Disclosures
I am on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, the Vaccine, Company, and Oncorus
I am an inventor on Fred Hutch licensed patents related to deep mutational scanning of viral proteins
Evolution was historically studied by examining changes in phenotype
~55 years ago, it became possible to also study evolution in terms of genotype
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."
We can now characterize evolution of genotype at high resolution
Phylogenetic tree of ~13 million 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 Coronavirus Is Mutating. What Does That Mean For Us?
"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."
Yesterday I described yeast display deep mutational scanning of SARS-CoV-2 RBD
Current deep mutational scanning used by our group and that of Yunlong Cao et al are based on yeast displaying libraries of the spike receptor-binding domain (RBD) and selecting for antibody or ACE2 binding.
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.
Two alternatives
- Lentiviral deep mutational scanning of viral entry proteins
- Estimation of mutation effects from natural sequences
Lentiviral pseudotyping
Bernadeta Dadonaite
Kate Crawford
Caelan Radford
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
Sequencing only measures relative amounts, so include "absolute standard"
Example: RBD antibody LY-CoV1404
Validates very well
Also works for non-RBD antbodies
We can also measure functional effects of mutations on viral entry
Method should be extensible to many other viral entry proteins
- Other viruses with pseudotype-able entry proteins: other coronaviruses, influenza viruses, Ebola virus, Nipah virus, Lassa virus
- System can provide safe way to do high-throughput mutational studies
- We need to be aware of information hazard
- Information hazard low for SARS-CoV-2 spike, which is already human adapted
- But maybe hazard of human-receptor adaptive mutations in potential pandemic viruses
- For such viruses, we propose to use natural host rather than human receptors
Estimating mutation effects from natural sequences
Estimating mutation effects from natural sequences
with Richard Neher
We now have a lot of SARS-CoV-2 sequences
Phylogenetic tree of ~13 million full-length SARS-CoV-2 genomes provided by GISAID
We first estimate mutation rates from four-fold degenerate sites
We count mutations at such sites along tree
Enough sequences that each neutral mutation expected to be observed many times
We then compare actual counts to expected counts (from four-fold degenerate sites) for each mutation to estimate effect
Conclusions
The Coronavirus Is Mutating. What Does That Mean For Us?
"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."
Understanding effects of mutations is long-standing but important problem
Two new approaches
- Lentiviral pseudotype deep mutational scanning
- Analysis of natural sequences