Interpreting SARS-CoV-2 evolution

Jesse Bloom

Fred Hutch Cancer Center / HHMI

These slides at https://slides.com/jbloom/sars2evol​

Panum (1847)

The Faroe Islands

"Measles had not prevailed on the Faroes since 1781, then it broke out early in April 1846."

Panum (1847)

"Of the 7782 inhabitants, about 6000 were taken with measles."

Panum (1847)

Panum (1847)

Panum was describing immune memory, which provides lifelong protection from measles

What was reason for thinking coronavirus immunity would not be affected by viral mutations?

Coronaviruses have lower mutation rate than other RNA viruses

Coronaviruses are only RNA viruses with proofreading activity in their polymerase, and so have ~5- to 10-fold lower mutation rate than influenza virus

The average single-nucleotide mutation to SARS-CoV-2 had occurred >10,000 independent times in human-transmitted SARS-CoV-2 by third year of the pandemic.

But evolution of SARS-CoV-2 is not mutation-limited

Evolution is not just mutation: also involves selection on phenotypic effects of mutations

My group studies virus evolution at the level of phenotype 

... atg ttt gtt ttt ctt gtt ...

... atg ttt ctt ttt ctt gtt ...

... atg ttt gtt tat ctt gtt ...

17,052,049 SARS-CoV-2 sequences are currently available

But what do they mean?

CoV-229E causes common colds and has been circulating in humans for a long time.

How do human coronaviruses evolve?

Evolution of CoV-229E spike

We experimentally generated CoV-229E spikes at ~8 year intervals to study in the lab:

- 1984

- 1992

- 2001

- 2008

- 2016

Eguia et al (2021)

Evolution of CoV-229E spike erodes neutralization by human serum antibodies

Eguia et al (2021)

Ideally vaccines would elicit evolution-resistant antibodies (like those made by person at right) rather than evolution-sensitive antibodies (like those made by person at left)

Evolution erodes neutralization by sera from different people at different rates

Eguia et al (2021)

We now know SARS-CoV-2 evolves to erode neutralizing antibodies, just like CoV-229E

Original vaccine induced high neutralizing antibody titers against early viral strains

Lasrado et al (2023)

We now know SARS-CoV-2 evolves to erode neutralizing antibodies, just like CoV-229E

Lasrado et al (2023)

newer viral variants

Can we predict evolutionary success of new viral variants?

SARS-CoV-2's spike binds receptor and mediates viral entry

viral membrane

cell membrane

spike

spike conformational change

Image adapted from here

ACE2

antibody

Image adapted from here

SARS-CoV-2's spike binds receptor and mediates viral entry

Deep mutational scanning to measure effects of mutations 

RBD

fluorescent ACE2

yeast

fluorescent tag on RBD

Yeast display of spike receptor-binding domain (RBD) to measure ACE2 binding

Starr et al (2020)

cell sorting

Deep mutational scanning: selections on libraries of mutants, read out by sequencing

Experiments identified N501Y as a mutation that increases ACE2 binding

Starr et al (2020)

RBD

fluorescently labeled antibody

yeast

fluorescent tag on RBD

Yeast display to measure how all RBD mutations escape antibody binding

Greaney et al (2021)

These anecdotes suggest experiments can identify mutations subsequently selected by evolution

Yeast display only measures binding, which is just part of spike's biological function

cell sorting

Viral infection is more biologically relevant measure of mutation effects

However, we are cognizant of biosafety concerns of mutating actual virus

actual SARS-CoV-2 virion: pathogen capable of spread in humans

pseudotyped lentiviral particle: not a pathogen, cannot spread in humans

actual SARS-CoV-2 virion: pathogen capable of spread in humans

pseudotyped lentiviral particle: not a pathogen, cannot spread in humans

Therefore we use pseudoviruses rather than actual replicative SARS-CoV-2

Pseudoviruses are modified viruses that can only undergo single round of infection

Crawford et al (2020)

Crawford et al (2020)

We need to link phenotype (spike mutant) and genotype (sequence encoded by virus)

To link genotype to phenotype, we encode spike in viral genome with barcode

Dadonaite et al (2023)

We then create genotype-phenotype linked libraries by two-step process

Dadonaite et al (2023)

Result is library of spike mutant pseudoviruses with identifying barcodes

• Captures full cell-entry function of spike
• Each mutant identified by short barcode
• Pseudoviruses safe at biosafety-level 2
• Broadly applicable to viral entry proteins

Dadonaite et al (2023)

Dadonaite et al (2024)

Measured how spike mutations affect three molecular phenotypes

Dadonaite et al (2024)

Measured how spike mutations affect three molecular phenotypes

How do these experimental measurements relate to actual evolution?

Estimating variant fitness (multinomial logistic regression)

With Trevor Bedford & Ben Murrell

Deep mutational scanning measurements correlate with SARS-CoV-2 variant fitness

Dadonaite et al (2024)

Combining phenotypes offers best predictions (because mutations can involve tradeoffs)

Dadonaite et al (2024)

We can explain ~55% of the variance in growth of different clades, with largest fraction of variance uniquely explained by sera escape.

Dadonaite et al (2024)

Can partially predict outcome of real-world evolutionary competition from experiments!

But human population in which SARS-CoV-2 evolves is changing...

Initially, most people were exposed to early strain, imprinting antibody response (     )

First exposed to early strain in original vaccine

First infected by an early strain (pre-Omicron)

Imprinting to early strain continues to shape antibody response after later exposures

But antibody response of infants is imprinted by more recent strain

Imprinted by recent strain

We measured which spike mutations erode neutralization by antibodies from people imprinted with different strains

Adults vaccinated with original strain followed by various exposures

Adult sera from Helen Chu's HAARVI study; infant sera from Mary Staat's IMPRINT cohort

6-12 month infants first infected by XBB*

Adult sera have similar escape (mostly in RBD)

site in spike

Sites of escape from sera of adults imprinted with original vaccine, then exposed to various infections and vaccinations.

adult 1

adult 2

adult 3

adult 4

adult 5

adult 6

2021

site in spike

Sites of escape from infants with only single infection with XBB*

infant 1

infant 2

infant 3

infant 4

infant 5

infant 6

2023

Infant sera have similar escape (mostly in NTD)

The approaches we have developed are applicable to many other viral entry proteins

• influenza hemagglutinin (Dadonaite et al, 2024)
• HIV envelope protein (Radford et al, 2023)
• Lassa virus glycoprotein (Carr et al, 2024)
• Nipah virus RBP and F proteins (Larsen et al, 2024)
• RSV G and F proteins
• Rabies G protein
• Chikungunya virus E proteins

Interpreting viral evolution

... atg ttt gtt ttt ctt gtt ...

... atg ttt ctt ttt ctt gtt ...

... atg ttt gtt tat ctt gtt ...

Lots of viral sequences

But what do they mean?

Bloom lab

These slides: https://slides.com/jbloom/sars2evol

Thanks

Fred Hutch Cancer Center

Trevor Bedford

University of Washington

Helen Chu and HAARVI cohort

Neil King

David Veesler

Cincinnati Children's Hospital

Mary Staat

David Haslam

Allie Burrell

Kate Crawford

William Hannon

Caelan Radford

Brendan Larsen