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.

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated mAbs against domain III of the WNV E protein. MAbs that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from human patients who had recovered from WNV infection also detected this epitope. One mAb, E16, neutralized 10 different strains in vitro, and demonstrated therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity, and neutralizing activity. In post-exposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and thus, may be a viable treatment option against WNV infection in humans.WNV is a single-stranded, positive-polarity RNA Flavivirus that is related to dengue fever, yellow fever, and Saint Louis, tick-borne, and Japanese encephalitis viruses. Humans infected with WNV develop a febrile illness that can progress to meningitis or encephalitis, and the elderly and immunocompromised are at greatest risk for severe disease 1 . At present, treatment is supportive and no vaccine exists for human use.The innate and adaptive immune responses prevent dissemination to and within the central nervous system (CNS) 2,3 . Recently, two groups demonstrated therapeutic efficacy of immune human γ-globulin in mice infected with WNV 4,5 . Even after virus had spread to the CNS, passive administration of immune heterologous γ-globulin improved survival 5 . In theory, a potently neutralizing mAb could have the same or better benefit with a lower dose and improved safety profile.Most neutralizing antibodies against flaviviruses recognize the envelope (E) protein. In general, virus-specific rather than cross-reactive antibodies have the strongest neutralizing activity in NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript vitro and greatest protection in vivo 6 . Crystallographic analysis of the soluble ectodomain of flavivirus E proteins has revealed three domains 7,8 . Domain I is an 8-stranded β-barrel 7-9 that participates in the conformational changes associated with the acidification in the endosome 10 . Domain II contains 12 β-strands and has roles in dimerization, trimerization, and fusion 7,8,10 . Domain III (DIII) adopts an immunoglobulin-like fold, and contains the loops that are most distal from the surface in the mature virion 11,12 and the site for putative receptor attachment 6,8,13,14 . Based on the sequencing of in vitro neutralization escape variants, many neutralizing antibodies against flaviviruses localize to DIII 15-22 .Here, we define further the molecular basis of antibody-mediated neutralization of WNV using a large panel of newly generated mAbs against WNV E protein. Humanized versions o...

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.

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The flavivirus nonstructural protein NS1 is a highly conserved secreted glycoprotein that does not package with the virion. Immunization with NS1 elicits a protective immune response against yellow fever, dengue, and tick-borne encephalitis flaviviruses through poorly defined mechanisms. In this study, we purified a recombinant, secreted form of West Nile virus (WNV) NS1 glycoprotein from baculovirus-infected insect cells and generated 22 new NS1-specific monoclonal antibodies (MAbs). By performing competitive binding assays and expressing truncated NS1 proteins on the surface of yeast (Saccharomyces cerevisiae) and in bacteria, we mapped 21 of the newly generated MAbs to three NS1 fragments. Prophylaxis of C57BL/6 mice with any of four MAbs (10NS1, 14NS1, 16NS1, and 17NS1) strongly protected against lethal WNV infection (75 to 95% survival, respectively) compared to saline-treated controls (17% survival). In contrast, other anti-NS1 MAbs of the same isotype provided no significant protection. Notably, 14NS1 and 16NS1 also demonstrated marked efficacy as postexposure therapy, even when administered as a single dose 4 days after infection. Virologic analysis showed that 17NS1 protects at an early stage in infection through a C1q-independent and Fc ␥ receptor-dependent pathway. Interestingly, 14NS1, which maps to a distinct region on NS1, protected through a C1q-and Fc ␥ receptor-independent mechanism. Overall, our data suggest that distinct regions of NS1 can elicit protective humoral immunity against WNV through different mechanisms.West Nile virus (WNV) is a single-stranded, positive-senseenveloped RNA virus that is maintained in nature through a mosquito-bird-mosquito transmission cycle. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks now occur annually in North America. Humans, which are dead-end hosts, can develop a febrile illness that progresses to a meningitis or encephalitis syndrome (32). At present, treatment is supportive, and no vaccine exists for human use.A member of the Flaviviridae family, WNV is closely related to other major human pathogens such as yellow fever (YF), dengue (DEN), tick-borne encephalitis (TBE), Japanese encephalitis (JEV), and Murray Valley encephalitis (MVE) viruses. The 10.7-kilobase genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by both virus-and host-encoded proteases (5). The NS proteins include an RNA-dependent RNA polymerase (NS5), a helicase/protease (NS3), and other proteins that form part of the viral replication complex (36, 37).NS1 is a highly conserved 48-kDa glycoprotein with 12 invariant cysteine residues. Although the disulfide linkage arrangement of MVE and DEN NS1 has been described (4, 66), structural analysis is currently lacking. NS1 is inserted into the lumen of the endoplasmic reticulum via a signal peptide that is cleaved cotranslationally by a cellular signalase to gene...