Some human RNA respiratory viruses evolve to escape immunity

Rate of viral antigenic evolution

Measles

Influenza

All enveloped viruses have one or more entry proteins that bind receptor and fuse the viral and cell membranes

• SARS-CoV-2 spike
• influenza hemagglutinin
• HIV envelope protein
• Lassa virus glycoprotein
• Nipah virus G and F proteins
• RSV G and F proteins

Deep mutational scanning of viral entry proteins (cell entry, antibody escape, receptor binding)

How can we rapidly and safely measure effects of mutations to viral entry proteins?

• General: can work for many viral entry proteins
• Comprehensive: measure mutations throughout the protein
• High-throughput: can be applied new variants and antibodies
• Safe: we want to avoid concerns with making novel replication-competent viruses
• Applicable directly to sera, to capture person-to-person heterogeneity

Lentiviral pseudotyping

• Many enveloped viruses have entry proteins amenable to lentiviral pseudotyping.
• Safe and well-established way to study cell entry function of these proteins
• However, traditional pseudotyping does not create genotype-phenotype link.

Two-step method to create genotype-phenotype linked pseudotype libraries

Dadonaite et al, Cell (2023)

Two-step method to create genotype-phenotype linked spike-pseudotypes

Dadonaite et al, Cell (2023)

Sequencing measures relative amounts, so include neutralization standard

Dadonaite et al, Cell (2023)

Example: RBD antibody LY-CoV1404

Dadonaite et al, Cell (2023)

Validates very well

Dadonaite et al, Cell (2023)

We mapped how all tolerated mutations to XBB.1.5 spike affect three key phenotypes

Cell entry: how well pseudovirus enters 293T-ACE2 cells

Sera escape: how pseudovirus is neutralized by human polyclonal serum

ACE2 binding: how pseudovirus is neutralized by soluble ACE2

Neutralization by soluble ACE2 is proportional to ACE2 binding affinity

How well do these spike phenotypes predict human SARS-CoV-2 clade growth?

change in clade growth

clade growth

Measured spike phenotypes correlate with changes in SARS-CoV-2 clade growth 

Multiple linear regression combining spike phenotypes predicts clade growth well

Extending this approach to additional viruses

Lassa virus glycoprotein complex (GPC)

Lassa virus causes thousands of human deaths each year, primarily from spillovers from Mastomys rodents. But there has been limited human-to-human spread.

There are efforts to develop antibodies and vaccines.

Valuable information about Lassa GPC

Defining functional constraint on GPC can identify regions that are unlikely to change, and aid immunogen design by informing protein engineering.

Prospective assessment of antibody escape can aid in choosing antibody therapeutics that will be robust to viral genetic variation and evolution.

Effects of mutations on GPC-mediated cell entry

Functional constraint on different GPC domains

Each antibody we analyzed is escaped by some well-tolerated mutations

Escape mutations for all antibodies are already observed in known natural GPC sequences

The natural GPC variants with these mutations indeed have increased escape

We can predict the escape of natural strains from adding up effects of mutations in deep mutational scanning

Conclusions

For both human endemic (SARS-CoV-2) and potential emerging (Lassa) viruses, we can safely measure how mutations to the entry proteins affect key molecular phenotypes.

For SARS-CoV-2, these measurements can help predict success of variants in humans.

For both viruses, we can predict extent of antibody escape of different variants.

These phenotypic maps can help inform the design of antibody and vaccine countermeasures that are more robust to viral escape.

We are now applying to: H5N1 HA, rabies G protein, HIV Env, Nipah RBP, RSV F