Generation of Neutralizing Monoclonal Antibodies Against SARS-CoV-2 for Prevention and Therapy in Patients with COVID-19

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

1. Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants A. Generation and characterization of anti-SARS-CoV-2 mAbs using phage-display libraries from COVID-19 convalescent patients. Peripheral blood samples were collected from 12 convalescent COVID-19 patients with high neutralizing serum antibody titers against SARS-CoV-2, and total RNA was extracted and used for the construction of four phage-display Fab libraries, derived from either a single donor or multiple donors combined. Three sequential cycles of panning were carried out to enrich for specific clones using a stabilized trimeric spike protein (S-2P) derived from the original SARS-CoV-2 strain, Wuhan-Hu-1. We obtained 18 Fabs that exhibited high-affinity binding to the viral spike with equilibrium dissociation constants (KD) in the picomolar or low nanomolar range. B. Potent neutralization of diverse SARS-CoV-2 variants of concern. The neutralizing activity of our 18 mAbs was evaluated using a pseudotype virus neutralization assay. Eleven showed potent neutralizing activity against the original SARS-CoV-2 strain (WA-1) and the Alpha variant, while 7 also neutralized the Delta variant in the picomolar range. In contrast, only 4 of the 11 mAbs were effective against the Beta variant. Of note, one of them, NA8, neutralized Beta, Omicron BA.1 and BA.2 sublineages with a picomolar potency, as well as BA.2.12.1 and BA.4 sublineages at nanomolar concentration. Since clinically approved mAbs are generally used in combination to ensure coverage against multiple variants, we compared side-by-side the neutralization potency of the combination of NA8 and NE12, used at equimolar concentrations (1:1), with that of 6 clinically approved antibodies used in paired combinations. Against the most recent variants, BA.2.12.1 and BA.4, the NA8/NE12 combination retained neutralizing activity at low nanomolar concentrations as did the REGN-10933/REGN-10987 combination, while COV2-2130/COV2-2196 was the most potent combination and LY-CoV555/LY-CoV016 was ineffective. These results suggest that NA8 and NE12, when used in combination, are one of the most broad and potent antibody pairs with picomolar or low nanomolar neutralizing activity against the major SARS-CoV-2 variants of concern. Based on these results, NA8 and NE12 were selected for further characterization and preclinical testing. C. Structural analysis of antibodies NE12 and NA8. To gain structural insight into the interactions of antibodies NE12 and NA8 with the SARS-CoV-2 spike glycoprotein, in collaboration with Peter Kwong and Yaroslav Tsybovsky, we obtained cryo-EM reconstructions of Fab NE12 and Fab NA8 bound to a stabilized spike trimer (S-6P) at nominal resolutions of 3.1 and 2.9 , respectively. The epitope of NE12 overlaps the ACE2 receptor footprint, indicating that NE12 directly blocks the spike-receptor interaction. Similar to Fab NE12, Fab NA8 shows extensive contacts with the receptor-binding ridge, but this antibody binds to a unique epitope along the outer side of the RBD distal from the tip. There is a moderate overlap between the NA8 epitope and the ACE2-binding interface of the RBD. These structural data further confirm that NE12 and NA8 provide a pair of potent complementary mAbs for potential clinical use. D. Antibodies NE12 and NA8 protect hamsters from SARS-CoV-2 infection. In collaboration with Ulla Buchholz, we tested the in vivo prophylactic efficacy of mAbs NE12 and NA8 in the golden Syrian hamster model, which closely mimics the severity of the disease in humans. Treatment with NA8 significantly protected hamsters from weight loss induced by two viral variants, WA-1 and Beta, while NE12 was highly effective against the WA-1 strain. Accordingly, the viral titers were significantly reduced in both the lungs and the nasal turbinates. Overall, these data demonstrate that mAb NA8 exerted strong prophylactic protection in vivo from two antigenically distinct SARS-CoV-2 variants, and NE12 exerted strong prophylactic protection against the original WA-1 strain in a highly susceptible animal model, despite the relatively low antibody doses used. In summary, these results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants. E. Development of a universal neutralizing mAb against all SARS-CoV2 variants and other sarbecoviruses The identification of potent, broadly neutralizing antibodies against SARS-CoV-2 variants of concern that resist neutralization by most mAbs, such as the B.1.351/Beta and the Omicron subvariants, is critical for creating an arsenal of therapeutic antibodies with high potency against both present and future variants of concern that will continue to emerge because of the sustained worldwide spread of this virus, leading to escape from current prophylactic and therapeutic interventions. Capitalizing on the data so far obtained, now we are focusing on the development of universal mAbs that are able to neutralize not only SARS-CoV-2 but also SARS-CoV-1 and other sarbecoviruses from other animal species such as bats. These antibodies are extremely important to prepare for potential new coronavirus-related pandemics caused by spill over from animals to humans. 2. Identification of Circulating Anti-PF4 Antibodies Associated with Disease Severity in Patients with COVID-19 The pathogenic mechanisms that trigger the most severe complications of COVID-19, which are often fatal, are still largely unknown. Severe COVID-19 is frequently associated with thrombosis and thrombocytopenia, which are reminiscent of the clinical presentation of two life-threatening syndromes: heparin-induced thrombocytopenia (HIT), which develops in patients treated with high doses of heparin after cardiac or orthopedic surgery, and vaccine-induced thrombosis with thrombocytopenia (VITT), which is a rare complication of adenovirus-vectored COVID-19 vaccines. The pathogenic mechanism of both the HIT and VITT syndromes involves the elicitation of autoantibodies that target partially cryptic epitopes in platelet factor 4 (PF4 or CXCL4), which are fully revealed upon binding to heparin or other polyanionic molecules. Using a clinically validated immunoassay, we evaluated the presence of antibodies to PF4 in 100 hospitalized patients with COVID-19 with moderate or severe disease, 25 acute COVID-19 cases visiting the Emergency Department and 65 convalescent individuals. Anti-PF4 antibodies were detected in 95 of 100 (95.0%) hospitalized COVID-19 patients irrespective of prior heparin treatment, while patients hospitalized for severe acute respiratory disease unrelated to COVID-19 had markedly lower levels. Higher antibody levels were detected in males than in females, and in African Americans and Hispanics than in White Caucasians (Liu Q, et al. 2022). Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In convalescent individuals from COVID-19, the antibody levels returned to near-normal values. Sera from COVID-19 patients induced higher levels of platelet activation than sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19, and have implications for the therapeutic management of the most severe cases.