SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

13SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determined the 14 X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient 15 infected with SARS-CoV-2, in complex with the receptor binding domain (RBD). The structure reveals 16 CV30's epitope overlaps with the human ACE2 receptor binding site thus providing the structural basis 17 for its neutralization by preventing ACE2 binding. 18 19 20

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%...

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and eleven bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

SUMMARY Epstein-Barr virus (EBV) is a cancer-associated pathogen for which there is no vaccine. Successful anti-viral vaccines elicit antibodies that neutralize infectivity; however, it is unknown whether neutralizing antibodies prevent EBV acquisition. Here we assessed whether passively delivered AMMO1, a monoclonal antibody that neutralizes EBV in a cell-type-independent manner, could protect against experimental EBV challenge in two animal infection models. When present prior to a high-dose intravenous EBV challenge, AMMO1 prevented viremia and reduced viral loads to nearly undetectable levels in humanized mice. AMMO1 conferred sterilizing immunity to three of four macaques challenged orally with rhesus lymphocryptovirus, the EBV ortholog that infects rhesus macaques. The infected macaque had lower plasma neutralizing activity than the protected animals. These results indicate that a vaccine capable of eliciting adequate titers of neutralizing antibodies targeting the AMMO1 epitope may protect against EBV acquisition and are therefore highly relevant to the design of an effective EBV vaccine.