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Antibodies targeting the spike protein of SARS-CoV-2 present a promising approach to combat the COVID19 pandemic; however, concerns remain that mutations can yield antibody resistance. We investigate the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared following in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Importantly, escape mutants were not generated following treatment with a non-competing antibody cocktail.

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola – one from genetically-humanized mice, and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the SARS-CoV-2 spike protein, yielding a large collection of fully-human antibodies that were characterized for binding, neutralization and three dimensional structure. Based on these criteria, we selected pairs of highly-potent individual antibodies that simultaneously bind the receptor-binding domain of the spike protein, providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single antibody treatment.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact, and that the conformation is shifted towards an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.

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Traditional approaches to antimicrobial drug development are poorly suited to combatting the emergence of novel pathogens. Additionally, the lack of small animal models for these infections hinders the in vivo testing of potential therapeutics. Here we demonstrate the use of the VelocImmune technology (a mouse that expresses human antibody-variable heavy chains and κ light chains) alongside the VelociGene technology (which allows for rapid engineering of the mouse genome) to quickly develop and evaluate antibodies against an emerging viral disease. Specifically, we show the rapid generation of fully human neutralizing antibodies against the recently emerged Middle East Respiratory Syndrome coronavirus (MERS-CoV) and development of a humanized mouse model for MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of the isolated antibodies. The VelocImmune and VelociGene technologies are powerful platforms that can be used to rapidly respond to emerging epidemics.

BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy to prevent coronavirus disease 2019 (COVID-19). Recently, we reported preliminary BNT162b2 safety and antibody response data from an ongoing placebo-controlled, observer-blinded phase 1/2 vaccine trial1. We present here antibody and T cell responses from a second, non-randomized open-label phase 1/2 trial in healthy adults, 19-55 years of age, after BNT162b2 prime/boost vaccination at 1 to 30 µg dose levels. BNT162b2 elicited strong antibody responses, with S-binding IgG concentrations above those in a COVID-19 human convalescent sample (HCS) panel. Day 29 (7 days post-boost) SARS-CoV-2 serum 50% neutralising geometric mean titers were 0.3-fold (1 µg) to 3.3-fold (30 µg) those of the HCS panel. The BNT162b2-elicited sera neutralised pseudoviruses with diverse SARS-CoV-2 S variants. Concurrently, in most participants, S-specific CD8+ and T helper type 1 (TH1) CD4+ T cells had expanded, with a high fraction producing interferon-γ (IFNγ). Using peptide MHC multimers, the epitopes recognised by several BNT162b2-induced CD8+ T cells when presented on frequent MHC alleles were identified. CD8+ T cells were shown to be of the early-differentiated effector-memory phenotype, with single specificities reaching 0.01-3% of circulating CD8+ T cells. In summary, vaccination with BNT162b2 at well tolerated doses elicits a combined adaptive humoral and cellular immune response, which together may contribute to protection against COVID-19.

AbstractVirus genome sequence variants that appear over the course of an outbreak can be exploited to map the trajectory of the virus from one susceptible host to another. While such variants are usually of no functional significance, in some cases they may allow the virus to transmit faster, change disease severity, or confer resistance to antiviral therapies. Since the discovery of SARS-CoV-2 as the cause of COVID-19, the virus has spread around the globe, and thousands of SARS-CoV-2 genomes have been sequenced. The rate of sequence variation among SARS-CoV-2 isolates is modest for an RNA virus but the enormous number of human-to-human transmission events has provided abundant opportunity for selection of sequence variants. Among these, the SARS-CoV-2 Spike protein variant, D614G, was not present in the presumptive common ancestor of this zoonotic virus but was first detected in late January in Germany and China. The D614G variant steadily increased in frequency and now constitutes >97% of isolates world-wide, raising the question whether D614G confers a replication advantage to SARS-CoV-2. Structural models predict that D614G would disrupt contacts between the S1 and S2 domains of the Spike protein and cause significant shifts in conformation. Using single-cycle vectors we showed that D614G is three to nine-fold more infectious than the ancestral form on human lung and colon cell lines, as well as on other human cell lines rendered permissive by ectopic expression of human ACE2 and TMPRSS2, or by ACE2 orthologues from pangolin, pig, dog, or cat. Nonetheless, monoclonal antibodies targeting the receptor binding domain of the SARS-CoV-2 Spike protein retain full neutralization potency. These results suggest that D614G was selected for increased human-to-human transmission, that it contributed to the rapidity of SARS-CoV-2 spread around the world, and that it does not confer resistance to antiviral therapies targeting the receptor binding domain.