Saw this paper by way of @apoorva_nyc:

"Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants"

https://www.biorxiv.org/content/10.1101/2020.07.21.214759v1.abstract

My instinct is to expect any preprint on this topic to be noise but I've seen great work from several of the authors, so let's dig in.

1/

Q: How easy it is for SARS-CoV-2 to escape neutralizing antibodies through RBD mutations?

Experiment: Culture rVSV/SARS-CoV-2/GFP in 293T/ACE2(B) cells in the presence of NAbs, sequence successful clones.

2/

What is rVSV/SARS-CoV-2/GFP?

It's a Vesicular Stomatitis Virus ( https://en.wikipedia.org/wiki/Indiana_vesiculovirus), but with the native glycoprotein replaced with the SARS-CoV-2 spike, as well as the addition of a fluorescent reporter.

(you might recognize VSV from the rVSV-EBOV vaccine)

3/

OK, but why rVSV/SARS-CoV-2/GFP and not just SARS-CoV-2?

(i) you can work with it in BSL2
(ii) it glows
(iii) no proofreading mechanism, faster mutations
(iv) do you really want researchers making NAb resistant SARS-CoV-2? A chimeric virus is safer.

4/

Neutralizing antibody sources: 3 monoclonals (C121, C135, C144) and convalescent serum from 4 patients, 3 of whom had neutralizing activity (COV-47, COV-72, COV-NY) and one that didn't (COV-107).

5/

Two serial passages later and there was clearly an escape mutation from all 3 monoclonals and COV-NY's polyclonal antibodies. They then did two more passages for COV-47, COV-72, COV-107 at 5x serum concentration.

6/

"Replication in the presence of COV72 or COV107 plasma did not lead to the clear emergence of escape mutations, suggesting that the neutralization by these plasmas was not due to one dominant antibody specificity... "

7/

"In the case of COV107, the failure of escape mutants to emerge may simply be due to the lack of potency of that plasma"

8/

For all the other antibodies (3 monoclonals and COV-47/COV-NY serum), however, they did get emergence of clear escape mutations.

C121: E484K, Q493K/R, & F490L (sub-clonal)
C144: E484, Q493
C135: R346K/S/L, N440K
COV-NY: K444R/N/Q, V445E
COV-47: N148S, K150R/E/T/Q, S151P

9/

These escape mutations are mostly in the RBD (and within those mostly in the RBM), except COV-47 all of whose escape mutations are in the N terminal domain (NTD).

This fits with other papers showing NTD neutralization.

10/

They then isolated individual clones and made sure they had the same fitness as the "wildtype" chimeric virus (they did).

They also found that a virus with escape mutations against one patient's serum could still be neutralized by serum from a different patient.

11/

Similarly, clones escaping neutralization by similar monoclonal antibodies (C121 & C144) could still be neutralized by a qualitatively different antibody (C135).

12/

Can the virus escape mixtures of monoclonal antibodies?

Combining C121+C135 and C144+C135 during culture: "viral spread was apparently completely suppressed and no replication competent rVSV/SARS-CoV-2/GFP was detected in p2 cultures".

13/

Lesson?

Don't use a single monoclonal antibody therapeutically, use a mixture instead.

Also, weirdly, convalescent serum seem to sometimes (often?) have a dominant neutralization target.