Interpreting the evolution

of SARS-CoV-2

Jesse Bloom

Fred Hutch Cancer Research Center / HHMI

Slides at https://slides.com/jbloom/activ-trace

@jbloom_lab

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.

There is no past behavior of SARS-CoV-2 to study, but we can look to its relatives

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.

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)

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/

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

Extent of focusing of polyclonal antibodies shapes antigenic evolution

For influenza virus, human polyclonal antibody immunity often highly focused, such that single mutations can reduce neutralization by >10 fold. (Lee et al, eLife, 2019)

For measles virus, polyclonal antibodies target many epitopes with similar potency, so no single mutation has a large effect. (Munoz-Alia, Cell Rep. Med., 2021)

RBD-binding antibodies are responsible for most neutralizing activty

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 activty

Within the RBD, how broad or narrow is the binding of infection- and vaccine-elicited polyclonal antibodies?

How do different antibody classes contribute to polyclonal responses?

We use the Barnes antibody classification scheme from Barnes et al Nature (2020). The extension of these antibody classes to escape mapping is described 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 often less neutralizing (see: Piccoli et al (2020), Dejnirattisai et al (2021), Liu et al (2020), Zost, et al (2020)).