Highlights d Early B cell responses to SARS-CoV-2 spike protein are analyzed from a COVID-19 patient d Most antibodies target non-neutralizing epitopes outside the RBD d A potent neutralizing mAb blocks the interaction of the S protein with ACE2 d Neutralizing antibodies are minimally mutated

SUMMARY Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1–2*02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.

22B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a 23 COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one 24 days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three 25 of which bound to the receptor binding domain (RBD). The antibodies were minimally 26 mutated from germline and were derived from different B cell lineages. Only two 27 antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most 28 potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, 29 while the other bound outside the RBD. Our study indicates that the majority of antibodies 30 against the viral envelope spike that were generated during the first weeks of COVID-19 31 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt 32 the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without 33 undergoing extensive maturation. Such antibodies have potential preventive/therapeutic 34 potential and can serve as templates for vaccine-design. 35 36 37 38 39 40 41 42 43 44 45 KEY WORDS 46 COVID-19, SARS, SARS-CoV-2, antibody, B cells, spike protein, receptor binding 47 RESULTS 105 Serology 106Serum and PBMC were collected twenty-one days after the onset of clinical disease. The 107 serum contained high titers of antibodies to the SARS-CoV-2 S2P (Fig. 1A). The 108 specificity of this response was confirmed by the absence of S2P reactivity by serum 109 antibodies isolated from donors collected prior to the SARS-CoV-2 pandemic, or donors 110 with confirmed infection by endemic coronaviruses. We also measured the serum 111 antibody response to RBD, and again observed specific high titers of binding antibodies 112 ( Fig. 1B). Isotype-specific ELISA revealed that the IgG titers were higher than the IgA and 113 the IgM titers to both S2P and RBD, suggesting a significant portion of the antibody 114 responses to SARS-CoV-2 S are IgG (Fig. 1C and D). The serum from the SARS-CoV-2 115 infected donor displayed potent neutralizing activity (Reciprocal ID50~3000)against a 116 pseudovirus expressing the S protein from SARS-CoV-2 isolate Wuhan-Hu-1 ( Fig 1E). 117 We concluded that this donor had developed strong binding and neutralizing antibody 118 responses within three weeks of disease onset. 119 B cell sorts and VH/VL sequencing 120 Fluorescently labeled S2P and RBD probes were used as baits to identify B cells specific 121 to the SARS-CoV-2 S protein that were circulating at this timepoint. S2P was labeled with 122 either phycoerythrin (PE) or brilliant violet 711 (BV711) and used to stain B cells 123 concurrently. This double labeling strategy helps to discriminate between bona fide S2P-124 specific B cells and non-specific background staining to the fluorophores. RBD was 125 labeled with alexa fluor 647 to identify B cells specific for that domain. : bioRxiv preprint 7Approximately 0.65% of total CD19+ B cells were S2P positive compared to 0.07%...