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

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%...

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

Background: 3,4-Dihydroxy-2-butanone-4-phosphate synthase (DHBPS) is essential for many pathogens and is absent in humans. Results: We have characterized DHBPS from Vibrio cholerae in the presence of substrate D-ribulose 5-phosphate (Ru5P) and inhibitor 4-phospho-D-erythronohydroxamic acid (4PEH). Conclusion: 4PEH inhibits DHBPS competitively and interacts with enzyme similarly to the substrate. Significance: 4PEH can be used as a lead molecule for designing novel antibiotics.

Abstractβ-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.

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Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two nonoverlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.