re-grown in

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SDCAA media and then switched to SGCAA medium as described earlier for repeating the sort.

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A total of five rounds of sorting and enrichment were performed. In each round of sort, a total of

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5 × 107 cells were analyzed. The first two rounds of sort were performed with 1 µM concentration

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of CR6261-IgG in the enrichment mode to recover all positive clones, and the remaining three

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rounds of sort were performed with 200 nM concentration of CR6261 IgG in purity mode to avoid

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coincident negative cells and achieve larger enrichment factors. The products of the final round of

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sort were plated onto a SDCAA agar plate to isolate individual clones.

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Analysis of clones isolated from sorting

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After the final round of sort, 20 randomly picked clones from the enriched population were

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individually analyzed for binding to CR6261 and for surface expression. All 20 clones were

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individually grown in SDCAA liquid medium followed by induction in SGCAA medium. Cells

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(~106) from each clone were separately first stained with 250 nM concentration of CR6261 and

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anti c-myc antibodies followed by fluorophore conjugated secondary antibody as described earlier

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and analyzed on a BDCanto-II flow cytometer.


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Biochemistry

Sequence analysis of isolated clones A total of 10 clones with improved binding and surface expression were selected for sequence 37

and transformed into E.coli DH5α cells.

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analysis. Plasmids were isolated from the yeast cells

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E.coli cultures were grown overnight in LB media containing 100 ug/mL of ampicillin and plasmid

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DNA was extracted. The H1HA6 ORF of all 10 plasmids was sequenced by Sanger sequencing at

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Macrogen, Korea.

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Conservation analysis of K314E and M317T mutations found in H1HA6P2

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Protein coding sequences of HA from H1N1 subtype (5996 in total) and all influenza A

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viruses (11489 in total) from human isolates were downloaded from the influenza data base

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(https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database) and aligned

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separately using Clustal Omega (www.ebi.ac.uk). After alignment, the frequency of every residue

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at each position was calculated using a Perl script software, developed in house.

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Generation of revertant mutants on H1HA6P2 background

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To revert back individual mutations in the H1HA6P2 mutant, thirty nucleotide-long

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overlapping primers were designed to generate revertants individually. Vector specific forward

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primer and reverse mutagenic primer (containing the wild-type codon) were used to amplify one

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overlapping fragment. Similarly, forward mutagenic primer (containing the wild-type codon) and

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the vector specific reverse primer were used to amplify the other overlapping fragment. All the

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PCR reactions were carried out with Phusion DNA polymerase (Finnzymes). pPNLS vector

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containing ~1 kb stuffer sequence was digested with SfiI (New England Biolabs) to remove the

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stuffer insert and gel purified. The digested vector and the two overlapping fragments, for each

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mutant, were transformed in S. cerevisiae EBY100 38. Single colonies were picked up, grown to 10 ACS Paragon Plus Environment

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saturation in 3 mL of liquid synthetic SDCAA medium and plasmid was extracted by a

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Phenol:Chloroform:Isoamylalcohol mixture (SRL Laboratories) as described elsewhere

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crude plasmid was transformed into E. coli DH5α cells to obtain enough plasmid for sequencing.

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The mutants were generated individually by this method and were sequence confirmed by Sanger

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sequencing (Macrogen Inc.).

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Cloning, expression and purification of the H1HA6 and H1HA6P2 proteins

37

. The

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The genes of the H1HA6, and the affinity matured protein H1HA6P2, that were initially

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cloned in the yeast display vector pPNLS, were PCR amplified with gene specific primers having

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NdeI and HindIII restriction sites to facilitate cloning into pET-22b(+) vector for expression in

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E.coli. After amplification, PCR products and the vector (pET-22b+) were digested with NdeI and

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HindIII for 3 hours at 37 oC. After digestion, the genes were individually ligated into pET-22b(+)

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vector using T4 DNA ligase and confirmed by DNA sequencing of individual clones (Macrogen,

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Inc.). Plasmids were transformed into BL21(DE3) cells for protein expression. The proteins were

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over-expressed in E.coli BL21(DE3) by growing the cells in 2 L terrific broth till an A600 of 0.8 at

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37 ºC and induced with 1 mM IPTG. After 18 hours of induction at 20 oC, cells were harvested

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and lysed by sonication in 50 mM Tris-HCl (pH 8.0) containing 1 mM EDTA. The cell lysate was

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centrifuged at 18,500 x g, 4 ºC for 30 min. The pellet was washed with 0.005% Triton X-100 in

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50 mM Tris-HCl (pH 8.0) followed by centrifugation at 18,500 x g, 4 ºC for 45 min. The pellet

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was solubilized using 8.0 M Gdm-HCl in 50 mM Tris-HCl (pH 8.0). Solubilized and clarified

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inclusion bodies were passed over a High-Performance Ni-NTA column (GE Healthcare Life

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Sciences) at room temperature and refolded on a column by serially diluting out Gdm-HCl.

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Proteins were finally eluted in 1M Gdm-HCl, 500 mM imidazole, 50 mM Tris-HCl (pH 8.0). The

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protein H1HA6 was dialyzed against deionized water containing 1 mM EDTA while H1HA6P2 11 ACS Paragon Plus Environment

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Biochemistry

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was dialyzed against 1× PBS (pH 7.4) containing 1 mM EDTA. All proteins were flash frozen in

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liquid nitrogen and stored at -80 oC.

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CD spectroscopy

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The circular dichroism (CD) spectra of both proteins were recorded on a Jasco J-715C

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Spectropolarimeter at 25 oC. The protein concentration used for acquiring the spectra was ~5 -10

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µM. The spectra were recorded using a 0.1 cm path length cuvette by scanning from 260 nm to

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190 nm with a spectral bandwidth of 2 nm, response time of 4 seconds and scan rate of 50 nm/min.

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Each spectrum is an average of 3 scans and the buffer control has been subtracted. Mean residue

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ellipticities (MRE) were calculated and plotted as a function of wavelength as described 39.

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Thermal unfolding by CD spectroscopy

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The thermal unfolding experiments of all proteins were monitored on a Jasco J-715C

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Spectropolarimeter at 222 nm in 1× PBS (pH 7.4) buffer. The data were collected between 20 oC

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and 90 oC with 1 oC/min gradient and a data pitch of 0.2 oC. Bandwidth was fixed at 2 nm and

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response time at 4 seconds. All spectra were collected using a 1 mm path-length quartz cuvette.

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The fraction unfolded (Fu) was plotted as a function of temperature.

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Fluorescence spectroscopy

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All fluorescence spectra were recorded on a Jasco FP-6300 Spectrofluorimeter at 25 oC. The

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intrinsic fluorescence emission spectrum of all the proteins was monitored at a concentration of 2

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μM using a 1-cm path length cuvette under native (1× PBS pH 7.4) or denaturing conditions (6M

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Gdm-HCl, 1× PBS pH 7.4). Proteins were excited at 280 nm and the emission spectra were

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recorded between 300 and 400 nm at 25 ºC using excitation and emission bandwidths of 3 nm and


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5 nm respectively. Each spectrum is an average of 3 scans and has been corrected for buffer

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fluorescence acquired under the same conditions.

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Size Exclusion Chromatography

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The oligomeric status of purified proteins was analyzed by size exclusion chromatography

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under native conditions at 4 oC on a Superdex-200 analytical gel filtration column (GE Healthcare).

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Briefly, the column was first equilibrated with ice-cold 1× PBS buffer (pH 7.4) at a flow rate of

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0.5 ml/min and then calibrated with broad range molecular weight markers (GE Healthcare). After

242

calibration, 100 μg of the protein at a conc. of 1 mg/ml in 1× PBS was loaded onto the column.

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Binding of recombinant HA to broadly neutralizing antibody CR6261 monitored by BLI

244

Affinities for CR6261 antibody were determined by Bi- Layer Interferometry (BLI) using an

245

Octet RED96 instrument (ForteBio, Inc.) CR6261 IgG was immobilized on AR2G biosensor tips

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by amine coupling at a concentration of 20 µg/mL in sodium acetate buffer (pH 4.5). Briefly,

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AR2G biosensor tips were activated as per the manufacturer’s instructions for 700 seconds,

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followed by incubation in an antibody solution (20 µg/mL of CR6261 in 10 mM sodium acetate

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buffer, pH 4.5) for 800 seconds. As a control, reference biosensor tips were activated and

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subsequently quenched without incubation in antibody solution. After ligand immobilization,

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biosensor tips were blocked with 1 M ethanolamine for 500 seconds and stored in PBST (1× PBS,

252

pH 7.4 plus 0.05% Tween-20) before further use. All steps were performed at 30 oC with an

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agitation speed of 1000 rpm.

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To obtain kinetic parameters of interaction, ligand (CR6261) immobilized biosensors were

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titrated with 5-6 varying concentrations of rHA (A/Puerto Rico/8/34) (Protein Science Corp.),. The

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association and dissociation phases were monitored for 300 and 1000 seconds respectively, for 13 ACS Paragon Plus Environment

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Biochemistry

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every analyte concentration in the interaction buffer (1× PBS, pH 7.4 plus 0.05% Tween 20 and

258

0.1% BSA). Six biosensors were used to monitor the kinetic titration series, whereas one biosensor

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recorded the buffer reference signal and one biosensor, which was activated and subsequently

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blocked (without any ligand), was used to monitor the non-specific binding of analyte to the sensor

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tip. All steps were performed at 30 oC with an agitation speed of 1000 rpm. The sensor surface

262

was regenerated after each binding experiment by dipping the biosensors into 2 M MgCl2 for 30

263

seconds. The sensograms were double referenced against the buffer reference signal and the non-

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specific signal to eliminate the effects of both non-specific binding and baseline drift using the

265

Data Analysis software 7.1 (ForteBio). After double referencing, the data were exported and fit to

266

a simple 1:1 Langmuir interaction model using the BiaEvaluation 3.1 software.

267

We also examined the thermal tolerance of H1HA6 and H1HA6P2 proteins by their ability to

268

bind CR6261 after heat stress. The protein samples were incubated at 80°C for 15 minutes in a

269

PCR cycler (BioRad) with heated lid to prevent evaporation. The samples were cooled to 25°C

270

and binding affinity to CR6261 was determined by BLI experiments as described above.

271

Binding of HA stem immunogens to broadly neutralizing antibody CR6261 monitored by

272

SPR

273

The binding affinity of H1HA6 and H1HA6P2 to the immobilized CR6261 IgG antibody by

274

SPR was performed on a Biacore2000 optical biosensor (Biacore, Uppsala, Sweden) at 25°C, at a

275

flow rate of 10μl/min. Briefly, 500-750 RU’s of the ligand CR6261 was immobilized on a CM5

276

sensor chip (GE Health Care, Uppsala, Sweden) by standard amine coupling. Ovalbumin

277

immobilized sensor channel served as a negative-control for each binding interaction. Multiple

278

concentrations of the analyte were passed over each channel in a running buffer of 1× PBS (pH 14 ACS Paragon Plus Environment

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Page 16 of 40

279

7.4) with 0.05% Tween-20 surfactant. The association and dissociation events, were monitored for

280

300 and 200 seconds respectively. After every binding event the sensor chip was regenerated by

281

repeated washes with 4M MgCl2. Each binding curve was analyzed after correcting for non-

282

specific binding by subtraction of signal obtained from the negative-control flow channel. The

283

trimeric concentration of all the proteins (analyte) was used for obtaining the kinetic parameters

284

25

285

interaction model using the BiaEvaluation 3.1 software.

286

Binding to bnAbs F10-ScFv and FI6v3-ScFv monitored by SPR

. The kinetic parameters were obtained by globally fitting the data to a simple 1:1 Langmuir

287

The binding affinity of H1HA6, H1HA6CC, H1HA6P2 and full-length rHA H1N1 A/Puerto

288

Rico/8/34 (Protein Science Corp.) to the single-chain variable fragment derivatives of stem-

289

directed bnAb F10-scFv and FI6v3-scFv was determined by SPR performed on a Biacore2000

290

optical biosensor (Biacore, Uppsala, Sweden) at 25°C, at a flow rate of 30μl/min. Plasmids

291

encoding F10-scFv and FI6v3-scFv were synthesized (GenScript, USA) based on the published

292

sequence

293

scFv or FI6v3-scFv was immobilized on a CM5 sensor chip (GE Health Care, Uppsala, Sweden)

294

by standard amine coupling. Ovalbumin immobilized sensor channel served as a negative-control

295

for each binding interaction. Multiple concentrations of the analyte were passed over each channel

296

in a running buffer of 1× PBS (pH 7.4) with 0.05% Tween-20surfactant. The association and

297

dissociation events were monitored for 100 and 200 seconds respectively. After every binding

298

event the sensor chip was regenerated by repeated washes with 4M MgCl2. Each binding curve

299

was analyzed after correcting for non-specific binding by subtraction of signal obtained from the

300

negative-control flow channel. Trimeric concentration of all the proteins (analyte) was used for

13, 17

and expressed in E.coli. For SPR experiments, 500-750 RU’s of the ligand F10-


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Biochemistry

301

obtaining the kinetic parameters 25. The kinetic parameters were obtained by globally fitting the

302

data to a simple 1:1 Langmuir interaction model using the BiaEvaluation 3.1 software.

303

Results

304

Construction of the H1HA6 library and yeast surface display

305

We have previously reported the design of a headless stem fragment immunogen (H1HA6)

306

from the HA of an H1 subtype (A/Puerto Rico/8/1934) 25. This immunogen is a circularly permuted

307

fragment of the HA stem. The circular permutant design comprises of residues 501 to 672 of HA2,

308

a 6-amino-acid linker (GSAGSA), residues 1 to 41 of HA1, and a 3-amino-acid linker (GSA)

309

followed by residues 290 to 325 of HA1. Mutations were incorporated into the construct to

310

stabilize the neutral pH conformation

311

immunogen binds to the stem directed bnAb CR6261 with a KD in the submicromolar range but is

312

quite unstable and aggregation prone. To improve antigenic properties and stability, we randomly

313

diversified the H1HA6 expression cassette by error-prone PCR and used yeast surface display to

314

isolate mutants with improved binding to the bnAb CR6261. We used two different approaches to

315

generate sequence variation in the H1HA6 gene – incorporation of mutagenic nucleotide analogues

316

(8-oxo GTP and dPTP) which are known to cause both transitions and transversions and use of

317

MnCl2 which causes the polymerase to become more error-prone 35, 36. Sequencing of 30 randomly

318

chosen clones from each library revealed between 3 to 8 amino acid substitutions per clone, as

319

well as insertions and deletions. Pooled libraries of H1HA6 variants were cloned into the yeast

320

surface display vector pPNLS by homologous recombination 34 yielding ~ 5 × 106 transformants.

321

The pooled library was subjected to FACS sorting and enrichment to isolate clones showing

and to remove exposed hydrophobic patches25.This


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322

improved surface expression and binding to the bnAb CR6261 as shown schematically in Figure

323

1.

324 325 326 327 328 329 330 331 332 333 334 335 336 337

Figure 1: Schematic of the mutant library construction and enrichment by FACS. First, the mutant DNA fragments were generated with flanking primers (red arrows) by error-prone PCR using either nucleotide analogues (8-oxo-dGTP and dPTP) or MnCl2 (1). The linear vector backbone was generated by digesting pPNLS-gp120 vector with SfiI. After gel band purification, PCR products and linear vector backbone were mixed and transformed into yeast to obtain a circular plasmid mutant library by homologous recombination (2). The library was passaged three times before expression of mutant clones (4) and a small amount was plated to obtain single clones for DNA sequencing to estimate the average number of mutations per insert (3). Cells expressing the mutant protein library were labeled with CR6261 (to detect tighter binders) and anti c-myc antibodies (to probe for better folders that result in better surface expression) and sorted by FACS (5). Better binding clones were sorted, re-grown and subjected to another round of sort (6). This process was repeated several times until no further increase in binding was observed. The symbol (X) represents the site of mutation, the ORF of the gene is colored in orange and the homologous sequences between vector backbone and amplified gene are colored in red.


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338

Biochemistry

Screening of H1HA6 mutant libraries and isolation of high affinity clones

339

H1HA6 libraries displayed on the yeast cell surface were screened by dual color labeling using

340

FACS. Binding to the c-myc tag is used to monitor surface expression while binding to the CR6261

341

MAb is used to monitor conformational integrity of the displayed molecule. The first two rounds

342

of sorting were performed with 1 µM concentration of CR6261 and the sort window was set to

343

collect ≤0.2% of cells from the total population to avoid contamination with parent H1HA6

344

expressing cells (Figures 2a and 2b). The library was then subjected to another three rounds of

345

sorting, with lower concentration of CR6261 (200 nM) and more stringent sort windows (Figures

346

2c, 2d and 2e). After five rounds of sort and enrichment, a highly enriched population with

347

improved binding to CR6261 and higher surface expression relative to H1HA6 was isolated

348

(Figure 2e).


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349 350 351 352 353 354 355

Figure 2: Screening of H1HA6 mutant library by yeast surface display. H1HA6 mutant library was sorted using 1 µM of CR6261 and 1:300 dilutions of anti c-myc antibody for round 1 and 2 (a & b). For round 3, 4 and 5, 200 nM of CR6261 and 1:300 dilution of anti c-myc antibody was used to stain the cells (c, d, & e). The positive population sorted by FACS is shown in the box (blue dots). After the initial two rounds of sort, the gate was made more stringent to collect better binding clones. (f) The dot plot of H1HA6 with 1 µM concentration of CR6261 and 1:300 dilution of anti c-myc antibody.

356

Cells from the final round of sort were plated to obtain single clones. Twenty clones were

357

chosen and further analyzed. These showed improved binding to CR6261 (~5-fold) and a ~2-fold

358

increase in expression as compared to the parent H1HA6 protein (Figure 3a and 3b). DNA

359

sequencing revealed that a single mutant (hereafter referred to as H1HA6P2) containing two amino

360

acid substitutions, K314E and M317T, dominated the enrichment process and hence only data for

361

this mutant is shown in Figure 3. All other mutants analyzed were identical to or differed only


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Biochemistry

362

slightly from this dominant mutant. Importantly, both the K314E and M317T mutations are outside

363

the CR6261 binding site as shown in Figure 3d.

364 365 366 367 368 369 370

Figure 3: Improvement in surface expression and binding of affinity matured mutant H1HA6P2. FACS histograms showing the improvement in surface expression monitored by antic-myc antibody, and binding to broadly neutralizing antibody CR6261 and FI6v3-ScFv compared to the H1HA6 surface expression, binding to CR6261 and FI6v3 Sc-Fv (a, b and c respectively). ‘US’ indicates unstained control (d) On the left, the regions of HA1 (dark green) and HA2 (light blue) included in our construct H1HA6, are mapped onto the crystal structure of the ectodomain of H1HA (PR8) (PDB ID: 1RU7). The CR6261 epitope is


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371 372 373

highlighted in red. The structure on the right is a model of H1HA6 with positions of mutated residues (K314 and M317) encircled and the side chains shown in ball and stick representation. Both residues are distant from the CR6261 binding site (red). The residues included in H1HA6 are shown above the model.

374

Improvement in FI6v3-ScFv binding

375

We also probed the binding of yeast surface displayed H1HA6P2 to FI6v3-ScFv, a

376

conformation specific broadly neutralizing antibody capable of neutralizing various subtypes from

377

both group 1 and group 2 influenza viruses. The affinity matured mutant clone H1HA6P2 showed

378

significant improvement in binding to FI6v3-Sc-Fv as compared to the parent H1HA6 protein

379

(Figure 3c), hence suggesting that the H1HA6P2 protein folds in a more native-like conformation

380

than H1HA6 and/or is less aggregation prone.

381

Expression, purification & biophysical characterization of purified H1HA6P2

382

To probe the effect of mutations on folding and stability, H1HA6 and the affinity-matured

383

mutant H1HA6P2 were over-expressed in E.coli and purified from inclusion bodies. The proteins

384

were refolded on-column by serially diluting out Gdm-HCl. Since H1HA6 protein aggregates

385

rapidly in most aqueous buffers, it was dialyzed against deionized water 8, 25. In contrast, H1HA6P2

386

dialyzed against 1× PBS (pH 7.4) buffer did not show any visible aggregation and remained

387

aggregate free for months upon storage at -20 oC. While the total amount of expressed protein was

388

similar, the yield of H1HA6P2 was ~2 fold lower (~10-15mg/mL) than the H1HA6 protein. This

389

is because only ~40-50% of the H1HA6P2 protein went into inclusion bodies and the remaining

390

protein was found in the soluble fraction, in contrast to H1HA6 which was entirely targeted to

391

inclusion bodies. The H1HA6P2 protein purified from inclusion bodies was of higher purity than

392

that from the soluble fraction, so only the former amount was used for yield estimates 40. Far UV

393

CD spectra of affinity matured H1HA6P2 protein showed a higher helix content compared to

394

H1HA6 (Figure 4a), though both proteins, were predominantly α-helical as expected. The intrinsic 21 ACS Paragon Plus Environment

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Biochemistry

395

tryptophan fluorescence of affinity matured H1HA6P2 mutant protein showed a slight blue shift

396

compared to the H1HA6 protein, further indicating that H1HA6P2 is better folded than H1HA6.

397

The intrinsic fluorescence of both proteins was also measured under native and denaturing

398

conditions. Both proteins showed a significant red shift in the emission maximum, from about 345

399

nm to 357 nm, upon denaturation with Gdm-HCl confirming loss of tertiary structure after

400

denaturant addition (Figure 4b).

401 402 403 404 405 406 407

Figure 4: Biophysical characterization of affinity matured mutant H1HA6P2. (a) Far-UV CD spectra of H1HA6 (solid line), H1HA6P2 (dash line) in 1× PBS buffer (pH 7.4) at 25 oC. (b) Fluorescence emission spectra of H1HA6, black and grey solid lines under native “N” (1× PBS, pH 7.4) and denaturing conditions “D” (6 M Gdm-HCl 1× PBS, pH 7.4) respectively and H1HA6P2 (black and grey dashed lines under native “N” and denaturing “D” conditions respectively) with excitation at 280 nm. (c) Thermal stability of H1HA6, H1HA6P2 and single mutants K314E and M317T monitored by CD spectroscopy signal at 222 nm. The


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Page 24 of 40

408 409 410 411 412 413 414 415 416

melting profile shows that the H1HA6P2 mutant (Tm= ~74 oC) has higher thermostability with a ~24 oC increase in the apparent Tm (the Tm at which Fu = 0.5) than the H1HA6 protein (Tm= ~49 oC). M317T (Tm= ~68 oC) shows higher thermal stability than K314E (Tm= ~60 oC). All proteins (~10 µM each) were heated from 20 oC to 100 oC with a gradient of 1 oC/min and change in ellipticity at 222 nm was measured as a function of temperature. (d) Gel filtration profiles of H1HA6 and H1HA6P2 on a Superdex-200 column in 1× PBS buffer (pH 7.4) at 4 oC. The insert shows the column calibration curve using broad range molecular weight markers (black dots). H1HA6 shows a large fraction that elutes as high molecular weight (HMW) aggregates. In contrast, H1HA6P2 does not show any aggregation and elutes predominantly as trimers (shown by encircled black triangle in the insert).

417

To further investigate the stabilizing effect of the K314E and M317T mutations on protein

418

stability, the melting temperatures (Tm) of H1HA6 and H1HA6P2 were measured using circular

419

dichroism (CD). H1HA6 showed a very broad transition, consistent with structural heterogeneity

420

or aggregation of the protein. In contrast, H1HA6P2 showed a more cooperative transition.

421

Compared to the H1HA6 protein, the affinity matured protein H1HA6P2 showed a significant

422

increase in Tm from 51 oC to 75.5 oC, a difference (∆Tm) of 24.5 oC (Figure 4c). When mapped onto

423

the native neutral pH structure of HA (PDB ID: 1RU7), the side chain of lysine 314 is solvent-

424

exposed and adjacent to the side chain of a second, positively charged, arginine residue (R316)

425

and thus might experience electrostatic repulsion (Figure 3d). When this lysine residue is mutated

426

to glutamic acid, the electrostatic repulsion is likely relieved and possible salt bridge formation

427

between E314 and R316 may contribute to the observed increase in stability. Mutating methionine

428

317 to threonine may provide additional stability because of the potential for threonine to form

429

hydrogen bonds with adjacent main chain or side chain groups at the inter-protomer region of the

430

trimeric molecule.

431

H1HA6P2 is a homogenous trimer in solution

432

The apparent molecular weight and oligomeric state of the affinity matured protein H1HA6P2

433

was assessed by analytical size exclusion chromatography experiments and compared with that of

434

the H1HA6 protein. Both proteins were purified and stored at -20 oC prior to use. Since H1HA6 is 23 ACS Paragon Plus Environment

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Biochemistry

435

aggregation prone in PBS, the protein was stored in water and diluted into PBS just before injection

436

into the size exclusion column, whereas H1HA6P2 could be stored in PBS as it is much less

437

aggregation prone. The data indicate that H1HA6P2 elutes predominantly as a trimer with a minor

438

fraction as monomer. Importantly, few higher molecular weight aggregates were seen (Figure 4d).

439

However, the H1HA6 protein is aggregation prone and elutes largely as higher molecular weight

440

aggregate with less than 50% as trimer and monomer (Figure 4d). Since all these constructs lack

441

the transmembrane domain and also do not contain any heterologous trimerization motif; we

442

believe that the long α-helices (LAH) of the HA2 domain of HA assemble together into a parallel,

443

coiled-coil helping the protein to attain a trimeric conformation that is stabilized by the additional

444

pair of mutations present in H1HA6P2.

445

H1HA6P2 adopts the desired neutral pH conformation and binds to conformation specific

446

antibodies

447

H1HA6 and the affinity matured H1HA6P2 contain the whole stem region of HA and hence

448

potentially contains the complete antibody footprint of various stem-directed broadly neutralizing

449

monoclonal antibodies (bnAbs) such as CR6261, F10 and FI6v3. These bnAbs bind to conserved

450

conformation-dependent epitopes on the prefusion HA stem. The epitopes are not present on the

451

low pH, fusion-competent conformation or as well as on misfolded stem constructs of HA.

452

Therefore, the binding of these bnAbs to the stem-derived immunogens offers a robust validation

453

of their conformational integrity. The binding of H1HA6P2, H1HA6 and full-length recombinant

454

HA (rHA) to the bnAb CR6261 was tested by SPR and Bi-layer Interferometry (BLI) (Table 1 and

455

Figure 5).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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456 457 458 459 460 461 462 463 464 465 466

Figure 5: Binding of conformation specific MAbs (CR6261, F10-ScFv and FI6v3-ScFv) to H1HA6P2, H1HA6 and rHA proteins. (a-c) Overlays show the binding kinetics of 20 nM, 10 nM, 5 nM and 2.5 nM (with analyte concentration increasing from the bottom to the top curve) of H1HA6P2 (a), 125 nM, 100 nM, 75 nM and 50 nM of H1HA6 (b), and 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM of rHA A/PR/8/34 (c) to immobilized Mab CR6261 IgG. (d-i) Binding kinetics to immobilized F10-ScFv (d-f) and FI6v3-ScFv (g-i) to H1HA6P2, H1HA6 and rHA . Overlays showing the binding kinetics of 500 nM, 250 nM, 125 nM and 62.5 nM (with analyte concentration increasing from the bottom to the top curve) of H1HA6P2 (d and g), H1HA6 (e and h), and 70 nM, 50 nM, 20 nM and 10 nM of rHA A/PR/8/34 (f and i) to F10-ScFv and to FI6v3-ScFv. Data obtained were fitted to 1:1 Langmuir model to obtain the kinetic parameters (Tables 1-3). The data points are represented by dots and the fits by solid lines.

467

Recombinant HA (from A/Puerto Rico/8/1934) bound the CR6261 antibody with a

468

dissociation constant (KD) of ~0.3 nM (Figure 5c). H1HA6 protein bound CR6261 with lower

469

affinity (KD ~22 nM) (Table 1 and Figure 5b). The affinity-matured, mutant protein H1HA6P2 25 ACS Paragon Plus Environment

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Biochemistry

470

showed a significant increase in binding affinity to CR6261 (KD ~1.3 nM) (~20-fold increase

471

compared to H1HA6 and only ~4-fold lower affinity than the full-length rHA) largely because of

472

an improvement in “kon” rate constant (Figure 5a and Table 1).

473 474

Table 1: Kinetic parameters for binding of rHA (A/PR/8/34), affinity matured mutant (H1HA6P2), and wild-type protein (H1HA6) to immobilized CR6261 IgG antibody.

475 476

The binding of H1HA6P2, H1HA6 and rHA to the single chain variable fragment (ScFv)

477

derivatives of F10 17 and FI6v3 13 antibodies were determined by surface plasmon resonance (SPR) Analyte

kon (M-1s-1)

koff (s-1)

KD (nM)

rHA A/PR/8/34

(4.0 ± 0.5) ×105

(1.7 ± 1.1) ×10-4

0.3 ± 0.1

H1HA6P2

(4.3 ± 0.1) ×105

(5.6 ± 7.8) ×10-4

1.3 ± 0.1

HA6wt

(4.0 ± 0.2) ×104

(8.8 ± 5.6) ×10-4

23 ± 2.8

478

using a Biacore 2000 instrument. The affinity-matured protein H1HA6P2 showed increased

479

affinity to both F10-ScFv (~5 fold) (Figure 5d) and FI6v3-ScFv (~3 fold) (Figure 5g) as compared

480

to H1HA6 (Figure 5e and 5h). The KD of H1HA6P2 binding to F10-ScFv was 19 nM, which is

481

about 5-fold weaker than that of the full length rHA (3.8 nM) (Figure 5f and Table 2).

482 483 484

Table 2: Kinetic parameters for binding of rHA (A/PR/8/34), affinity matured mutant (H1HA6P2), and wild-type protein (H1HA6) to immobilized F10-ScFv antibody fragment as determined by SPR (Biacore).

485


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Page 28 of 40

Analyte

kon(M-1s-1)

koff(s-1)

KD (nM)

rHA A/PR/8/34

(8.1 ± 1.2) ×104

(3.0 ± 0.5) ×10-4

3.7 ± 0.4

H1HA6P2

(5.8 ± 0.2) ×104

(1.1 ± 0.1) ×10-3

19 ± 1.0

HA6wt

(9.8 ± 1.3) ×103

(8.9 ± 0.9) ×10-4

95 ± 16

486 487

However, the KD of H1HA6P2 binding to FI6v3 is 26 nM, which is similar to that of the full-

488

length rHA (23 nM) (Figure 5i and Table 3).

489 490 491

Table 3: Kinetic parameters for binding of rHA (A/PR/8/34), affinity matured mutant (H1HA6P2), and wild-type protein (H1HA6) to immobilized FI6v3-ScFv antibody fragment as determined by SPR (Biacore).

492 Analyte

kon(M-1s-1)

koff (s-1)

KD (nM)

rHA A/PR/8/34

(5.2 ± 0.9) ×104

(1.0 ± 0.1) ×10-3

23 ± 5.7

H1HA6P2

(5.2 ± 1.1) ×104

(1.2 ± 0.1) ×10-3

26 ± 4.2

HA6wt

(2.0 ± 0.4) ×104

(1.5 ± 0.3) ×10-3

84 ± 20

493 494

H1HA6P2 retains binding to bnAb CR6261 after heat-stress

495

The binding of both H1HA6 and H1HA6P2 protein to CR6261 antibody after heat stress was

496

also determined. The proteins were heated for 15 minutes at 80 oC and binding to CR6261 was

497

monitored by BLI at 25 oC. H1HA6P2 protein retained binding to CR6261 while very little binding

498

was observed with the H1HA6 protein after heat stress, further confirming that the affinity-

499

matured H1HA6P2 protein is more thermostable than the H1HA6 protein (Figure 6).


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Biochemistry

500 501 502 503 504 505 506 507

Figure 6: Binding isotherms of conformation specific antibody CR6261 to H1HA6P2 and H1HA6 proteins before and after heat stress (80 oC for 15 min) measured by BLI (Octet). The ligand CR6261 IgG was immobilized on AR2G biosensors by standard amine coupling and the binding was measured by dipping the antibody bound sensors into varying concentrations of the analyte before and after heat stress. The overlays show the binding kinetics of 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM (with analyte concentration increasing from the bottom to the top curve) of H1HA6P2 before heat stress (a), after heat stress (b) and H1HA6 before heat stress (c) and after heat stress (d).

508

The above results clearly indicate that mutations in H1HA6P2 have significantly improved its

509

ability to attain a trimeric, native-like pre-fusion conformation. Future immunization studies will

510

be carried out with this affinity-matured mutant protein to examine if improved stability and

511

conformation enhance the ability of the immunogen to provide enhanced protection against

512

multiple strains of influenza virus relative to that observed previously for H1HA6 25.


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Page 30 of 40

513

Residue conservation and contribution of K314E and M317T mutations to improved

514

stability and antibody binding

515

We examined the residue conservation and frequency of K314E and M317T mutations in

516

natural, human isolates of influenza A. A lysine at position 314 is dominant, occurring at

517

frequencies of 99.7% and 54.6% in H1 and all influenza A subtypes, respectively. The K314E

518

mutation is found in only one isolate (A/Qingdao/1269/2009/HA), At position 317, Leucine is the

519

most common amino acid occurring at frequencies of 80.1and 89.3% in H1 and all human

520

influenza A sequences respectively. Methionine at position 317 is present at frequencies of 19.7%

521

and 10.2% in H1 and all influenza A subtypes, respectively. The M317T mutation is not found in

522

any natural human isolate of influenza A virus. Thus, it would not have been possible to identify

523

these mutations through widely used consensus sequence-based approaches for protein

524

stabilization.

525

To determine the effect of each individual mutation on surface expression and binding to bnAbs,

526

we reverted both mutations (K314E and M317T) in H1HA6P2 individually. Surface expression of

527

each mutant on the yeast surface was as good as H1HA6P2 (Figure 7a). Both mutants, however,

528

showed weaker binding to bnAb CR6261 relative to H1HA6P2 with K314E showed relatively

529

better binding to CR6261as compared to the M317T single mutant (Figure 7b).


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Biochemistry

530 531 532 533 534

Figure 7: FACS histograms showing the surface expression and binding of reverted mutants. (a) Surface expression of HA6, K314E and M317T monitored by anti c-myc antibody and (b) binding to bnAb CR6261 of HA6, K314E and M317T. Both mutants showed better surface expression compared to H1HA6 but the K314E mutant shows relatively higher binding to CR6261 compared to the M317T mutant.

535

Further, to determine the effect of each individual mutation on thermal stability of the protein, we

536

expressed reverted single mutants in E.coli and the melting temperature (Tm) of purified protein

537

for each mutant was measured using CD spectroscopy. The data show that the M317T mutation

538

contributed more to the thermal stability (Tm: ~70 oC) of the protein compared to the K314E

539

mutation (Tm: ~62 oC) as shown in Figure 4c.

540

Discussion

541

The constant threat of an influenza pandemic has fueled efforts to develop a universal

542

influenza vaccine that can provide heterosubtypic protection or at least protection against multiple

543

strains within a subtype or group. The stem domain of HA is more conserved than the globular

544

head that contains the receptor binding site 8, 9. However, the stem domain is poorly immunogenic


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 40

545

in the presence of the immunodominant head, and neutralizing MAbs against the stem typically

546

have lower potency than head directed ones.

547

During the past few years, much progress has been made in the isolation of human monoclonal

548

antibodies capable of neutralizing a wide range of influenza viruses. Very recently, several non-

549

neutralizing antibodies that confer protection against group I influenza viruses have also been

550

isolated 41, 42. Structural studies of protective stem-directed antibodies have shown that they target

551

conserved regions on the stem domain of HA

552

guide approaches for the design of new vaccine candidates capable of eliciting protective

553

antibodies against diverse strains of influenza A.

13-21

and have provided a wealth of knowledge to

554

Since the discovery of such stem-binding bnAbs, several groups have tried different

555

innovative approaches through active immunization to steer the immune response toward the stem

556

domain of HA in an attempt to elicit antibodies with similar breadth and specificity. Immunization

557

with a series of chimeric HA (cHA) molecules having a globular head domain from one HA

558

subtype and a stem domain from a different HA subtype were shown to protect against homologous

559

and heterologous (with respect to the stem) virus challenge in immunologically naïve animals 43-

560

46

561

been shown to enhance heterologous protection, albeit against only a few viruses. The use of

562

ferritin nanoparticles displaying repetitive arrays of full-length HA

563

and trimeric headless stem fragments of HA in which monomers are held together by trimerization

564

domains were recently shown to induce stronger immune responses against the homologous and

565

heterologous (though within the same group) viruses 26-28. These new approaches were able to steer

566

the immune response toward the stem domain of HA and induced cross-reactive anti-stem

567

antibodies, thus conferring broader cross-protection against different viruses.

. Masking the immunodominant regions of the head domain of HA by hyperglycosylation 47 has

48

or HA stem fragments

32

However, all 31

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Biochemistry

568

approaches have their own limitations. Smaller headless HA stem fragments from H1 and H5

569

subtypes that we have previously designed, were able to fold in a native-like conformation and

570

provided complete protection against homologous and partial protection against heterologous viral

571

challenge. However, these immunogens lack the complete antibody footprint of the bnAbs and do

572

not attain a native like trimeric conformation in the absence of a heterologous trimerization motif

573

28, 30

574

the mini-HA stem constructs reported by Impagliazzo et al.,26 in that it contains most of the HA

575

stem and forms a soluble trimer. Important differences include the fact that H1HA6P2 is circularly

576

permuted and so retains the native N terminus of the HA2 subunit in cleaved HA, whereas the

577

mini-HA retains the connectivity found in uncleaved HA. The mini-HA also contains a

578

heterologous GCN4 trimerization domain and was expressed as a glycosylated protein in 293F

579

mammalian cells whereas the H1HA6P2 does not contain a heterologous trimerization domain and

580

is not glycosylated. How these differences will impact immunogenicity and protective ability

581

remains to be evaluated in future studies. We had also previously designed HA-stem fragment

582

immunogens (from H3N2 and H1N1 subtypes) which include the entire HA stem domain and

583

therefore could potentially present more protective epitopes to the immune system

584

these bacterially expressed HA stem fragment immunogens conferred complete protection against

585

virus challenge in mice, they were relatively unstable and aggregation prone. To overcome the

586

aforementioned problems, we attempted to improve upon one of our previously designed

587

immunogens from the H1 subtype, H1HA6, by directed evolution and yeast surface display. The

588

resulting protein, H1HA6P2, has just two additional mutations located outside of the CR6261

589

binding site. Sequence analysis suggests that these mutations might stabilize other HA stem

590

designs, especially those from the H1 subtype. H1HA6P2, when expressed in E.coli, was

. The H1HA6P2 immunogen described in the present study is similar in several respects to

8, 25

. While


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 40

591

predominantly trimeric in the absence of any heterologous trimerization motif and was stable,

592

aggregation-resistant, and bound strongly to several bnAbs. H1HA6P2 showed an improvement

593

of ~20 fold in KD for the bnAb CR6261 compared to H1HA6 and retained binding even after being

594

subjected to heat stress. These results suggest that the mutations in H1HA6P2 have likely stabilized

595

the inter-protomer interactions, helping the protein to fold and attain a trimeric, native-like

596

prefusion conformation, thus enabling binding to stem-directed bnAbs with high affinity. The

597

improvements in physio-chemical properties, such as resistance to thermal denaturation, combined

598

with the ability to be expressed in E. coli would be useful for mass production, storage and

599

pandemic preparation. Since the mutations are located outside of the major (CR6261)

600

neutralization epitope on the HA stem, we do not expect them to decrease the response to this

601

epitope, however this can only be confirmed in future immunization studies. This stabilized

602

‘headless’ HA stem fragment immunogen can also potentially be used specifically to boost the low

603

levels of pre-existing, stem-directed antibodies in previously exposed human populations. It can

604

also act as an important reagent to unambiguously detect HA-stem specific antibody responses in

605

various immunological assays. Overall, our study provides a promising strategy to improve HA

606

stem fragment immunogens for the development of HA stem-based ‘universal’ influenza vaccine.

607

For improved mucosal immunity, the efficacy of intranasal administration of H1HA6P2 alone or

608

on VLPs, can be evaluated. In future studies, immunogenicity and breadth of protection of

609

conferred by this immunogen will be assessed in animal models.

610

Author Contributions:

611

T.A.N. and R.V. designed the study and analyzed and interpreted data. T.A.N. performed most

612

of the experiments except for some of the data in Figure 5 and Table 1 which were generated by


Page 35 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

613

U.K. T.A.N. and R.V. wrote the manuscript. J.F. provided the CR6261 Ab and analyzed and

614

reviewed the data.

615

Acknowledgements:

616

We thank Dr. Dennis R. Burton (The Scripps Research Institute, La Jolla, California, USA)

617

and Dr. K. Dane Wittrup (Massachusetts Institute of Technology, MA, USA) for providing us

618

pPNLS vector and EBY100 strain of S. cerevisiae respectively. We acknowledge technical support

619

from Dr. Shruti Khare, for assistance out with sequence analysis and Ms Srilatha and Dr.

620

Kavyashree Manjunath for assistance with the Biacore experiments respectively.

621

Funding:

622

Financial support for these studies was provided by the Department of Biotechnology,

623

Government of India (BT/BIPP/0213/04/09) and a JC Bose Fellowship from the Department of

624

Science and Technology to RV. We also acknowledge funding for infrastructural support from the

625

following programs of the Government of India: DST-FIST, UGC Center for Advanced Study,

626

and the DBT-IISc Partnership Program.

627

Conflict of Interest:

628 629 630

The authors declare no competing financial interests. Supplementary Information: Supplementary tables (S1, S2 and S3).

631

References:

632 633

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794 795

TOC graphic: Schematic of the mutant library construction and enrichment by FACS.

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