Jesse Bloom PRO
Scientist studying evolution of proteins and viruses.
Interpreting the evolution
of SARS-CoV-2
Jesse Bloom
Fred Hutch Cancer Research Center / HHMI
Slides at https://slides.com/jbloom/sars-cov-2_activ
Only sometimes does virus evolution erode antibody immunity
- Measles virus: Does not evolve to escape immunity. People infected at most once in their lives. Vaccine developed in the 1960s still works today.
- Influenza virus: Evolves to escape immunity. People infected every ~5 years. Vaccine updated annually.
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.
Antibody immunity recognizes CoV-229E strains that circulated during a person's life
For complete data on all sera, see
Viral evolution erodes antibody immunity of different people at different rates
We are studying basis of differences, as ideally vaccines would elicit evolution-resistant sera as on the right.
Most mutations in RBD and to lesser extent NTD
Plot of sequence variability across CoV-229E spike taken from Eguia, ..., Bloom, bioRxiv (2020). See also Wong, ..., Rini, Nature Communications (2017) and Li, ..., Rini, eLife (2019) for detailed structural studies of evolution in CoV-229E receptor-binding loops.
We decided to focus on prospectively mapping effects of mutations in SARS-CoV-2 RBD
Most neutralizing activity of most convalescent human sera is from RBD-binding antibodies
Data shown here from Greaney et al (2021); similar results were obtained by Piccoli et al (2020).
Note that neutralizing antibodies can also target other spike domains such as NTD (e.g., McCallum et al, 2021).
We wanted to map how each mutation affects key biochemical phenotypes
Impossible to measure true viral fitness in the lab, so we focus on three biochemical phenotypes that crucially contribute to fitness:
1) Does RBD fold properly?
2) Does RBD bind ACE2 with high affinity?
3) Is RBD bound by anti-viral antibodies?
Impossible to measure true viral fitness in the lab, so we focus on three biochemical phenotypes that crucially 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 wanted to map how each mutation affects key biochemical phenotypes
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
Click here for details on how library is made.
Phenotypic maps of how mutations affect RBD folding & ACE2 affinity
We map escape mutations by sorting for RBD variants that don't bind antibody
We map escape mutations by sorting for RBD variants that don't bind antibody
We map escape mutations by sorting for RBD variants that don't bind antibody
We map escape mutations by sorting for RBD variants that don't bind antibody
We map escape mutations by sorting for RBD variants that don't bind antibody
In maps, tall letters indicate strong escape mutations
Escape map for one antibody (COV2-2499)
Interactive structural view of escape map (click here)
To test if phenotypic maps explain evolution, we grew virus with antibody
Spike-expressing VSV (Case*, Rothlauf*, ..., Whelan. Cell Host & Microbe, 2020) in a real-time cell analysis assay (Gilchuk, ..., Crowe. Immunity, 2020).
See here for more details.
Antibody COV2-2499 selected escape mutations in 5 of 16 replicates
mutation
G446D
Q498R
count
3
2
mutation
G446D
Q498R
count
3
2
Escape map has many mutations, why were these two selected?
mutation
G446D
Q498R
count
3
2
Only some mutations accessible by single-nucleotide changes
mutation
G446D
Q498R
count
3
2
Many mutations deleterious for ACE2 binding, but ones selected in virus aren't
ACE2 binding
weak strong
Selected mutations are single-nt changes that escape antibody, retain ACE2 binding
effect on escape
ACE2 affinity
effect on escape
ACE2 affinity
Phenotypic maps also explain mutations selected by other antibodies
Antibody COV2-2165 did not select any escape mutations in repeated trials
0 escape mutants in 56 attempts
Initially appear to be many escape mutations from this antibody
But only a few are accessible by single-nucleotide changes
Accessible mutations impair folding or ACE2 binding, explaining why virus can't escape
RBD expression
ACE2 binding
RBD expression /
ACE2 binding
weak strong
We now have escape maps for many antibodies, including lead clinical ones
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, bioRxiv, 2020)
We mapped how all RBD mutations affect binding by convalescent human sera
For more maps see
The maps define main epitopes as receptor-binding ridge and secondarily 443-450 loop
Most important site is E484, which is mutated in 501Y.V2 and 501Y.V3 lineages
501Y.V2 also known as B.1.351, originally identified in South Africa.
501Y.V3 also known as P1, originally identified in Brazil.
But as for CoV-229E, people affected differently by viral mutations
E484 mutations have huge effect
G446/G447 mutations have large effect
No mutations have a large effect
These differences between people validate in neutralization assays
For instance, E484K greatly reduces neutralization by subject C. Subject G is unaffected by E484K, but affected by G446V.
We can use maps to assess which current mutations have biggest antigenic effect
Chu lab (Univ Wash): Helen Chu, Caitlin Wolf
Crowe lab (Vanderbilt): James Crowe, Seth Zost, Pavlo Gilchuk
King lab (Univ Wash): Neil King, Dan Ellis
Veesler lab (Univ Wash): David Veesler, Alexandra Walls, Ale Tortorici
Whelan lab (Wash U)
Boeckh lab (Fred Hutch): Terry Stevens-Ayers
Alex Greninger (Univ Wash)
Janet Englund (Seattle Children's)
These slides: https://slides.com/jbloom/sars-cov-2_activ
Andrea Loes
Tyler Starr
Allie Greaney
Rachel Eguia
Bloom lab (Fred Hutch)
Sarah Hilton
Kate Crawford
sars-cov-2_ACTIV
By Jesse Bloom
sars-cov-2_ACTIV
Presentation to ACTIV group, Feb 2021
