Towards a Universal Flu Vaccine:�Glycan-shielding of H7 hemagglutinin head domain

7/23/19

Ria Kolli

We need� - Universal Influenza Vaccine �- to be ready for pandemics

1918 "Spanish flu"

1957-58 "Asian flu"

1968-69 "Hong Kong flu"

1997 "Bird flu"

2009 "Swine flu"

H1N1, World wide, 50 million

H3N2, China, 2 million

H3N2, world wide, 1 million

H5N1, Hong Kong, Egypt, Indonesia,

Vietnam, China, 2000

H1N9_pdm, World wide, 18000

Slide from Mrinmoy Sanyal

Vaccine effectiveness by year (2004-2016)

flugen.com/about-influeza/

Slide from Mrinmoy

H7 is potential candidate for scaffolding for universal vaccine design

• General human population is not exposed to H7 protein

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• H7 elicits cross-group stem specific immune response

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• Influenza H7N9 epidemic is gaining momentum

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Slide from Mrinmoy

Candidate for universal influenza vaccine

Slide from Mrinmoy

Five epidemic waves of H7N9 infection

Su et al, Trends in Microbiol (2017) 25:9-p713-728

Background

• HA has head and stem domains
• Head: strain-specific, immunodominant
• Stem: highly conserved, immunosubdominant
• We can shield head epitopes from antibodies using engineered glycans

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Adding more glycans to the H7 head

HA1

HA2

340

513

19

30

46

249

421

493

30

46

89

100

134

141

148

167

172

234

249

285

294

421

493

WT

+10

A/Shanghai/02/2013(H7N9)

Slide from Steven

Glycosylate immunogenic HA epitopes in order to design an immunogen that redirects immune responses towards the conserved stem region of HA

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Design of H7+10 GlcNAc

Engineered glycan (10)

Native glycan (5)

front

back

top

Slide from Steven

Outline

1. Test the TrimerTag for H7 expression
2. Use the TrimerTag to express 7 different Has
3. Characterize the H7 head- and stem-only proteins

Part 1: TrimerTag

• Current trimerization strategies in the lab:
• HA-foldon
• HA-IZ
• HA-Ferritin
• In this project, we wanted to test a new trimerization domain, the TrimerTag

https://www.genhunter.com/trimer-tag/

The TrimerTag

• Origin: Human collagen
• Structure

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(the entire human C-propeptide of α1(I) collagen containing 11 glycine-repeat triple helical region)

Ria Kolli & Steven Tang, July 2019

Expression and purification of Trimer-Ctag H7

Using the CaptureSelect C-tagXL Affinity Matrix

• Ctag: 4-amino acid peptide tag (E-P-E-A)
• Elution: 2M MgCl₂ into 20mM Tris 8

- H7

Size Exclusion Chromatography of Trimer-Ctag H7 in PBS buffer

Ria Kolli & Steven Tang, July 2019

-H7

-H7+10

Using Biotin Anti-C-tag Conjugate to load C-tagged proteins

onto Octet Streptavidin Tips

CaptureSelect™ Biotin Anti-C-tag Conjugate consists of a 13 kDa camelid antibody fragment (affinity ligand) with high affinity and selectivity for "C-tag" peptide tag E‑P‑E‑A (glutamic acid - proline - glutamic acid - alanine).

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Octet test on the H7 Trimer-Ctag

Loading: SAV tips into 5 μg/mL Biotinylated antiCtag Nanobody to 3.0 nm (~3 min)

Ria Kolli & Steven Tang, July 2019

Octet 20190711

Head

Stem

4FQV

5JW3

CR9114

MEDI8852

H7 Stem antibodies with available pseudoatomic structures

Andrews et al. Sci Immunol. 2017

Patrick Wilson’s H7 antibodies

Dunand et al. Cell Host & Microbe 2016

Part 1 Conclusion

• H7-TrimerTag binds to well-characterized stem and head antibodies
• The current H7+10 constructs have unprotected head epitopes
• Steven is testing new H7+X constructs
• The TrimerTag can be used as an alternative trimerization strategy for HA expression

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Part 2: Testing 7 different HAs

• Test whether well-characterized antibodies against H7 demonstrate expected cross-reactivity against other HAs
• H7: A/Shanghai/02/2013(H7N9)
• H1: A/California/07/2009 (H1N1)
• H2: A/Japan/305+/1957 (H2N2)
• H3: A/Singapore/INFIMH-16-0019/2016 (H3N3)
• H5: A/Viet Nam/1203/2004(H5N1)
• B-Phuket: B/Phuket/3073/2013
• B-Colorado: B/Colorado/06/2017

H7

H1

H2

H3

H5

B-Phuket

B-Colorado

Steven Tang, July 2019

Purification of 7 HA from Influenza A Groups 1 & 2 and Influenza B

TrimerTag-Ctag

+ βme

Ria Kolli & Steven Tang, July 2019

Octet 20190722

Influenza A Group 1 HA-TrimerTag-Ctag binding to bNAbs

Ria Kolli & Steven Tang, July 2019

Octet 20190722

Influenza A Group 2 HA-TrimerTag-Ctag binding to bNAbs

Ria Kolli & Steven Tang, July 2019

Octet 20190722

Influenza B HA-TrimerTag-Ctag binding to bNAbs

Part 2 Conclusions

• The TrimerTag can be used to express HAs from diverse strains of influenza
• The purified HAs bind to well-characterized bNAbs
• The purified HAs will be used as a substrate for testing sera binding from H7-immunized mice

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Part 3: H7 Head and Stem-only proteins

• To test the binding epitopes of uncharacterized antibodies, we attempted to purify H7 head and stabilized stem proteins

Corbett et al. mBio 2019

H7 Head

H7 Stem

Expression and purification of H7 Head and stem-only proteins using Ni-NTA Columns

• H7^ss-Foldon-Thrombin-Avi-His₆
• H7^WT head-Thrombin-Avi-His₆
• H7^+10GlcNAc head-Thrombin-Avi-His₆

H7ss-Foldon

H7 WT

H7+10

- H7 WT head

- H7+10 head

Expression and purification of H7 head domain using Size Exclusion

Ria Kolli & Steven Tang, July 2019

-H7 Head

-H7+10 Head

H7ss-Fdn

H7-head

H7+10-head

Octet test on the H7 head constructs

Ria Kolli & Steven Tang, July 2019

Octet 20190711

Part 3 Conclusions

• The purified head proteins showed specificity to head antibodies compared to the full length H7 foldon, which bound to both head and stem antibodies
• Use the purified samples in high-throughput screens to profile antibodies.

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Acknowledgements

• Steven Tang
• Mrinmoy Sanyal
• Peter Kim

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More antibody stuff

• L4A-14
• FluA-20
• S5V2-29

H7 Head antibodies L4A-14 (PDB: 6II4)

Huang et al. Nat Microbiol. 2019

FluA-20

H7 head bNAb binding to the HA receptor binding site (PDB: 6OCB)

Bangaru et al. Cell 2019

S5V2-29

Influenza A Group 2 H3 head antibody (PDB: 6E4X)

Watanabe et al. Cell 2019