Interpreting the evolution

of SARS-CoV-2

Jesse Bloom

Fred Hutch Cancer Research Center / HHMI

Slides at https://slides.com/jbloom/sars-cov-2-evolution

@jbloom_lab

Disclosures

I am on the scientific advisory boards of Flagship Labs 77 and Oncorus
I consult for Moderna
I am an inventor on Fred Hutch licensed patents related to deep mutational scanning of viral proteins
My lab has unfunded research collaborations with Vir Biotechnology

Evolution was historically studied by examining changes in phenotype

Haeckel. Pedigree of Man (1866)

~55 years ago, it became possible to also study evolution in terms of genotype

Zuckerkandl and Pauling (1965)

How changes in genotype affect phenotype is complex

"There is no reason to expect that the extent of functional change in a polypeptide chain is proportional to the number of amino-acid substitutions... It is the type rather than the number of substitutions that is decisive."

Zuckerkandl and Pauling (1965)

We can now characterize evolution of genotype at high resolution

Phylogenetic tree of 5,152,481 full-length SARS-CoV-2 genomes provided by GISAID

We want to know how changes in genotype affect phenotype

The Coronavirus Is Mutating. What Does That Mean For Us?

The New York Times (2021)

The Coronavirus Is Mutating. What Does That Mean For Us?

The New York Times (2021)

Zuckerkandl and Pauling (1965)

biochemical phenotypes

- protein folding

- affinity for ACE2

- binding by antibodies

Protein structure matters implicitly, as it largely determines mapping from mutation to phenotype.

-Asn-Ile-Thr-Asn-Leu-

-Asn-Ile-Thr-Glu-Leu-

-Asn-Lys-Thr-Asn-Leu-

My lab studies how changes in genotype affect phenotype for viral proteins

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)
How do coronavirus spikes evolve?

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)
How do coronavirus spikes evolve?
How mutations affect function of RBD

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)
How do coronavirus spikes evolve?
How mutations affect function of RBD
How mutations affect antibody recognition of RBD

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)
How do coronavirus spikes evolve?
How mutations affect function of RBD
How mutations affect antibody recognition of RBD

SARS-CoV-2 virions are decorated by spike proteins that mediate viral entry

virion image from https://phil.cdc.gov/Details.aspx?pid=23312

See Tortorici and Veesler (2019) for general review on coronavirus spikes.

See Walls et al (2020) and Wrapp et al (2020) for details on structure of SARS-CoV-2 spike.

A key portion of the spike is the receptor-binding domain (RBD)

The RBD binds ACE2 receptor on cells

Neutralizing antibodies target spike; often bind RBD and block interaction with ACE2

RBD-binding antibodies are responsible for most neutralizing activity

Data from Greaney et al (2021a, 2021b) using lentiviral pseudotypes on ACE2-overexpressing cells. Similar results seen by Piccoli et al (2020). Neutralizing antibodies can target other spike regions such as NTD (e.g., McCallum et al, 2021).

RBD-binding antibodies are responsible for most neutralizing activity

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)
How do coronavirus spikes evolve?
How mutations affect function of RBD
How mutations affect antibody recognition of RBD

Only sometimes does virus evolution lead to changes in antigenic phenotype

Measles virus: Does not evolve to escape immunity. People are infected at most once in their lives. A vaccine developed in the 1960s still works today.

Influenza virus: Evolves to escape immunity. People are infected every ~5 years. The vaccine needs to be updated annually.

Initially, many people suggested coronaviruses didn't evolve antigenically

Coronaviruses are the only RNA viruses with a proofreading polymerase (Denison et al, 2011), meaning they have a lower mutation rate than influenza or measles virus

The Washington Post, March 4, 2020

Not a good argument

Evolution isn't just mutation. Rather, it involves three related factors:

The rate at which mutations arise.
The phenotypic impact of these mutations.
How selection (and drift) act on these mutations.

We studied CoV-229E, a coronavirus that causes common colds and has been circulating in humans since at least the 1960s.

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

Eguia, ..., Bloom. PLoS Pathogens (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. PLoS Pathogens (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. PLoS Pathogens (2021)

Most mutations in RBD & NTD

Plot of sequence variability across CoV-229E spike taken from Eguia, ..., Bloom, PLoS Pathogens (2021) . See also Wong, ..., Rini, Nature Communications (2017) and Li, ..., Rini, eLife (2019) for detailed structural studies of evolution in receptor-binding loops.

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)
How do coronavirus spikes evolve?
How mutations affect function of RBD
How mutations affect antibody recognition of RBD

For SARS-CoV-2 RBD, want to prospectively map how each mutation affects phenotype

Impossible to measure true viral fitness in the lab, so we focus on three biochemical phenotypes that contribute to fitness:

1) Does RBD fold properly?

2) Does RBD bind ACE2 with high affinity?

3) Is RBD bound by anti-viral antibodies?

For SARS-CoV-2 RBD, want to prospectively map how each mutation affects phenotype

Impossible to measure true viral fitness in the lab, so we focus on three biochemical phenotypes that contribute to fitness:

1) Does RBD fold properly?

2) Does RBD bind ACE2 with high affinity?

3) Is RBD bound by anti-viral antibodies?

Evolutionary pressure is to maintain these two phenotypes...

... while changing this phenotype.

We use yeast display to enable high-throughput experiments

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.

Library of yeast, each expressing different RBD mutant with identifying 16 nt barcode

Starr*, Greaney*, ..., Bloom. Cell (2020)

Click here for details on how library is made.

Phenotypic maps of how mutations affect RBD folding & ACE2 affinity

Starr*, Greaney*, ..., Bloom. Cell (2020)

Outline

SARS-CoV-2 spike and its receptor-binding domain (RBD)
How do coronavirus spikes evolve?
How mutations affect function of RBD
How mutations affect antibody recognition of RBD

We map escape mutations by sorting for RBD variants that don't bind antibody

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

RBD

fluorescently labeled antibody

yeast

fluorescent tag on RBD

https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_LY-CoV555/

https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Vir_mAbs/

Escape map from a single antibody

Escape maps from lots of antibodies

https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/

Infection / vaccination elicit polyclonal antibodies that can bind many epitopes

Monoclonal antibodies bind one epitope, so can usually be escaped by single mutation

Polyclonal antibodies can bind many epitopes, so often more resistant to escape

If polyclonal antibodies bind multiple epitopes, escape needs multiple mutations

We use the antibody classification scheme from Barnes et al Nature (2020). The extension of these antibody classes to escape mapping is in Greaney et al (2021), and escape maps are available here. Class 1, 2, and 3 antibodies are often potently neutralizing, while class 4 antibodies are less neutralizing (see: Piccoli et al (2020), Dejnirattisai et al (2021), Liu et al (2020), Zost, et al (2020)).