sars2-dms

Disclosures

I am on the scientific advisory boards of Apriori Bio, Aerium Therapeutics, Invivyd, and the Vaccine Company

I am an inventor on Fred Hutch licensed patents related to deep mutational scanning of viral proteins

Panum, 1846

The Faroe Islands

History offers natural experiment with influenza like Panum's study of measles

In 1977, old H1N1 strain from ~1954 was inadvertently re-released and caused a global pandemic.

Influenza infection elicits multi-decade immunity, but only if virus is evolutionarily frozen

Only some human RNA respiratory viruses evolve to escape immunity

Why some viruses evolve to escape immunity while others don't is a deep question outside scope of this talk. See here for some possible explanations.

Rate of viral antigenic evolution

Measles

Influenza

Rate of viral antigenic evolution

Measles

Where do human coronaviruses fall on this spectrum?

Coronaviruses

???

Influenza

Rate of viral antigenic evolution

Measles

Coronaviruses

???

The Washington Post, March 4, 2020

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Initially, people speculated SARS-CoV-2 would evolve slowly. But we weren't so sure...

Influenza

We decided to study another human coronavirus: CoV-229E causes common colds and has been circulating in humans for a long time.

Reconstructing evolution of CoV-229E spike

We experimentally generated CoV-229E spikes at ~8 year intervals so we could study them in the lab:

- 1984

- 1992

- 2001

- 2008

- 2016

Note "ladder-like" shape of tree

Eguia, ..., Bloom (2021)

Evolution of CoV-229E spike erodes neutralization by human antibody immunity

Serum collected in 1985 neutralizes virus with spike from 1984, but less effective against more recent viruses.

Eguia, ..., Bloom (2021)

Viral evolution erodes antibody immunity of different people at different rates

We are studying basis of these differences, as ideally vaccines would elicit more evolution-resistant sera as on the right.

Eguia, ..., Bloom (2021)

Rate of viral antigenic evolution

Measles

CoV-229E

CoV-OC43

SARS-CoV-2

Most human coronaviruses undergo rapid antigenic evolution

Influenza

Eguia, ..., Bloom (2021) and Kistler and Bedford (2021)

Phylogenetic tree shape & vaccine strategy

CoV-229E has ladder-like tree:

new variants displace old ones
new variants descend from recent successful ones

Human influenza A evolves this way too. It's theoretically possible to pick single well-matched vaccine strain.

See Volz et al (2013), Kistler and Bedford (2021), and Kucharski (Twitter, 2022)

Phylogenetic tree shape & vaccine strategy

See Volz et al (2013), Kistler and Bedford (2021), and Kucharski (Twitter, 2022)

CoV-229E has ladder-like tree:

new variants displace old ones
new variants descend from recent successful ones

Human influenza A evolves this way too. It's theoretically possible to pick single well-matched vaccine strain.

CoV-OC43 split into two ladder-like lineages. Influenza B evolves this way too. It's theoretically possible to pick well-matched bivalent vaccine.

Phylogenetic tree shape & vaccine strategy

See Volz et al (2013), Kistler and Bedford (2021), and Kucharski (Twitter, 2022)

CoV-229E has ladder-like tree:

new variants displace old ones
new variants descend from recent successful ones

Human influenza A evolves this way too. It's theoretically possible to pick single well-matched vaccine strain.

CoV-OC43 split into two ladder-like lineages. Influenza B evolves this way too. It's theoretically possible to pick well-matched bivalent vaccine.

In non-ladder-like tree, next variant not descended from recent successful one. Makes picking vaccine strains difficult.

Bloom (2022)

The molecular clock identifies unusual patterns in Omicron evolution

During typical acute infections, random transmission bottlenecks disrupt selection

But in chronic infections of 100+ days, selection can fix antibody-escape mutations without bottlenecks

Strongest evolutionary selection is in RBD

Sites of evolutionary change in the spike of CoV-229E over the last four decades

Strongest evolutionary selection is in RBD

Sites of evolutionary change in the spike of CoV-229E over the last four decades

Sites of mutations in SARS-CoV-2 Omicron BQ.1.1 spike relative to Wuhan-Hu-1

Majority of neutralizing antibody response in vaccinated/infected humans targets RBD

Greaney et al (2021)

Importance of RBD

Human CoVs, which evolve to escape transmission-blocking immunity, show strongest selection in RBD.

So virus is telling us RBD antibodies matter most for blocking transmission. Other antibodies and T-cells still may reduce disease severity while putting less selection on virus.

Most neutralizing activity from RBD antibodies (although antibodies to other domains can also be neutralizing).

SARS-CoV-2 variants like Omicron have a lot of mutations in RBD

RBD mutations in Omicron BA.1

How does Omicron even bind ACE2 with so many mutations?

To answer this question, we measure how RBD mutations affect ACE2 affinity

RBD

fluorescent ACE2

yeast

fluorescent tag on RBD

Importantly, we use ACE2 titrations to measure true affinities, not just relative FACS binding signal; see here for details.

Deep mutational scanning: measurements for a library of all RBD mutations

Library of yeast each expressing a different RBD mutant. Click here for details on how library is made.

Interactive heatmaps are available here, and are from Starr et al, 2022 and unpublished work.

Starr et al (2022)

ACE2 affinity-enhancing mutations buffer antibody-escape mutations

RBD will not run out of evolutionary space

25 of 31 residues in CoV-229E RBD that contact receptor varied during virus's evolution in humans over last ~50 years (Li et al, 2019)

There are lots of mutations to SARS-CoV-2 RBD that retain (and sometimes even enhance) ACE2 affinity (Starr et al, 2020; Starr et al, 2022)

We use deep mutational scanning to map all RBD mutations that escape antibody binding

Greaney*, Starr*, Gilchuk*, Zost*, ..., Crowe, Bloom. Cell Host & Microbe (2021)

RBD

fluorescently labeled antibody

yeast

fluorescent tag on RBD

Experiments combine flow cytometry and deep sequencing of a library of yeast expressing all RBD mutants

For interactive escape map, see: https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_LY-CoV555/

Escape map from one antibody (LY-CoV555, ie bamlanivimab). Peaks indicate sites where mutations escape binding.

484

452

490

Infection / vaccination elicit polyclonal antibodies

How do mutations affect polyclonal antibodies? First, consider an equal mix of three monoclonal antibodies.

Interactive version of this mini example is at https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/mini-example-escape-calc/

LY-CoV555 is escaped at both sites 484 and 490, so mutating either site has same overall effect

Average escape across all antibodies

Mutating site 484 or 490 eliminates neutralization by antibody LY-CoV555, as reflected in thick black line showing average

Interactive version of this mini example is at https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/mini-example-escape-calc/

Antibody-escape calculator extends this principle to deep mutational scanning data for ~3,000 different human antibodies

Escape calculator is described in Greaney et al (2022), and is available at https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/escape-calc/

36 antibodies mapped by Tyler Starr & Allie Greaney in Bloom lab, from early SARS-CoV-2 strains

Antibody-escape calculator extends this principle to deep mutational scanning data for ~3,000 different human antibodies

Escape calculator is described in Greaney et al (2022), and is available at https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/escape-calc/

36 antibodies mapped by Tyler Starr & Allie Greaney in Bloom lab, from early SARS-CoV-2 strains

3,000 (!) antibodies mapped by Y unlong Cao et al at Peking University. From early strains, BA.1, & patients with prior SARS-CoV-1 infection. See here.

Antibodies elicited by early SARS-CoV-2 that neutralize Wuhan-Hu-1 heavily focus on sites like 484, 417, 446

417

446

484

417

446

484

Omicron BA.1 is mutated at many of these sites, which is why it was neutralized substantially less well by current vaccines

417

446

484

Omicron BA.2 has some different RBD mutations than BA.1, but similar overall escape from antibodies from early SARS-CoV-2

Can identify mutations that mediate further escape. In Dec 2021 we predicted 486 as likely site of future evolution--in April 2022, mutation F486V was identified in Omicron BA.4 and BA.5.

486 is largest site of escape for antibodies not already escaped by mutations in BA.2

Limitations of RBD yeast-display deep mutational scanning

Only examines mutations in RBD, which is just part of spike
Measures antibody binding, not neutralization. They are unequal in polyclonal serum.
Only works for viral entry proteins with domains amenable to yeast display.

Lentiviral pseudotyping

Many viruses have entry proteins amenable to lentiviral pseudotyping.
However, traditional pseudotyping does not create genotype-phenotype link.

Two-step method to create genotype-phenotype linked spike-pseudotypes

Dadonaite et al, Cell (2023)

Example: RBD antibody LY-CoV1404

Dadonaite et al, Cell (2023)

This new deep mutational scanning approach is extensible to many viruses

Applicable to entry proteins from other coronaviruses, influenza viruses, Ebola virus, Nipah virus, Lassa virus, RSV, rabies virus, HIV, etc

Safe way to do high-throughput mutational studies

There is potential information hazard: as we can increasingly map the functional and antigenic effects of viral mutations, we need to ensure such information is not used to inform high-risk gain-of-function studies using actual human pathogens.