Anti-yellow fever virus antibodies and methods for their production and use

Abstract

To provide antibodies and antigen-binding fragments thereof specific to the Yellow Fever Virus (YFV) E protein and with neutralizing potency against YFV, wherein these antibodies and antigen-binding fragments are useful in treating YFV.SOLUTION: Provided are antibodies and antigen-binding fragments thereof that bind to YFV protein and exhibit neutralizing activity, in particular antibodies that bind to domain III (DIII) of the E protein and exhibit high neutralization potency. The antibodies of the present invention may also cross-react with other flaviviruses, for example, displaying binding reactivity to DENV-2, DENV-4, WNV, and / or ZIKV E proteins. The panel of antibodies described herein provides promising therapeutic candidates and a framework for the rational design of YFV vaccines.SELECTED DRAWING: Figure 1a

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 940,049, filed November 25, 2019, which is incorporated herein by reference in its entirety.

[0002] AN ASCII file containing a "Sequence Listing," table, or computer program listing appendix reference The sequence listing set forth in file MAB-501001WO_SequenceListing_ST25.txt, 493,241 bytes, machine format IBM-PC, MS Windows operating system, created on November 24, 2020, is incorporated herein by reference.

[0003] The present disclosure relates to anti-yellow fever virus (YFV) antibodies and antigen-binding fragments thereof, compositions containing such antibodies and antigen-binding fragments thereof, and therapeutic and diagnostic uses for the antibodies, antigen-binding fragments, and compositions. [Background technology]

[0004] Yellow fever virus (YFV) is a mosquito-borne flavivirus found in tropical and subtropical regions of Africa and South America. It is transmitted to humans primarily through the bite of infected Aedes or Haemagogus mosquitoes and has three distinct transmission cycles: 1) the jungle or forest cycle, 2) the African savanna (intermediate) cycle, and 3) the urban cycle (www.cdc.gov / yellowfever / transmission / index.html). While many people infected with YFV are asymptomatic, others develop symptoms following a 3-6 day incubation period, including fever, chills, headache, backache, muscle pain, loss of appetite, nausea, vomiting, and / or fatigue (www.who.int / news-room / fact-sheets / detail / yellow-fever). Approximately 15% of infected individuals develop severe YFV symptoms, including high fever, bleeding diathesis, abdominal pain, renal failure, cardiovascular instability, and liver failure, and up to 50% of patients with severe YFV will die (McGuinness et al, Neurohospitalist 2017,7(4);157-158).

[0005] YFV has an RNA genome of 10,862 nucleotides that encodes three structural proteins and seven nonstructural proteins. From the 5' end, the encoded proteins are C, prM / M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The three structural proteins include the C (capsid) protein, the membrane protein M, and the envelope protein E. The envelope proteins play important roles in cell tropism, pathogenicity, and immunity.

[0006] The live-attenuated 17D vaccine is considered one of the safest and most effective vaccines ever developed. However, despite the availability of the vaccine, yellow fever remains a serious public health problem. Some data suggest that, while protective, immunity may wane over time in certain populations. Additionally, outbreaks of YFV in non-endemic countries (such as the 11 imported cases in China in 2016) and concurrent outbreaks that deplete 17D stockpiles highlight the importance of developing treatments.

[0007] Indeed, to date, there is currently no approved YFV treatment (the only available treatment is supportive care), and despite decades of research, the development of safe and effective therapeutic antibodies against YFV remains elusive. YFV E-specific serum antibody responses have been shown to be predominantly mediated by antibodies targeting domain I (DI) and / or domain II (DII) of the E protein, while antibodies targeting domain III (DIII) are absent or present at very low titers (DVratskikh et al. PLoS pathogens 9, e1003458 (2013)). Correspondingly, all six YFV E-specific human monoclonal antibodies described to date target overlapping epitopes within the DIII of the E protein (Lu et al. Cell Reports 26, 438-446 e435 (2019); Daffis et al. Virology 337, 262-272 (2005)). Recently, the crystal structure of one of these mAbs (5A) in complex with a soluble YFV E dimer was determined, showing that this mAb binds to a conserved neutralizing epitope within the D1I of one E monomer (Lu et al. Cell Reports 26, 438-446 e435 (2019)). Thus, there remains a need for highly specific, high-affinity, and potent neutralizing anti-YFV antibodies and their antigen-binding fragments. Summary of the Invention

[0008] The present disclosure relates to the discovery of antibodies and antigen-binding fragments thereof that bind to YFV proteins and exhibit neutralizing activity, particularly antibodies that bind to domain III (DIII) of the E protein, which exhibit high neutralizing activity. The antibodies of the present disclosure may also cross-react with other flaviviruses, e.g., DENV-2, DENV-4, WNV, and / or ZIKV E proteins, exhibiting binding reactivity. A broad panel of YFV-specific monoclonal antibodies is described. Binding studies demonstrated that neutralizing antibody responses to YFV-17D are primarily mediated by antibodies that recognize FL-proximal epitopes within the DIII of the YFV E protein. A small set of DIII-targeting antibodies with potent neutralizing activity was also identified. In addition, binding assays revealed that YFV-17D vaccination appears to induce a subset of antibodies that exhibit broad flavivirus binding activity, the majority of which target the highly conserved FL and exhibit little or no cross-neutralizing activity. Neutralization studies demonstrated that a proportion of antibodies exhibit very potent neutralizing activity. Overall, the panel of antibodies described herein provides a framework for the rational design of potential therapeutic candidates and YFV vaccines.

[0009] Such antibodies may be useful when administered prophylactically (prior to exposure to and infection with the virus) to reduce the severity or duration of primary infection with YFV or to ameliorate at least one symptom associated with the infection. The antibodies may be used alone or in conjunction with a second agent useful for treating YFV infection. In certain embodiments, the antibodies may be given therapeutically (after exposure to and infection with the virus), either alone or in conjunction with a second agent, to reduce the severity or duration of primary infection or to ameliorate at least one symptom associated with the infection. In certain embodiments, the antibodies may be used prophylactically as a stand-alone treatment to protect patients at risk of contracting infection with YFV, such as those described above. Any of these patient populations may benefit from treatment with the antibodies of the present disclosure, either alone or when given in conjunction with a second agent, including, for example, antiviral therapy or other antiviral vaccines.

[0010] In certain embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to YFV is provided, wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of such an antibody or antigen-binding fragment is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween, to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 3 of an antibody selected from antibody numbers 1 to 152 disclosed in Table 3.

[0011] The antibody or antigen-binding fragment thereof may also have one or more of the following properties: a) the antibody or antigen-binding fragment thereof exhibits a clean or low polyreactivity profile, b) the antibody or antigen-binding fragment thereof exhibits an in vitro neutralization capacity (IC50) of about 0.5 micrograms / milliliter (μg / ml) to about 5 μg / ml, about 0.05 μg / ml to about 0.5 μg / ml, or less than about 0.05 mg / ml, c) the antibody or antigen-binding fragment thereof binds to YFV-17D particles, or d) the antibody or antigen-binding fragment thereof binds to the envelope protein of YFV. In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises at least two, at least three, or four of the above properties a) to d).

[0012] In certain other embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a) the CDRH1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; b) the CDRH2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; c) the CDRH3 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; d) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; f) the CDRL3 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; and / or g) any combination of two or more of a), b), c), d), e), and f).

[0013] In certain other embodiments, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of antibodies that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween to at least one of the antibodies designated Antibody No. 1 through Antibody No. 152 disclosed in Table 3.

[0014] In certain other embodiments, the isolated antibody or antigen-binding fragment thereof comprises a) the heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3, and / or b) the light chain (LC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0015] The present disclosure also contemplates nucleic acids encoding the described anti-YFV antibodies and expression vectors comprising the nucleic acids, as well as host cells that express such antibodies via the nucleic acids and / or expression vectors.

[0016] In one embodiment, an isolated nucleic acid sequence encoding an antibody or antigen-binding fragment thereof disclosed herein is provided.

[0017] In other embodiments, an expression vector is provided comprising an isolated nucleic acid sequence encoding an antibody or antigen-binding fragment disclosed herein.

[0018] In other embodiments, a host cell transfected, transformed, or transduced with a nucleic acid sequence encoding an antibody or antigen-binding fragment disclosed herein, or an expression vector comprising an isolated nucleic acid sequence encoding an antibody or antigen-binding fragment disclosed herein, is provided.

[0019] In other embodiments, pharmaceutical compositions are provided comprising one or more of the isolated antibodies or antigen-binding fragments thereof disclosed herein and a pharmaceutically acceptable carrier and / or excipient.

[0020] In other embodiments, pharmaceutical compositions are provided comprising one or more nucleic acid sequences encoding the antibodies or antigen-binding fragments disclosed herein, or one or more expression vectors comprising nucleic acid sequences encoding the antibodies or antigen-binding fragments disclosed herein, and a pharmaceutically acceptable carrier and / or excipient.

[0021] In other embodiments, an expression vector comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment disclosed herein, or a host cell comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment disclosed herein, is provided.

[0022] The present disclosure further contemplates methods of prevention and / or treatment using the described anti-YFV antibodies (or nucleic acids encoding or expression vectors containing such nucleic acids).

[0023] In one embodiment, a method of treating or preventing Yellow Fever Virus (YFV) infection or at least one symptom associated with a YFV infection is provided, comprising administering to a patient in need of or suspected of needing treatment or prevention, such that the YFV infection is treated or prevented or at least one symptom associated with a YFV infection is treated, alleviated, or reduced in severity, a) one or more antibodies or antigen-binding fragments thereof according to other embodiments disclosed herein; b) one or more nucleic acid sequences encoding one or more antibodies or antigen-binding fragments thereof disclosed herein, an expression vector comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment disclosed herein, or a host cell comprising an expression vector comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment disclosed herein; or c) a pharmaceutical composition according to other embodiments disclosed herein.

[0024] In other embodiments, the method further comprises administering to the patient a second therapeutic agent.

[0025] In embodiments, the second therapeutic agent is selected from an antiviral drug, a vaccine specific for YFV, a vaccine specific for a flavivirus, an siRNA specific for a YFV antigen, and a secondary antibody specific for a YFV antigen.

[0026] In certain embodiments, pharmaceutical compositions are provided for use in preventing YFV infection in a patient in need of prevention or suspected of needing prevention, or for treating a patient suffering from YFV infection, or for ameliorating at least one symptom or complication associated with the infection, wherein such use results in either the infection being prevented, or at least one symptom or complication associated with the infection being prevented, ameliorated, or reduced in severity and / or duration. In certain embodiments, pharmaceutical compositions are provided for use in preventing YFV infection in a patient in need of prevention or suspected of needing prevention. In certain embodiments, pharmaceutical compositions are provided for use in treating a patient suffering from YFV infection. In certain embodiments, pharmaceutical compositions are provided for use in ameliorating at least one symptom or complication associated with the infection. In certain embodiments, the infection is prevented. In certain embodiments, at least one symptom or complication associated with the infection is prevented, ameliorated, or reduced in severity and / or duration as a result of such use.

[0027] In certain embodiments, pharmaceutical compositions are provided for use in treating or preventing YFV infection, or at least one symptom associated with said YFV infection, in a patient in need of or suspected of being in need of treatment or prevention, wherein such use results in either prevention of the infection, or prevention, or prevention, or reduction in severity and / or duration of at least one symptom or complication associated with the infection.

[0028] In certain other embodiments, there is provided the use of a pharmaceutical composition in the manufacture of a medicament for preventing YFV infection in a patient in need thereof, or for treating a patient suffering from YFV infection, or for ameliorating at least one symptom or complication associated with the infection, wherein the infection is prevented, or at least one symptom or complication associated with the infection is either prevented, ameliorated, or reduced in severity and / or duration.

[0029] In certain other embodiments, there is provided the use of a pharmaceutical composition in the manufacture of a medicament for preventing YFV infection, or at least one symptom associated with said YFV infection, in a patient in need of or suspected of needing prevention, wherein such use results in either the infection being prevented, or at least one symptom or complication associated with the infection being prevented, ameliorated, or reduced in severity and / or duration.

[0030] In certain other embodiments, an antibody that binds to the YFV E protein is provided. The antibody can bind to at least one of an epitope within the FL of domain II of the YFV E protein, a protein proximal to the FL of domain II of the YFV E protein, and a protein within domain III of YFV. The antibody can also have one or more of the following properties: a) the antibody or antigen-binding fragment thereof exhibits a clean or low polyreactivity profile; b) the antibody or antigen-binding fragment thereof exhibits an in vitro neutralization capacity (IC50) of about 0.5 micrograms per milliliter (μg / ml) to about 5 μg / ml, about 0.05 μg / ml to about 0.5 μg / ml, or less than about 0.05 mg / ml; c) the antibody or antigen-binding fragment thereof binds to YFV-17D particles; and d) the antibody or antigen-binding fragment thereof binds to the envelope protein of YFV. [Brief explanation of the drawings]

[0031] [Figure 1a-1b] Illustrated are donor serum analyses following YFV-14D vaccination. Figure 1a: Serum neutralizing activity against YFV-17D on day -5 (pre-vaccination), 10, 14, 28, 90, 180, 270, and 360 days post-vaccination. Mean ± SD (n=6) from two independent experiments is shown. Figure 1b: Neutralization IC50 of serum samples at each time point post-vaccination, expressed as reciprocal serum dilution. [Figures 2a-2d]Figure 2 shows the characterization of YFV-17D vaccination-induced plasmablast responses on days 10 and 14. Figure 2a: Frequency of plasmablasts among CD19+CD20- / lo B cells in peripheral blood on days 0, 10, and 14 post-vaccination. Plasmablasts are defined herein as CD19+CD3-CD8-CD14-CD16-CD20- / loCD38hiCD27hi cells. Figure 2b: Percentage of PB-derived mAbs that showed ELISA binding reactivity to whole YFV-17D particles at 100 nM. Figure 2c: Neutralizing activity of PB-derived mAbs against YFV-17D at 100 nM and 10 nM concentrations. Green dots indicate the number of nucleotide substitutions in VH+VL. Figure 2d: Percentage of YFV-17D-reactive PB-derived mAbs with the indicated neutralizing potency (IC50). [Figure 3] Figure 1 illustrates the binding activity of germline-reverted plasmablast monoclonal antibodies. Binding traces and affinities of three somatically mutated PB-derived mAbs (ADI-46184, ADI-46185, and ADI-42168) and their corresponding UCAs as determined by Biacore. UCA is the unmutated common ancestor. [Figure 4] Neutralization screening of PB-derived mAbs. Representative YFV-17D neutralization titration curves of PB mAbs screened by microtiter neutralization assay. Means ± SD (n=6) from two independent experiments are shown. [Figure 5a-5b] Figure 5a shows the presence of swIg+ B cells showing reactivity to YFV-17D. Figure 5a: YFV E reactivity of swIg+ B cells at each sampling time point. Fluorescence-activated cell sorting (FACS) plots shown are gated on CD19+CD20+IgD-IgM- B cells. YFV E was labeled with two different colors to reduce background binding. Figure 5b: Percentage of swIg+ B cells showing YFV E reactivity at each sampling time point. [Figures 6a-6e]Figure 6 illustrates that YFV E-specific antibodies exhibit preferential usage of the VH3-72 germline gene. Figure 6a: VH germline gene usage of YFV E-specific mAbs isolated from each sampling time point. The VH germline gene frequency of the unselected human MBC repertoire ("Unselected") is also included for comparison. Sequencing data for unselected human MBCs was obtained from multiple high-throughput sequencing studies. Figure 6b: VL germline gene usage of mAbs utilizing the VH3-72 germline gene. MAbs from all sampling time points were pooled for this analysis. The number in the center of the pie chart represents the total number of VH3-72 mAbs. Figure 6c: CDR H3 length distribution in YFV E-specific mAbs utilizing the VH3-72 germline gene, mAbs utilizing all other VH germline genes, or unselected Abs from MBCs. Figure 6d: SHM burden (expressed as the number of nucleotide substitutions in VH) of YFV E-specific mAbs utilizing the VH3-72 germline gene or all other VH germline genes. Figure 6e: Apparent binding affinity to YFV E protein of mAbs utilizing the VH3-72 germline gene or all other VH germline genes as determined by BLI. Black bars indicate median values. Affinity KDApp is plotted for mAbs isolated from day 14 MBCs because only a small fraction of these mAbs showed detectable binding to YFV E in the monovalent orientation. Statistical comparisons were performed using the Mann-Whitney test (***P<0.001, **P<0.01, *P<0.05). [Figures 7a-7d]Figure 7 illustrates that antibodies targeting epitopes within or proximal to the FL dominate memory B cell responses to YFV-17D vaccination. Figure 7a: Percentage of mAbs within each of the major competitor groups at each sampling time point. Figure 7b: VH3-72 utilizing mAbs are shaded according to competitor group, and the naturally paired light chain germline gene is indicated. Figure 7c: Percentage of mAbs competing with 4G2 and using the VH3-72 germline gene. Figure 7d: Apparent affinity of 4G2-competing mAbs using either the VH3-72 germline gene or all other germline genes. Statistical comparisons were performed using the Mann-Whitney test (**P<0.01). [Figures 8a-8d] Figure 8a illustrates that the majority of highly potent neutralizing antibodies recognize FL-proximal epitopes. Figure 8a: Percentage of mAbs with neutralizing IC50s against YFV-17D (<1, 1-10, >10-100, >100 nM) in each epitope bin (non-neutralizing - non-binding). Figure 8b: Neutralizing IC50s of individual mAbs against YFV-17D across the indicated epitope bins. Black bars indicate median values. Figure 8c: Percentage of highly potent neutralizing antibodies (IC50 <1 nM) targeting the indicated antigenic site on YFV E. Numbers in the center of the pie chart indicate the number of highly potent neutralizing antibodies. Figure 8d: Use of VH and VL germline genes of 5A alone or 5A / ADI-45107 competing neutralizing antibodies. MAbs from both donors were combined for all analyses shown. [Figures 9a-9c]Figure 9 shows that a subset of monoclonal antibodies exhibits broad flavivirus cross-reactivity. Figure 9a: Percentage of mAbs reacting with one or more of the flavivirus E proteins tested (YFV, DENV-1, DENV-2, ZIKV, and WNV). Recombinant E protein binding was measured in an affinity-directed manner by BLI. The numbers in the center of the pie chart indicate the number of mAbs analyzed. Figure 9b: Percentage of cross-reactive mAbs recognizing the indicated antigenic site. Cross-reactive mAbs from both donors were combined for this analysis. Figure 9c: Heatmap showing the cross-reactivity profiles of 50 mAbs that showed binding to at least one flavivirus E protein other than YFV E. Apparent affinity (KDApp) was determined in an affinity-directed manner using BLI. Heatmaps showing virus neutralization activity against YFV-17D and ZIKV are shown below the binding heatmap. Competitor group assignments for individual mAbs are shown above the heatmap. NB is non-binding, nn is non-neutralizing, and neut. is neutralizing. DETAILED DESCRIPTION OF THE INVENTION

[0032] A deeper understanding of human antibody responses to YFV infection will aid in the development and evaluation of YFV vaccines and therapeutic and / or prophylactic antibodies for the treatment and / or prevention of YFV infection. A high-throughput antibody isolation platform was used to dissect the human memory B cell response to YFV in two vaccinated adult donors, and highly potent and selective YFV-neutralizing antibodies were isolated and characterized.

[0033] High-throughput epitope mapping studies revealed that epitopes within or proximal to the FL on the D1I of the YFV E protein are immunodominant. While many mAbs that bound to FL-specific epitopes were non-neutralizing, most mAbs targeting FL-proximal epitopes overlapping with the 5A epitope exhibited neutralizing activity. Furthermore, the majority of potent nAbs recognized this antigenic site, suggesting that the nAb responses induced by YFV-17D vaccination were primarily mediated by this class of Abs. A subset of these mAbs exhibited exceptionally potent neutralizing activity, with IC50 values ​​approximately 10-fold lower than those of previously described YFV mAbs. Given the recent YFV outbreaks in Brazil and the Democratic Republic of the Congo, coupled with the limited supply of YFV-17D vaccine and the lack of effective treatments for YFV disease, these mAbs represent promising candidates for prophylaxis and / or therapy.

[0034] Thus, disclosed herein are highly selective and potent anti-YFV antibodies for the treatment and / or prevention of YFV infection, as well as potential vaccine candidates. In addition, the disclosed reagents provide a set of useful tools for the evaluation of clinical trials, which will be important for selecting the optimal YFV vaccination or antibody-based treatment strategy from those currently under investigation.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0036] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0038] The term "about," when used before a numerical designation, such as temperature, time, amount, concentration, and the like, including ranges, indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, when used in reference to an amount, the term "about" means that the amount may vary by + / - 10%.

[0039] "Comprising" or "comprising" is intended to mean that compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, shall mean excluding any other elements that are essential to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" shall mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0040] "Yellow fever virus," also referred to as "YFV," is an RNA virus that is typically spread by the bite of an infected Aedes or Haemagogus species of mosquito.

[0041] The term "YFV-17D" refers to an attenuated YFV vaccine strain developed by passaging the wild-type Asibi strain in chicken and mouse tissues. There are currently three 17D substrains in production: 17DD produced in Brazil, 17D-213 produced in Russia, and 17D-204 produced in China, France, Senegal, and the United States. While the mechanism of attenuation is poorly understood, the limited genetic diversity of the 17D vaccine virus is hypothesized to contribute to the vaccine's attenuation and safety. There is evidence that 17D replication is less error-prone than wild-type RNA viruses. See Pugachev et al., J Virol. 78(2):1032-8 (2004).

[0042] The term "envelope protein" or "E protein" refers to the structural YFV protein, a primary immunogen that plays a central role in receptor binding and membrane fusion. The structure of the E protein extracellular domain (the soluble N-terminal portion consisting of 395 residues) contains three distinct structural domains designated domains I, II, and III. (Volk et al., Virology 2009, 394(1):12-18) Domain II contains a highly conserved SS-bridge stabilization loop at its distal end that functions as a fusion loop (FL). Upon virus entry into target host cells, the FL of domain II is exposed and inserted into the host cell membrane. (Zhang et al., Viruses 2017, 9(11):338) In some embodiments, antibodies and antigen-binding fragments thereof bind to the FL of domain II YFV E protein. In other embodiments, antibodies and antigen-binding fragments thereof bind to domain III of YFV E protein.

[0043] The development of effective YFV treatments has presented several unique challenges. The detailed analysis of human antibody responses to YFV vaccines performed here provides insights for the development of such therapeutic treatments. The antibody repertoire analysis disclosed herein reveals that while the majority of neutralizing YFV-specific antibodies target FL-proximal epitopes that overlap with the 5A epitope, a small number of potently neutralizing antibodies target the DIII domain, a region of the E protein that has not previously been an epitope for any effective anti-YFV antibodies.

[0044] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B and / or T cells respond. It also refers to the region of an antigen bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural and / or specific charge characteristics. The term "antibody" (or "Ab") as used herein is intended to refer to an immunoglobulin molecule (i.e., a "full antibody molecule") composed of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers thereof (e.g., IgM) or antigen-binding fragments thereof.

[0045] As used herein, the terms "antigen-binding portion," "antigen-binding fragment," and equivalents include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. In certain embodiments, the term "antigen-binding portion" or "antibody fragment" as used herein refers to one or more fragments of an antibody that retain the ability to bind to YFV.

[0046] Antibody fragments can include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard techniques, such as, for example, proteolysis or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0047] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular molecule immunopharmaceuticals (SMIPs), and other genetically engineered molecules such as shark variable IgNAR domains are also encompassed within the term "antigen-binding fragment" as used herein.

[0048] Antigen-binding fragments of antibodies will typically contain at least one variable domain, which may be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L V associated with the domain H For antigen-binding fragments containing domains, V H and V L The domains can be arranged relative to each other in any suitable arrangement. For example, the variable region is a dimer and has a V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0049] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a VH -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (V)V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -CL, (viii)V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -CH1-CH2-CH3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to each other or can be linked by a full or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which provides a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure can be linked to each other (e.g., by disulfide bonds) and / or to one or more monomeric V H Or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, covalently linking the domains.

[0050] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in conjunction with the antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0051] Each heavy chain comprises a heavy chain variable region ("HCVR" or "V H ") and heavy chain constant region (domain C H 1. C H 2, and C H Each light chain is composed of a light chain variable region ("LCVR" or "V L ") and the light chain constant region (C L ). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs. Thus, the CDRs in the heavy chain are designated "CDRH1," "CDRH2," and "CDRH3," respectively, and the CDRs in the light chain are designated "CDRL1," "CDRL2," and "CDRL3."

[0052] In some embodiments, the antibody or antigen-binding fragment thereof contains a CDRL3 binding domain comprising a consensus motif having the sequence QQX1X2X3X4X5X6T, where X1 is Y, F, or A, X2 is N, H, or Y, X3 is R, S, T, or D, X4 is D, F, Y, W, or P, X5 is P or S, and X6 is Y, F, K, or W. The following clones contain this consensus motif: ADI-50211, ADI-48899, ADI-45136, ADI-45078, ADI-49162, ADI-49141, ADI-42844, ADI-48910, ADI-45074, ADI-49041, ADI-50220, ADI-42172, ADI-42178, ADI-50218, and ADI-49194.

[0053] In some embodiments, the disclosure provides an antibody comprising a YFV binding domain CDRL3, wherein the CDRL3 binding domain comprises a consensus motif, the consensus motif comprising the sequence QX1X2X3X4TX5X6T, where X1 is Q or H, X2 is A or S, X3 is S or Y, X4 is T or S, X5 is R or P, and X6 is Y, L, W, or R. The following clones: ADI-42201, ADI-45164, ADI-46729, ADI-42223, ADI-46718, ADI-45076, ADI-48968, ADI-45156, ADI-50536, and ADI-50537 contain this consensus motif.

[0054] In some embodiments, the disclosure provides an antibody comprising a YFV binding domain CDRL3, wherein the CDRL3 binding domain comprises a consensus motif, the consensus motif comprising the sequence GTWDX1SX2X3SAGX4V, where X1 is S or T, X2 is S or is not an amino acid, X3 is L or P, and X4 is K, G, or R. The following clones contain this consensus motif: ADI-45083, ADI-42225, ADI-42210, ADI-42198, ADI-42809, ADI-42830, ADI-42818, ADI-42151, and ADI-50533.

[0055] In some embodiments, an antibody is provided comprising a YFV binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif, the consensus motif comprising the sequence AX1X2YDSX3X4YYX5X6X7X8, wherein X1 is K or R, X2 is Y, F, T, A, G, Y, or H, X3 is S, N, or R, X4 is A or G, X5 is W or Y, X6 is F, L, I, A, or E, X7 is D, E, or H, and X8 is Y, H, or S. The following clones contain this consensus motif: ADI-45085, ADI-50211, ADI-45078, ADI-49162, ADI-45136, ADI-42172, ADI-49194, ADI-50203, ADI-42178, ADI-48908, ADI-42844, ADI-48910, and ADI-49168.

[0056] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL2, wherein the CDRL2 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1X2X3X4RPS, wherein X1 is D or E, X2 is N, V, or D, X3 is K, N, D, or S, and X4 is K, E, or R. The following clones i.e. ADI-49039, ADI-42229, ADI-45097, ADI-45083, ADI-42225, ADI-49139, ADI-48969, ADI-4 8900, ADI-42786, ADI-42210, ADI-42198, ADI-49154, ADI-49188, ADI-42188, ADI-42809, ADI-46596, ADI ADI-42830, ADI-46591, ADI-48955, ADI-42818, ADI-46586, ADI-42151, ADI-45140, ADI-46722, ADI-45128, ADI-45127, ADI-46739, ADI-46724, ADI-50539, ADI-42114, ADI-50533, and ADI-49205 contain this consensus motif.

[0057] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL2, wherein the CDRL2 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1X2X3X4LX5X6, where X1 is A, G, or R, X2 is A or T, X3 is S or T, X4 is T, G, S, or I, X5 is Q or R, and X6 is S or R. The following clones contain this consensus motif: ADI-49133, ADI-49033, ADI-48895, ADI-42201, ADI-42230, ADI-48916, ADI-42211, ADI-5164, ADI-42191, ADI-49145, ADI-46729, ADI-42189, ADI-46718, ADI-45076, ADI-48968, ADI-50203, ADI-42227, ADI-48894, ADI-50218, ADI-45156, ADI-50536, ADI-50537, ADI-46737, ADI-45123, and ADI-50200.

[0058] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL2, wherein the CDRL2 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1X2SX3RAX4, wherein X1 is G, D, R, or A, X2 is A or S, X3 is S, T, or N, and X4 is T or A. The following clones contain this consensus motif: ADI-49147, ADI-50201, ADI-45113, ADI-50219, ADI-48897, ADI-42194, ADI-42847, ADI-48908, ADI-42231, ADI-42233, ADI-45148, ADI-42187, ADI-42787, ADI-49141, ADI-42213, ADI-42192, ADI-49590, ADI-48462, ADI-42200, ADI-42181, ADI-49037, ADI-49137, and ADI-42817.

[0059] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL2, wherein the CDRL2 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1VX2X3RPS, wherein X1 is D, E, or R, X2 is S, T, N, or A, and X3 is N, K, or Q. The following clones contain this consensus motif: ADI-42228, ADI-42190, ADI-49183, ADI-49189, ADI-50205, ADI-50531, ADI-49138, ADI-45154, ADI-49161, ADI-49561, ADI-42219, ADI-48435, ADI-45161, ADI-42193, ADI-42149, ADI-42216, ADI-42810, ADI-48890, ADI-42206, ADI-48950, and ADI-42124.

[0060] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL2, wherein the CDRL2 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1ASX2LEX3, where X1 is R, Q, or K, X2 is T, S, G, R, or I, and X3 is T or S. The following clones contain this consensus motif: ADI-42821, ADI-45085, ADI-50211, ADI-48899, ADI-49168, ADI-45136, ADI-45078, ADI-42844, ADI-48910, ADI-49041, ADI-42172, ADI-42178, ADI-49032, and ADI-49194.

[0061] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif, the consensus motif having the sequence X1X2X3HX4X5X6X7X8YX9PX 10 X 11 X 12X1 is D, E, or S, X2 is I or V, X3 is F or Y, X4 is X or T, X5 is G or E, X6 is S, G, or T, X7 is T or A, X8 is N, S, H, K, or T, X9 is N or S, and X 10 is S or F, and X 11 is L or V, and X 12 is K or E. The following clones contain this consensus motif: ADI-45083, ADI-42225, ADI-49139, ADI-48900, ADI-42232, ADI-42786, ADI-42210, ADI-42198, ADI-49154, ADI-42188, ADI-42809, ADI-42818, ADI-42151, ADI-46722, ADI-46742, ADI-49141, ADI-46739, ADI-46724, ADI-50539, ADI-48951, ADI-50538, and ADI-50533.

[0062] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif, the consensus motif having the sequence X1X2X3X4DX5X6X7KX8X9ADSX 10 X 11 X1 is V or L, X2 is I or M, X3 is S, W, or L, X4 is F or Y, X5 is E or G, X6 is S or T, X7 is K, N, or Y, X8 is F, W, or Y, X9 is Y or F, and X 10 is V or L, and X 11is K or R. The following clones contain this consensus motif: ADI-45097, ADI-42144, ADI-49138, ADI-45154, ADI-49561, ADI-42189, ADI-42844, ADI-45161, ADI-48462, ADI-42172, ADI-42178, ADI-42217, ADI-46737, ADI-49205, ADI-45151, and ADI-46728.

[0063] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif, the consensus motif comprising the sequence RX1SX2X3X4X5X6X7X8X9, wherein X1 is A or T, X2 is Q or R, X3 is S or T, X4 is I or V, X5 is S or T, X6 is S, N, T, F, D, or G, X7 is N, Y, W, F, or K, X8 is L or V, and X9 is A or N. The following clones i.e. ADI-49147, ADI-50201, ADI-45113, ADI-42201, ADI-42194, ADI-42847, ADI-45085, ADI-48908, ADI-5 0211, ADI-42231, ADI-45164, ADI-48899, ADI-46729, ADI-49168, ADI-49040, ADI-45136, ADI-45078, ADI-46718, ADI ADI-49141, ADI-42844, ADI-42192, ADI-48910, ADI-42200, ADI-50203, ADI-42181, ADI-49041, ADI-50220, ADI-42172, ADI-42178, ADI-49032, ADI-49137, ADI-42817, ADI-45156, ADI-50536, ADI-50537, and ADI-49194 contain this consensus motif.

[0064] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif, the consensus motif comprising the sequence SGSX1SNX2GX3X4X5VX6, wherein X1 is N or S, X2 is I or F, X3 is S or N, X4 is N, Y, S, or D, X5 is Y, F, or D, and X6 is S or A. The following clones, namely, ADI-49039, ADI-42229, ADI-45097, ADI-45083, ADI-42225, ADI-48900, ADI-42786, ADI-42210, ADI-42198, ADI-49154, ADI-42188, ADI-42809, ADI-46596, ADI-4283 0, ADI-46591, ADI-48955, ADI-42818, ADI-46586, ADI-42151, ADI-45140, ADI-46722, ADI-45128, ADI-46739, ADI-46724, ADI-50539, ADI-42114, and ADI-50533 contain this consensus motif.

[0065] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1GTX2X3DX4GX5X6X7X8VS, where X1 is A or T, X2 is S, G, or R, X3 is S or T, X4 is V, F, or I, X5 is G or A, X6 is Y, D, or F, X7 is K or N, and X8 is Y or F. The following clones contain this consensus motif: ADI-48969, ADI-42228, ADI-42190, ADI-49183, ADI-49189, ADI-50205, ADI-50531, ADI-49138, ADI-45154, ADI-49161, ADI-49561, ADI-42219, ADI-48435, ADI-45161, ADI-45127, ADI-42149, ADI-42216, ADI-42810, ADI-48890, ADI-42206, ADI-48950, ADI-42124, and ADI-49205.

[0066] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1X2FX3X4X5X6X7X8, where X1 is F, Y, or L, X2 is T, A, S, or N, X3 is S or T, X4 is S, T, or R, X5 is Y or L, X6 is G, A, T, W, S, or D, X7 is M, I, or L, and X8 is H, S, N, or T. The following clones, namely ADI-45090, ADI-49044, ADI-45113, ADI-42144, ADI-50026, ADI-45075, ADI-42230, ADI-42154, ADI-45085, ADI-42211, ADI-50211, ADI-42231, ADI-42233, ADI-49168, ADI-42187, ADI-49561, ADI-42219, ADI-50535, ADI-45136, ADI-42189, ADI-48435, ADI ADI-46718, ADI-42844, ADI-45161, ADI-48910, ADI-48462, ADI-42200, ADI-50203, ADI-42149, ADI-42172, ADI-42178, ADI-50197, ADI-42810, ADI-50218, ADI-45156, ADI-50536, ADI-50537, ADI-46737, ADI-42114, ADI-49194, ADI-42124, and ADI-46728 contain this consensus motif.

[0067] In some embodiments, the present disclosure provides an antibody comprising a YFV binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif, the consensus motif comprising the sequence X1SIX2X3X4X5X6WX7, where X1 is G or I, X2 is S or T, X3 is S, T, G or is not an amino acid, X4 is D, S, T, or G, X5 is Y, N, or D, X6 is W or Y, and X7 is S or T. The following clones contain this consensus motif: ADI-45083, ADI-42225, ADI-48900, ADI-42786, ADI-42210, ADI-49188, ADI-42188, ADI-42818, ADI-42151, ADI-48913, ADI-46722, ADI-49141, ADI-46741, ADI-46739, ADI-50539, ADI-50538, and ADI-50533.

[0068] In some embodiments, the present disclosure provides an antibody comprising a YFV-binding domain CDRH1, wherein the CDRH1-binding domain comprises a consensus motif, the consensus motif comprising the sequence FX1FSDX2YMX3, where X1 is I or T, X2 is H or Y, and X3 is A or D. The following clones contain this consensus motif: ADI-42191, ADI-49040, ADI-42223, ADI-42193, ADI-48968, ADI-42212, ADI-45126, ADI-42141, ADI-49140, ADI-48894, ADI-42226, ADI-49137, ADI-48890, ADI-42206, and ADI-49030.

[0069] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be omitted for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0070] CDR residues that do not contact the antigen can be identified based on previous studies by molecular modeling and / or empirically from regions of the Kabat CDRs outside the Chothia CDRs (e.g., residues H60-H65 in CDRH2 are often not required). When a CDR or its residues are omitted, they are usually replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the amino acid to be substituted can also be selected empirically.

[0071] The fully human monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues in the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence.Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0072] The present disclosure also includes fully monoclonal antibodies comprising variants of any of the CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes antibodies having CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any of the amino acid sequences disclosed herein. In some embodiments, the disclosed anti-YFV antibodies and antigen-binding fragments are human antibodies. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, within the CDRs, particularly within CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation).

[0073] In some embodiments, the disclosed anti-YFV antibodies and antigen-binding fragments are recombinant antibodies. The term "recombinant" generally refers to any protein, polypeptide, or cell expressing a gene of interest produced by genetic engineering methods. When used with respect to a protein or polypeptide, the term "recombinant" refers to a polypeptide produced by expression of a recombinant polynucleotide. The proteins used in the immunogenic compositions of the present disclosure can be isolated from natural sources or produced by genetic engineering methods.

[0074] The antibodies of the present disclosure may, in some embodiments, be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all antibodies, including human or humanized antibodies, prepared, expressed, created, or isolated by any recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that are derived from and related to human germline VH and VL sequences, but which may not naturally exist within the human antibody germline repertoire in vivo.

[0075] In some embodiments, anti-YFV antibodies and antigen-binding fragments thereof are isolated antibodies. As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to YFV is substantially free of Abs that specifically bind to antigens other than YFV). In some embodiments, anti-YFV antibodies and antigen-binding fragments specifically bind to the YFV E protein, e.g., the DII domain or the FL of DIII. The terms "specifically bind" or "specifically binds to," or equivalents, mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is defined as a binding activity of at least about 1 x 10 -6 The binding can be characterized by an equilibrium dissociation constant of M or less (e.g., a smaller KD indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies can be analyzed by surface plasmon resonance, e.g., BIACORE™. (商標) , for example, by biolayer interferometry using a ForteBio Octet HTX instrument (Pall Life Sciences) that specifically binds to YFV. Furthermore, multispecific antibodies that bind to a YFV protein and one or more additional antigens, or bispecific antibodies that bind to two different regions of YFV, are still considered "specifically binding" antibodies as used herein. In certain embodiments, the antibodies disclosed herein bind to a YFV protein at a concentration of about 1×10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, about 1 x 10 -10 M, about 1 x 10 -6 M ~ approx. 1×10 -7 M, about 1 x 10 -7 M ~ approx. 1×10 -8 M, about 1 x 10 -8 M ~ approx. 1×10 -9 M, about 1 x 10 -9 M ~ approx. 1×10 -10M, or approximately 1 x 10 -9 M ~ approx. 1×10 -10 indicates the equilibrium dissociation constant (and therefore specificity) of M.

[0076] In some embodiments, the anti-YFV antibodies and antigen-binding fragments are high affinity binders. The term "high affinity" refers to a binding affinity, expressed as a KD, of at least 10 as measured by surface plasmon resonance, e.g., a BIACORE™ instrument, e.g., a biolayer interferometry instrument using a ForteBio Octet HTX instrument (Pall Life Sciences), or a solution affinity ELISA. -9 M, more preferably 10 -10 M, more preferably 10 -11 M, more preferably 10 -12 This refers to a mAb with binding affinity to YFV of M.

[0077] By "deceleration rate", "Koff" or "kd" refers to a rate of deceleration of 1×10 as determined by surface plasmon resonance, e.g., a BIACORE™ or ForteBio Octet HTX instrument (Pall LifeSciences). -3 s -1 Less than 1 x 10, preferably -4 s -1 This means that the antibody dissociates from YFV with the following rate constant:

[0078] In specific embodiments, the antibodies or antibody fragments of the present disclosure may be conjugated to a therapeutic moiety (an "immunoconjugate") such as an antibiotic, a second anti-YFV antibody, a vaccine, or a toxoid, or any other therapeutic moiety useful for treating YFV.

[0079] Also contemplated are antibodies and antigen-binding fragments that are substantially identical to the antibodies provided herein. The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity of at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP, as discussed below. Thus, nucleic acid sequences that exhibit a particular percentage of "identity" share that percentage of identity and / or are "identical" to each other. A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of such antibody or antigen-binding fragment thereof that is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 3 of an antibody selected from antibody Nos. 1 to 152 disclosed in Table 3.

[0081] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the CDRH3 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0082] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the CDRH2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0083] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the CDRH1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0084] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the CDRL3 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0085] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0086] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0087] In some embodiments, the anti-YFV antibodies and antigen-binding fragments thereof are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween to at least one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0088] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0089] In certain embodiments, antibodies and antigen-binding fragments thereof of the present invention comprise the light chain (LC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3. In certain embodiments, antibodies and antigen-binding fragments thereof comprise the heavy chain (HC) amino acid sequence and light chain (LC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0090] In certain embodiments, the antibodies and antigen-binding fragments thereof are selected from the group consisting of the antibodies designated Antibody No. 1 to Antibody No. 152 as disclosed in Table 3, respectively.

[0091] Nucleic acids encoding the antibodies described herein are also provided. In certain embodiments, isolated nucleic acid sequences are provided that encode antibodies that specifically bind to YFV and antigen-binding fragments thereof, wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the antibody or antigen-binding fragment thereof is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween, to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 3 of an antibody selected from Antibody Nos. 1 to 152 disclosed in Table 3.

[0092] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the CDRH3 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0093] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the CDRH2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0094] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the CDRH1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0095] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the CDRL3 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0096] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0097] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0098] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3.

[0099] In certain embodiments, isolated nucleic acid sequences encoding antibodies and antigen-binding fragments thereof are provided, including sequences encoding the light chain (LC) amino acid sequence of any one of the antibodies designated ANTIBODY NO. 1 through ANTIBODY NO. 152 disclosed in Table 3. When applied to polypeptides, the term "substantial identity" or "substantially identical" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Accordingly, amino acid sequences exhibiting a particular percentage of "identity" share that percentage of identity and / or are "identical" to one another. Accordingly, amino acid sequences exhibiting a particular percentage of "identity" share that percentage of identity and / or are "identical" to one another.

[0100] In certain embodiments, the disclosed antibody amino acid sequences are, for example, at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percent identities therebetween to other sequences and / or share such percent identities with each other (or with specific subsets of the antibody sequences disclosed herein).

[0101] Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions will not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331). Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0102] The sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using a scale of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type proteins and their muteins. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters (programs in GCG version 6.1). FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. (See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403 410 and (1997) Nucleic Acids Res. 25:3389 402).

[0103] In certain embodiments, antibodies or antibody fragments for use in the methods of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for epitopes of more than one target polypeptide.

[0104] The anti-YFV antibodies disclosed herein can be obtained from human B cells using techniques available to those skilled in the art, for example, as described in the Examples below. Methods for generating human antibodies in transgenic animals, such as mice, are also known in the art and can be employed to derive antibodies according to the present disclosure. Any such method can be used in the context of the present disclosure to generate human antibodies that specifically bind to YFV (see, e.g., US Pat. No. 6,596,541).

[0105] In certain embodiments, the antibodies of the present disclosure have a binding activity of about 1.0 x 10 as measured by binding to an antigen immobilized either on a solid phase or in solution phase. -7 M ~ approx. 1.0×10 -12 In certain embodiments, antibodies of the disclosure possess an affinity (KD) ranging from about 1×10 to about 1×10 M, as measured by binding to antigen immobilized either on a solid phase or in solution phase. -7 M ~ approx. 6×10 -10 In certain embodiments, antibodies of the disclosure have an affinity (KD) ranging from about 1 x 10 M, as measured by binding to antigen immobilized either on a solid phase or in solution phase. -7 M ~ approx. 9×10 -10 It has an affinity (KD) ranging from M.

[0106] In addition to the specific anti-YFV antibodies and antibody fragments disclosed herein, the present disclosure also contemplates variants of these antibodies and antibody fragments that maintain biological equivalence. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit essentially the same biological activity as the described antibody. Similarly, DNA sequences encoding the antibodies of the present disclosure encompass sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present disclosure.

[0107] Two antigen-binding proteins or antibodies are considered bioequivalent if they are pharmaceutical equivalents or substitutes that do not show significant differences in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions, e.g., in either a single or multiple doses. Some antibodies may be considered equivalents or substitutes if they are equivalent in their extent of absorption but not in their rate of absorption, and may also be considered bioequivalent because such differences in absorption rate are intentional, reflected in the label, and are not considered medically significant for the particular pharmaceutical being studied, e.g., not essential to achieving effective body drug concentrations in chronic use.

[0108] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0109] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or decreased efficacy, compared to continued therapy without such switching.

[0110] In one embodiment, two antigen binding proteins are bioequivalent if they both operate by one or more mechanisms of action for one or more conditions of use, to the extent that such mechanisms are known.

[0111] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo testing in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro testing that correlates with and reasonably predicts human in vivo bioavailability data, (c) in vivo testing in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability, or bioequivalence, of the antibody.

[0112] Biologically equivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antibodies can include antibody variants containing amino acid changes that can modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0113] Biological and biophysical properties of antibodies In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention specifically bind to YFV, and at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween, to the corresponding CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequence disclosed in Table 3 of an antibody selected from antibody numbers 1 to 152 disclosed in Table 3.

[0114] In some embodiments, anti-YFV antibodies and antigen-binding fragments thereof are neutralizing antibodies, i.e., exhibit neutralizing ability. As used herein, a "neutralizing antibody" (or "antibody that neutralizes YFV activity" or "antibody with neutralizing activity") refers to an antibody whose binding to an antigen, such as the YFV E protein in some cases, results in inhibition of at least one biological activity, as disclosed herein. For example, an antibody of the present disclosure may help block YFV fusion to host cells, prevent syncytium formation, or prevent primary disease caused by YFV. Alternatively, an antibody of the present disclosure may demonstrate the ability to ameliorate at least one symptom of YFV infection. This inhibition of YFV biological activity can be assessed by measuring one or more indicators of YFV biological activity by one or more of several standard in vitro assays (such as neutralization assays described herein) or in vivo assays known in the art (e.g., animal models for protection from YFV challenge following administration of one or more of the antibodies described herein).

[0115] In certain embodiments, the antibodies and antigen-binding fragments thereof have an in vitro neutralization capacity (IC) of about 0.5 micrograms per milliliter (μg / ml) to about 5 μg / ml, about 0.05 μg / ml to about 0.5 μg / ml, or less than about 0.05 mg / ml. 50 ) is shown.

[0116] "I C 50 The term "half-maximal inhibitory concentration" refers to the value that measures the effectiveness of a compound (e.g., an anti-YFV antibody) inhibiting a biological or biochemical utility. This quantitative measurement indicates the amount of a particular inhibitor required to inhibit a given biological process by half. In certain embodiments, the YFV neutralizing ability of the anti-YFV neutralizing antibodies disclosed herein is measured using a neutralizing IC 50 Of the antibodies described herein, those that bind to DIII of the YFV E protein generally have the highest neutralizing capacity.

[0117] In some embodiments, the antibodies and antigen-binding fragments thereof cross-react with DENV-2, DENV-4, WNV, or ZIKV E protein, i.e., bind to YFV E protein and E proteins from one or more of the other flaviviruses. In certain embodiments, such antibodies and antigen-binding fragments thereof have high apparent affinity (K D Apps <10 nM) and bind to DENV-2, DENV-4, WNV, YFV, and ZIKV E proteins. In certain embodiments, the cross-reactive antibody or antigen-binding fragment thereof has neutralizing activity against YFV-17D and another flavivirus. In certain embodiments, the cross-reactive antibody and antigen-binding fragment thereof bind to the FL epitope. In certain embodiments, the cross-reactive antibody and antigen-binding fragment thereof bind to DIII. In certain embodiments, the cross-reactive antibody is ADI-48905.

[0118] Epitope binning and related techniques As described above and demonstrated in the Examples, the applicant has characterized the epitope binning of the antibodies and antigen-binding fragments thereof of the present invention. In addition to methods for performing such characterization, various other techniques are available to those skilled in the art that can be used to perform such characterization or otherwise confirm whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63), peptide truncation analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuterium-labeling a protein of interest and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface retain deuterium and therefore may exhibit a relatively high mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0119] As those skilled in the art will appreciate, epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, and more commonly at least five or 8-10, amino acids in a unique spatial conformation.

[0120] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying multiple monoclonal antibodies (mAbs) against the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (US 2004 / 0101920). Each category may reflect a unique epitope that is either distinct or partially overlapping with the epitopes represented by other categories. This technology allows for rapid filtering of genetically identical antibodies so that characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired properties. MAP can be used to sort the antibodies of the present disclosure into groups of antibodies that bind to different epitopes.

[0121] As one skilled in the art will appreciate, routine methods available in the art can easily be used to determine whether an antibody binds to the same epitope as a reference anti-YFV antibody or competes for binding with the reference anti-YFV antibody. For example, to determine whether a test antibody binds to the same epitope as a reference YFV antibody of the present disclosure, the reference antibody is allowed to bind to a YFV protein or peptide under saturating conditions. The ability of the test antibody to bind to a YFV molecule is then evaluated. If the test antibody is able to bind to YFV following saturation binding with the reference anti-YFV antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-YFV antibody. On the other hand, if the test antibody is unable to bind to a YFV molecule following saturation binding with the reference anti-YFV antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-YFV antibody of the present disclosure.

[0122] To determine whether an antibody competes for binding with a reference anti-YFV antibody, the binding methodology described above is performed in two ways. In the first way, the reference antibody is allowed to bind to YFV molecules under saturating conditions, followed by evaluation of the binding of the test antibody to the YFV molecules. In the second way, the test antibody is allowed to bind to YFV molecules under saturating conditions, followed by evaluation of the binding of the reference antibody to the YFV molecules. If, in both ways, only the first (saturating) antibody is able to bind to YFV molecules, it is concluded that the test antibody and the reference antibody compete for binding to YFV. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0123] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antibody inhibits the binding of the other antibody by at least 50%, preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. (1990) 50:1495-1502). Alternatively, two antigens have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0124] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art.

[0125] immune complex The present disclosure encompasses human YFV monoclonal antibodies ("immunoconjugates") conjugated to a therapeutic moiety, such as an agent, capable of reducing the severity of a primary YFV infection or ameliorating at least one symptom associated with YFV infection, including fever, muscle pain, headache, vomiting, diarrhea, and bleeding, or its severity. Such an agent may be a second, different antibody or a vaccine against YFV. The type of therapeutic moiety that may be conjugated to an anti-YFV antibody will take into consideration the condition to be treated and the desired therapeutic effect to be achieved. Alternatively, if the desired therapeutic effect is to treat sequelae or symptoms associated with YFV infection or any other condition resulting from such infection, such as, but not limited to, disseminated intravascular coagulation, acute renal failure, and acute respiratory distress syndrome, it may be advantageous to conjugate an appropriate agent to treat the sequelae or symptoms of the condition or to alleviate any side effects of the antibodies of the present disclosure. Examples of suitable agents for forming immunoconjugates are known in the art; see, for example, WO 05 / 103081.

[0126] multispecific antibodies The antibodies of the present disclosure can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of a single target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical bonding, genetic fusion, noncovalent association, or other methods) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

[0127] Therapeutic Administration and Formulations The present disclosure provides therapeutic compositions comprising the anti-YFV antibodies or antigen-binding fragments thereof of the present disclosure. Therapeutic compositions according to the present disclosure will be administered with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, and the like. Many suitable formulations can be found in formularies known to all pharmacists, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0128] The dosage of each of the antibodies of the present disclosure may vary depending on the age and size of the subject, the target disease, condition, route of administration, and the like. When the antibodies of the present disclosure are used to treat YFV infection, or to treat one or more symptoms associated with YFV infection, such as fever, nausea, or myalgia in a patient, or to reduce the severity of the disease, it is advantageous to administer each of the antibodies of the present disclosure intravenously or subcutaneously. Typically, each of the antibodies will be administered in a single dose of about 0.01 to about 30 mg / kg body weight, more preferably about 0.1 to about 20 mg / kg body weight, or about 0.1 to about 15 mg / kg body weight, or about 0.02 to about 7 mg / kg body weight, or about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight, or about 1 mg / kg body weight, or about 3.0 mg / kg body weight, or about 10 mg / kg body weight, or about 20 mg / kg body weight. Multiple doses may be administered as necessary. The frequency and duration of treatment may be adjusted depending on the severity of the condition. In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 300 mg, or about 10 to about 150 mg, about 100 mg, or about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof, in an amount that may be about the same as or less than the initial dose, with the subsequent doses separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0129] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. It can be delivered as an aerosolized formulation (see, e.g., US2011 / 0311515 and US2012 / 0128669). Delivery of drugs useful for treating respiratory diseases by inhalation is becoming more widely accepted (see AJ Bitonti and JADumont, (2006), Adv. Drug Deliv. Rev, 58:1106-1118). In addition to being effective in treating localized pulmonary diseases, such delivery mechanisms may also be useful for the systemic delivery of antibodies (see Maillet et al. (2008), Pharmaceutical Research, Vol. 25, No. 6, 2008).

[0130] The pharmaceutical composition can also be delivered in a vesicle, in particular a liposome (see, eg, Langer (1990) Science 249:1527-1533).

[0131] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

[0132] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable formulations may be prepared by publicly known methods. For example, injectable formulations may be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, and the like, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mole) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil, soybean oil, and the like, which may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, and the like. The injectable formulations prepared in this manner are preferably filled into appropriate ampoules.

[0133] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily used to deliver the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Rather, disposable pen delivery devices come pre-filled with the pharmaceutical composition held in a reservoir within the device. When the reservoir no longer contains the pharmaceutical composition, the entire device is discarded.

[0134] A number of reusable pen and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC, to name just a few. (商標) Pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ Pen, HUMALOG (商標) Pen, HUMALIN 70 / 30 (商標) Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN (商標) I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (商標) (Novo Nordisk, Copenhagen, Denmark), BD (商標) Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN (商標) , OPTIPEN PRO (商標) , OPTIPEN STARLET (商標) , and OPTICLIK (商標) (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices that have application for subcutaneous delivery of pharmaceutical compositions of the present disclosure include, but are certainly not limited to, the SOLOSTAR, to name just a few. (商標) Pen (Sanofi-Aventis), FLEXPEN (商標) (Novo Nordisk), and KWIKPEN (商標) (Eli Lilly), SURECLICK (商標) Automatic injector (Amgen, Thousand Oaks, CA), PENLET (商標) (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA (商標) Penn (Abbott Labs, Abbott Park, IL), but certainly not limited to:

[0135] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms with unit doses suitable for the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in unit doses. In particular, in the form of injection, the amount of the antibody contained is preferably about 5 to about 100 mg, and for other dosage forms, about 10 to about 250 mg.

[0136] Management Plan In some embodiments, a therapeutically effective amount of an anti-YFV antibody or antigen-binding fragment thereof is provided to a subject in need thereof, for example, a subject infected with or at risk of being infected with YFV. The phrase "therapeutically effective amount" refers to the amount for which it is administered that produces the desired effect. The exact amount will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0137] According to certain embodiments, multiple doses of an antibody against YFV may be administered to a subject over a defined time course. A method according to this aspect of the present disclosure includes sequentially administering multiple doses of an antibody against YFV to a subject. As used herein, "sequentially administering" means that each dose of an antibody against YFV is administered to a subject at different time points, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods that include sequentially administering a single initial dose of an antibody against YFV to a patient, followed by one or more secondary doses of an antibody against YFV, and optionally, one or more tertiary doses of an antibody against YFV.

[0138] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of antibodies against YFV. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of antibodies against YFV, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antibodies against YFV contained in the initial, secondary, and / or tertiary doses varies from each other during the course of treatment (e.g., adjusted up or down as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as "loading doses," followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0139] In one exemplary embodiment of the present disclosure, each secondary and / or tertiary dose is administered within 1 to 26 weeks (e.g., 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 61 weeks, 62 weeks, 63 weeks, 64 weeks, 65 weeks, 66 weeks, 67 weeks, 68 weeks, 69 weeks, 70 weeks, 71 weeks, 72 weeks, 73 weeks, 74 weeks, 75 weeks, 76 weeks, 77 weeks, 78 weeks, 79 weeks, 80 weeks, 81 weeks, 82 weeks, weeks, 13.5 weeks, 14 weeks, 14.5 weeks, 15 weeks, 15.5 weeks, 16 weeks, 16.5 weeks, 17 weeks, 17.5 weeks, 18 weeks, 18.5 weeks, 19 weeks, 19.5 weeks, 20 weeks, 20.5 weeks, 21 weeks, 21.5 weeks, 22 weeks, 22.5 weeks, 23 weeks, 23.5 weeks, 24 weeks, 24.5 weeks, 25 weeks, 25.5 weeks, 26 weeks, 26.5 weeks, or more). The phrase "immediately preceding dose" as used herein refers to the dose of antibody to YFV that, in a multiple administration sequence, is administered to a patient prior to administration of the next dose in that sequence, without any intervening doses.

[0140] Methods according to this aspect of the disclosure can include administering any number of secondary and / or tertiary doses of antibodies against YFV to a patient. For example, in certain embodiments, only a single secondary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to a patient. Similarly, in certain embodiments, only a single tertiary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to a patient.

[0141] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient following clinical testing.

[0142] Thus, in certain embodiments, pharmaceutical compositions are provided that comprise one or more of the antibodies or antigen-binding fragments thereof of the invention disclosed herein and throughout, and a pharmaceutically acceptable carrier and / or one or more excipients. In certain other embodiments, pharmaceutical compositions are provided that comprise one or more nucleic acids encoding one or more of the antibodies or antigen-binding fragments thereof of the invention, or one or more expression vectors comprising such nucleic acids, and a pharmaceutically acceptable carrier and / or one or more excipients.

[0143] Therapeutic Uses of Antibodies The anti-YFV antibodies disclosed herein can be used to treat subjects with YFV and / or to prevent YFV infection.

[0144] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of YFV infection or a symptom or condition associated therewith (fever, chills, headache, back pain, muscle pain, loss of appetite, nausea, vomiting, fatigue, or a combination thereof) resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In certain embodiments, such terms refer to a reduction or inhibition of YFV replication, an inhibition or reduction of the spread of YFV to other subjects, an inhibition or reduction of infection of cells by YFV, or an amelioration of one or more symptoms associated with YFV infection.

[0145] As used herein, the terms "prevent," "preventing," and "prevention" refer to preventing or inhibiting the onset or development of YFV infection or a condition associated therewith in a subject, preventing or inhibiting the progression of YFV infection or a condition associated therewith due to administration of a therapy (e.g., a prophylactic or therapeutic agent), preventing symptoms of YFV infection or a condition associated therewith, or administering a combination of therapies (e.g., a combination of prophylactic or therapeutic agents). As used herein, the terms "ameliorate" and "alleviate" refer to a reduction or lessening of the severity of a condition or any of its symptoms.

[0146] Due to their binding to and interaction with YFV, the antibodies and antigen-binding fragments of the present invention are believed to be useful, without wishing to be bound by any theory, for preventing viral fusion with host cell membranes, preventing cell-to-cell viral spread, and inhibiting syncytia formation. Alternatively, the antibodies of the present disclosure may be useful for ameliorating at least one symptom associated with an infection, such as fever, diarrhea, and bleeding, or for reducing the severity, duration, and / or frequency of an infection. The antibodies of the present disclosure are also contemplated for prophylactic use in patients at risk of developing or acquiring a YFV infection. It is contemplated that the antibodies of the present disclosure may be used alone or in conjunction with a second or third agent to treat a YFV infection or to reduce at least one symptom or complication associated with or resulting from such an infection, such as fever, nausea, or muscle pain. The second or third agent may be delivered simultaneously with the antibodies of the present disclosure, or they may be administered separately, either before or after the antibodies of the present disclosure. The second or third agent can be an antiviral agent, an NSAID or other agent for reducing fever or pain, another second but different antibody that specifically binds to YFV, an agent (e.g., an antibody) that binds to another YFV antigen, a vaccine against YFV, and an siRNA specific for a YFV antigen.

[0147] In yet a further embodiment of the disclosure, the antibody is used in the preparation of a pharmaceutical composition for treating a patient suffering from a YFV infection. In yet another embodiment of the disclosure, the antibody is used in the preparation of a pharmaceutical composition for reducing the severity of a primary YFV infection, or shortening the duration of the infection, or reducing at least one symptom associated with a YFV infection. In a further embodiment of the disclosure, the antibody is used as an adjunct therapy with any other agent useful for treating a YFV infection, including an antiviral agent, a toxoid, a vaccine, a second YFV antibody, or any other antibody specific for a YFV antigen, or any other palliative therapy known to those of skill in the art.

[0148] Thus, in certain embodiments, methods are provided for treating or preventing YFV infection or at least one symptom associated with YFV infection, comprising administering one or more of the antibodies of the present invention or antigen-binding fragments thereof disclosed herein and throughout, such as, for example, one or more of the anti-YFV antibodies disclosed in Table 3, to a patient in need of or suspected of being in need of treatment or prevention, such that YFV infection is treated or prevented, or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0149] In certain other embodiments, methods are provided for treating or preventing YFV infection or at least one symptom associated with YFV infection, comprising administering to a patient in need of or suspected of needing treatment or prevention a nucleic acid encoding one or more of the antibodies or antigen-binding fragments thereof of the present invention, such as a nucleic acid sequence encoding the amino acid sequence disclosed in Table 3 and its complement, such that YFV infection is treated or prevented or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0150] In a further embodiment, a method for treating or preventing YFV infection or at least one symptom associated with YFV infection is provided, comprising administering to a patient in need of treatment or prevention or suspected of being in need of treatment or prevention a host cell comprising a nucleic acid sequence or an expression vector comprising such a nucleic acid sequence, wherein such nucleic acid sequence encodes an amino acid sequence selected from the sequences disclosed in Table 3, such that YFV infection is treated or prevented, or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0151] In a further embodiment, a method is provided for treating or preventing YFV infection or at least one symptom associated with YFV infection, comprising administering to a patient in need of treatment or prevention or suspected of needing treatment or prevention a pharmaceutical composition comprising an antibody of the present invention or an antigen-binding fragment thereof disclosed in Table 3, one or more nucleic acid sequences or expression vectors comprising such nucleic acid sequences, wherein such nucleic acid sequences encode amino acid sequences selected from the sequences disclosed in Table 3 and their complements, one or more host cells comprising one or more nucleic acid sequences, wherein such nucleic acid sequences encode amino acid sequences selected from the sequences disclosed in Table 3 and their complements, or expression vectors comprising such one or more nucleic acid sequences, and a pharmaceutically acceptable carrier and / or one or more excipients, such that YFV infection is treated or prevented or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0152] In certain embodiments, methods are provided for treating or preventing YFV infection or at least one symptom associated with YFV infection, comprising administering to a patient in need of or suspected of needing treatment or prevention one or more of the antibodies or antigen-binding fragments thereof disclosed herein and throughout, such as, for example, one or more of the anti-YFV antibodies disclosed in Table 3, such that YFV infection is treated or prevented or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0153] In certain other embodiments, methods are provided for treating or preventing YFV infection or at least one symptom associated with YFV infection, comprising administering to a patient in need of or suspected of needing treatment or prevention a nucleic acid encoding one or more of the antibodies or antigen-binding fragments thereof of the present invention, such as a nucleic acid sequence encoding the amino acid sequence disclosed in Table 3 and its complement, such that YFV infection is treated or prevented or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0154] In a further embodiment, a method for treating or preventing YFV infection or at least one symptom associated with said YFV infection is provided, comprising administering to a patient in need of treatment or prevention or suspected of being in need of treatment or prevention a host cell comprising a nucleic acid sequence or an expression vector comprising such a nucleic acid sequence, wherein such nucleic acid sequence encodes an amino acid sequence selected from the sequences disclosed in Table 3 and its complement, such that YFV infection is treated or prevented, or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0155] In a further embodiment, a method is provided for treating or preventing YFV infection or at least one symptom associated with YFV infection, comprising administering to a patient in need of treatment or prevention or suspected of requiring treatment or prevention a pharmaceutical composition comprising an antibody of the present invention or an antigen-binding fragment thereof disclosed in Table 3, one or more nucleic acid sequences or expression vectors comprising such nucleic acid sequences, wherein such nucleic acid sequences encode amino acid sequences selected from the sequences disclosed in Table 3 and their complements, one or more host cells comprising one or more nucleic acid sequences, wherein such nucleic acid sequences encode amino acid sequences selected from the sequences disclosed in Table 3 and their complements, or expression vectors comprising such one or more nucleic acid sequences, and a pharmaceutically acceptable carrier and / or one or more excipients, such that YFV infection is treated or prevented or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity.

[0156] Combination therapy As described above, according to certain embodiments, the disclosed methods include administering to a subject one or more additional therapeutic agents in combination with an antibody against YFV. As used herein, the term "in combination with" means that the additional therapeutic agent is administered before, after, or simultaneously with a pharmaceutical composition comprising an anti-YFV antibody. The term "in combination with" also includes sequential or simultaneous administration of an anti-YFV antibody and a second therapeutic agent.

[0157] For example, when administered "before" a pharmaceutical composition comprising an anti-YFV antibody, the additional therapeutic agent can be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before administration of the pharmaceutical composition comprising an anti-YFV antibody. When administered "after" a pharmaceutical composition comprising an anti-YFV antibody, the additional therapeutic agent can be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after administration of the pharmaceutical composition comprising an anti-YFV antibody. "Concurrent" administration or administration with a pharmaceutical composition comprising an anti-YFV antibody means that the additional therapeutic agent is administered to the subject in a separate dosage form within 5 minutes of (before, after, or simultaneously with) administration of the pharmaceutical composition comprising the anti-YFV antibody, or is administered to the subject as a single combined dosage form containing both the additional therapeutic agent and the anti-YFV antibody.

[0158] The combination therapy can include an anti-YFV antibody of the present disclosure and any additional therapeutic agent that can be advantageously combined with an antibody of the present disclosure or a biologically active fragment of an antibody of the present disclosure.

[0159] For example, a second or third therapeutic agent, such as an antiviral agent, can be employed to help reduce the viral load in the liver. The antibody can also be used in conjunction with other therapies, such as toxoids, YFV-specific vaccines, YFV-specific secondary antibodies, or antibodies specific for another YFV antigen.

[0160] Diagnostic Uses of Antibodies The anti-YFV antibodies and antigen-binding fragments thereof of the present invention can also be used to detect and / or measure YFV in a sample, for example, for diagnostic purposes. It is envisioned that confirmation of an infection believed to be caused by YFV can be performed by measuring the presence of the virus through the use of any one or more of the antibodies of the present disclosure. An exemplary diagnostic assay for YFV can include, for example, contacting a sample obtained from a patient with an anti-YFV antibody of the present disclosure, where the YFV antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate virus-containing proteins from the patient sample. Alternatively, an unlabeled YFV antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The antibody may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure YFV in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0161] Samples that can be used in YFV diagnostic assays according to the present disclosure include any tissue or fluid sample obtained from a patient that contains a detectable amount of YFV protein or fragment thereof under normal or pathological conditions. Generally, the level of YFV in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with the presence of YFV) will be measured to first establish a baseline or standard level of YFV protein. This baseline level of YFV can then be compared to the level of YFV measured in a sample obtained from an individual suspected of having YFV infection or symptoms associated with such infection. [Example]

[0162] The human antibody response to YFV was comprehensively profiled by isolating and characterizing 152 YFV-specific monoclonal antibodies from memory B cells of two flavivirus-naive donors following immunization with YFV-17D. These antibodies were then used to map the antigenic topology of YFV. The obtained anti-YFV antibodies were found to bind to several antigenic sites, most commonly targeting epitopes within or proximal to the FL of domain II of the YFV E protein, thus providing support for the development of YFV antibodies targeting domain II. However, a second, less common class of antibodies with highly potent neutralizing activity was found to target the viral DIII. Such DIII-directed antibodies may be particularly important in the context of therapeutic applications of monoclonal antibodies or cocktails, as this epitope is subdominant in the innate immune response. Collectively, these results have implications for the design and evaluation of YFV vaccine and antibody-based therapeutic candidates and offer new options for passive prevention.

[0163] Study Design: Two flavivirus-naive healthy adult donors ("Donor 8" and "Donor 9") were immunized with YFV-17D vaccine (Stamaril, Sanofi), and blood samples were collected at 10, 14, 28, 90, 180, 270, and 360 days post-vaccination. Serum neutralizing activity against YFV-17D appeared in both donors by day 14 post-vaccination and persisted throughout the course of the study (Figure 1a). Pre-vaccination sera from both donors lacked reactivity with YFV-17D and showed no detectable neutralizing activity against YFV-17D (data not shown), as well as with E and NS1 proteins from other commonly circulating flaviviruses, namely, dengue virus serotypes 1-4 (DENV1-4), JEV, TBEV, West Nile virus (WNV), and Zika virus (ZIKV), confirming that both donors were likely flavivirus-naive at the time of vaccination (data not shown).

[0164] Molecular and functional characterization of YFV-17D-induced plasmablast responses Plasmablast responses in both donors were monitored 10 and 14 days after vaccination. Expanded plasmablast populations approximately 10-fold greater than prevaccination levels were observed in both donors at both 10 and 14 days (Figure 2a). Approximately 300 plasmablasts from each donor were sorted, and the corresponding VH and VL regions were amplified by single-cell PCR. 161 and 210 naturally paired antibodies were cloned from donors 8 and 9, respectively, and expressed as full-length IgG in engineered strains of Saccharomyces cerevisiae. Sequence analysis showed that the plasmablast responses were highly diverse in both donors, with only approximately 15% of clones belonging to the expanded clonal lineage (data not shown). The majority of plasmablast-derived antibodies from both donors contained high levels of somatic hypermutation (SHM), suggesting efficient recruitment of MBCs into the PB response (data not shown). The median SHM among PB-derived mAbs was significantly higher at day 10 than at day 14. Correspondingly, the majority of mAbs cloned from PB at day 14 lacked SHM, suggesting increased recruitment of cells from the naive B cell compartment at this time point (data not shown).

[0165] To analyze whether somatic mutations in PB-derived mAbs contribute to binding activity, putative unmutated common ancestor (UCA) mAbs were generated from three somatically mutated PB clones, and their binding affinities to recombinant YFV E protein were measured. In all three cases, the UCA mAbs showed substantially reduced binding affinities compared with the mature mAbs, suggesting that somatic mutations in PB mAbs are important for recognition of YFV E (Fig. 3).

[0166] PB-derived mAbs were then tested for binding reactivity to YFV-17D particles using a sandwich ELISA assay (Figure 2b). The frequency of YFV-17D-binding mAbs isolated from day 10 and day 14 PB ranged from 8 to 41%. Forty-five and 46 YFV-17D-binding mAbs were recovered from the expanded PB populations of donors 8 and 9, respectively, and then analyzed for neutralizing activity in a microtiter neutralization assay at 100 and 10 nM concentrations. Neutralizing activity ranged from complete neutralization at 10 nM to no detectable neutralization at 100 nM (Figure 2c). A higher percentage of mAbs isolated from day 14 PB exhibited neutralizing activity compared with those isolated from day 10 PB, consistent with the increased serum neutralizing activity at day 14 compared with day 10 in both donors (data not shown). Neutralization titration experiments for mAbs showing at least 50% infection inhibition at 100 nM revealed that 9–12% of YFV-17D-binding mAbs isolated from day 14 PB showed moderate to high neutralizing activity (IC 50 ≤10 nM) (Figures 2d and 4). Sequence analysis revealed that 12.5–33% of PB-derived nAbs utilized the VH4-4 / VL1-51 germline gene pairing, suggesting recognition of a common antigenic site (data not shown).

[0167] Approximately 50% and 22% of the neutralizing antibodies isolated from donors 8 and 9, respectively, lacked somatic mutations, indicating that YFV-17D neutralizing antibodies are present in the naive B cell repertoire, suggesting that YFV-17D vaccination induces PB responses arising from both naive and MBC B cells, and that only a small fraction of these B cells encode Abs that exhibit neutralizing activity (see Figures 1a and 1b).

[0168] Molecular and functional characterization of YFV-17D-induced MBC responses MBC responses in both donors were monitored by collecting PBMCs on days 14, 28, 90, 180, 270, and 360 postvaccination, and purified B cells were stained with a panel of previously described B cell surface markers (CD19, CD20, CD27, IgM, IgD, CD21, and CD71) as well as fluorescently labeled recombinant YFV E protein (Figure 5a). YFV E-specific swIg+ MBCs appeared in both donors by days 14–28, peaked between days 90–180, and slowly declined between days 180–360 (Figure 5b).

[0169] Between 100 and 400 YFV E-reactive B cells were sorted from both donors at each sampling time point. Non-binding mAbs derived from naive B cells were captured via the sorting strategy employed but were excluded from subsequent analysis. Analysis of B cell surface markers expressed on single-cell sorted YFV E-reactive B cells revealed that the MBC response to YFV E was highly heterogeneous at all time points (data not shown). At the earliest sampling time point (day 14), activated naive B cells and IgM+CD27+ MBCs dominated the response in both donors, but these B cell populations rapidly declined over time. By day 90, less than 15% of the YFV E-specific response was composed of IgM+CD27+ MBCs, and by day 360, only approximately 5% of YFV E-specific B cells belonged to this MBC population (Figure 8b). In contrast, the swIg+ MBC population, composed of both CD27+ and CD27- B cells, expanded from day 14 to day 90 and then remained stable throughout the course of the study. The MBC responses observed following YFV-17D vaccination were also observed following natural infection with PUUV (data not shown).

[0170] SHM loading, apparent binding affinity (K D Apps), and neutralizing capacity were tracked at each sampling time point. In both donors, median levels of SHM were low at day 14, with over 50% of Abs lacking somatic mutations, gradually increasing over a period of 6 to 9 months and plateauing in both donors by 9 months post-vaccination, with a median of 9 and 7 nucleotide substitutions in the VH of donors 8 and 9, respectively (data not shown). Binding studies using recombinant YFV E protein confirmed the K of MBC-derived mAbs. D Apps showed that the KD was very weak at early time points and gradually improved over the 6-9 months following vaccination (data not shown). At 14 and 28 days post-vaccination, the majority of YFV E-specific mAbs were KD Apps >50 nM, whereas by day 180, 50% of YFV E-specific mAbs showed a K D Apps In parallel with the increase in affinity, starting from day 90, highly potent neutralizing antibodies (IC 50 The emergence of neutralizing mAbs (<1 nM) was observed (data not shown). These neutralizing antibodies were derived from multiple MBC subsets, including atypical IgM+ and / or IgD+ MBC (data not shown). Table 2 summarizes the affinity and neutralization data of the isolated and characterized neutralizing mAbs.

[0171] Ongoing B cell activation was assessed by analyzing the expression of CD71 and CD21 on YFV E-specific MBCs. CD71 was expressed on 75–85% of YFV E-specific B cells at day 14 and remained elevated for approximately 6 months in both donors (data not shown). In both donors, YFV E-specific CD21 lo CD71 cells comprised approximately 40–80% of the YFV E-specific response and were present at high frequencies on days 14 and 28 post-vaccination, then rapidly declined by day 90. + Populations and CD21 lo There is a high degree of overlap between the YFV E-specific activated B cell population (CD71 + and / or CD21 lo50-80% of the cells (defined as CD71+) were CD71+ at day 14 + CD21 lo By day 28–90, the CD71 phenotype was + CD21 lo The population was attenuated to <50% of the activated B cell response in both donors, and the majority of YFV E-specific activated B cells were CD71 + CD21 + or CD71 - CD21 lo phenotype and were heterogeneous with respect to isotype and CD27 expression (data not shown).

[0172] Isolation and characterization of anti-YFV antibodies Approximately 152 neutralizing monoclonal antibodies were isolated and characterized. Antibody variable heavy (VH) and variable light (VL) chain genes were rescued by single-cell PCR. Tiller et al. (2008) J Immunol Methods 329, 112-124. Cognate heavy and light chain pairs were subsequently cloned and expressed as full-length IgG in engineered strains of Saccharomyces cerevisiae for further characterization. Bornholdt et al. (2016) Science 351, 1078-1083.

[0173] The germline gene usage of the isolated mAbs was analyzed. In both donors, mAbs utilizing the VH3-72 germline gene dominated the response at all time points (Figure 6a). The majority of these mAbs also utilized one of the five dominant light chain (LC) germline genes and exhibited shorter-than-average heavy chain (HC) complementarity-determining region 3 (CDRH3) lengths, suggesting a shared mode of antigen recognition (Figure 6b-c). The binding affinity of mAbs utilizing VH3-72 was significantly higher than that observed for mAbs utilizing other VH germline genes, despite containing similar levels of SHM (Figure 6d-e). Table 1 summarizes the germline usage and number of nucleotide substitutions for the isolated mAbs.

[0174] To investigate the epitope coverage of the isolated mAb, pairwise competition experiments were performed using the newly isolated mAb and two well-characterized control mAbs, 4G2 and 5A, which recognize proximal but non-overlapping epitopes within the DIII of the YFV E monomer. 4G2 is a pan-flavivirus mAb targeting FL, while 5A is a YFV E-specific mAb that binds to an FL-proximal epitope overlapping with the proposed prM-associated region. Competition experiments were performed using high-throughput surface plasmon resonance (SPR) on a Carterra LSA instrument. The reactivity of the mAb with recombinant YFV-17D DIII protein by BLI was also assessed. The majority of mAbs recognized one of eight distinct antigenic sites defined based on their reactivity with DIII and competition with 4G2, 5A, and three of the newly isolated mAbs (ADI-49147, ADI-44112, and ADI-45107) (Fig. 7a). A subset of mAbs competed with both 5A and ADI-45107, suggesting that these two antigenic sites are in close proximity. A small subset of mAbs (6 of 772) recognized epitopes within DIII. Five of the DIII-directed mAbs cross-competed, while the sixth, ADI-48945, may recognize a unique epitope. More than half of the mAbs from both donors competed with 4G2 and / or 5A, suggesting that the majority of YFV E-specific responses are mediated by Abs targeting epitopes within or proximal to the FL on DIII (Fig. 7a). Nearly all mAbs utilizing the VH3-72 germline gene competed with 4G2 (Figure 7b). Accordingly, analysis of the sequence characteristics of mAbs clustered by competitor group revealed that more than half of the mAbs that competed with 4G2 utilized the VH3-72 germline gene (Figure 7c). 4G2-competing mAbs utilizing VH3-72 exhibited significantly higher affinity compared with those utilizing other VH germline genes (Figure 7d). While the proportion of mAbs targeting each antigenic site did not change dramatically over time, suppression of 4G2 / 5A-competing mAbs was observed at later time points (days 270 and 360) in donor 8.Furthermore, in both donors, mAbs competing with both 5A and ADI-45107 did not emerge until days 28 to 90. The results suggest that the majority of YFV E-specific responses were directed against epitopes within or proximal to the FL on domain II and that there was only a minor shift in the Ab immunodominance hierarchy during maturation of B cell responses to YFV-17D.

[0175] Highly potent neutralizing antibodies recognize FL-proximal epitopes. The relationship between antigenic site and neutralizing ability was investigated. More than 90% of the mAbs that competed with either 5A alone or both 5A and ADI-45107 exhibited neutralizing activity (Fig. 8a). The most potent neutralizing antibodies (IC 50 The vast majority (78%) of the 5A-only or 5A / ADI-45107-competitive neutralizing antibodies (<1 nM) belonged to these two competing groups (Figures 8b-8c). Table 2 shows the binning data for these antibodies. Analysis of the sequence characteristics of these 5A-only or 5A / ADI-45107-competitive neutralizing antibodies revealed that nearly 40% utilized the VH4-4 / VL1-51 germline gene pairing and showed no evidence of convergent CDRH3 sequences, suggesting a common mode of germline-encoded antigen recognition (Figure 8d). Consistent with previous studies, most DIII-directed mAbs also exhibited highly potent neutralizing activity. In contrast to the 5A competitors and DIII-directed mAbs, only a few mAbs belonging to the other competing groups exhibited neutralizing activity. For example, only 12% and 20% of mAbs competing with 4G2 alone or both 4G2 and 5A, respectively, exhibited neutralizing IC50s <100 nM. The results demonstrate that the nAb response to YFV-17D is primarily mediated by Abs that recognize FL-proximal epitopes within DII of the YFV E protein.

[0176] A subset of mAbs shows cross-reactivity with E proteins from other flaviviruses. The isolated mAbs were evaluated for binding reactivity to recombinant DENV-2, DENV-4, WNV, or ZIKV E proteins. In both donors, approximately 6% of the YFV E-reactive mAbs showed cross-reactivity to at least one heterologous flavivirus E protein (Figure 9a). The majority of these cross-reactive mAbs targeted highly conserved FL epitopes and exhibited high apparent affinity (K D Apps <10 nM) to all five flavivirus E proteins (Fig. 9b-9c). Correspondingly, the small subset of mAbs that bound to epitopes outside of FL generally exhibited more restricted cross-reactivity profiles and lower K D Apps Only 6 of the 50 cross-reactive mAbs showed neutralizing activity against YFV-17D, and only a single mAb, the DIII binder ADI-48905, showed detectable but weak neutralizing activity against ZIKV (IC of approximately 100 nM). 50 ). None of the mAbs had measurable neutralizing activity against West Nile virus or Japanese encephalitis virus reporter virus particles. YFV-17D vaccination therefore appears to induce a subset of Abs with broad flavivirus binding activity, the majority of which target highly conserved FLs and show little or no cross-neutralizing activity.

[0177] Table 1 below provides germline usage and amino acid sequence information for 152 anti-YFV antibodies as described herein. The sequences provided in Table 1 include, for each listed antibody, the CDRH3 sequence (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 162, 164, 166, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, and 302), and CDRL3 sequences (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 1, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, and 303).

[0178] Table 2 below shows the affinity and neutralization data for the 152 anti-YFV antibodies listed in Table 1.

[0179] Table 3 below provides partial amino acid sequences for the heavy and light chain CDRs of each of the 152 anti-YFV antibodies listed in Table 1. The CDRs are shown in bold / underlined. Each CDR amino acid sequence is also listed separately in the sequence listing (CDRH1 and CDRH2 correspond to SEQ ID NOs: 607-840, and CDRL1 and CDRL2 correspond to SEQ ID NOs: 841-1005). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

Table 3-36

Table 3-37

Table 3-38

[0180] material and method research design Study subjects, ages 30 and 31, received the YFV-17D Stamaril vaccine. Heparinized blood (50–100 cc) was obtained from subjects before vaccination and on days 10, 14, 28, 90, 180, 270, and 360 following vaccination. Samples were processed at the Geisel School of Medicine at Dartmouth's Immune Surveillance and Flow Cytometry Core Laboratory to obtain plasma and isolate peripheral blood-derived B cells. Isolated cells and plasma were stored frozen in aliquots at -80°C.

[0181] Cells: Huh 7.5.1 cells (received from Dr. Jan Carette, originally received from Dr. Frank Chisari) were passaged every 3–4 days using 0.05% trypsin / EDTA solution (Gibco) and maintained in Dulbecco's modified Eagle's medium (DMEM high glucose, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals), 1% penicillin / streptomycin (P / S, Gibco), 1% Gluta-MAX (Gibco), and 25 mM HEPES (Gibco). Vero African riveted monkey kidney cells (obtained from ATCC) were passaged every 3–4 days using 0.05% trypsin / EDTA solution (Gibco) and maintained in Dulbecco's modified Eagle's medium (DMEM high glucose, Gibco) supplemented with 2% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals), 1% penicillin / streptomycin (P / S, Gibco), 1% Gluta-MAX (Gibco), and 25 mM HEPES (Gibco).

[0182] Yellow fever virus 17D production: YFV-17D was obtained from BEI Resources (catalog number NR-115). At 80% confluency, 15 cm plates with Huh 7.5.1 were infected with 90 μL of a second-passage stock of YFV-17D supernatant in 3 mL of infection medium (DMEM low glucose (Gibco), 7% FBS, 1% Pen-Strep, 1% Gluta-MAX (Gibco), 25 mM HEPES (Gibco)) for 1 hour at 37°C and 5% CO2. After 3 days, the supernatant was harvested and centrifuged twice at 4,000 rpm for 15 minutes at 4°C to remove cell debris. YFV-17D virus stock solutions for neutralization assays were generated by ultracentrifugation of the pre-cleared supernatant through 2 mL of 30% (v / v) D-sucrose / PBS buffer for 4 hours at 28,000 rpm using an SW28 rotor (Beckman Coulter) in a Beckman Coulter Optima LE-80K ultracentrifuge. The pellet was resuspended in 300 μl of PBS overnight on ice, then aliquoted and frozen at -80°C.

[0183] Zika virus production: Zika virus strain MR 766 was obtained from ATCC (ATCC (登録商標) VR-84 (商標) ). For the neutralization assay, 15 cm plates with Vero cells at 80% confluency were infected with 90 μL of a first-passage stock of Zika supernatant in 3 mL of infection medium (DMEM low glucose (Gibco), 2% FBS, 1% Pen-Strep, 1% Gluta-MAX (Gibco), 25 mM HEPES (Gibco)) for 1 hour at 37°C and 5% CO2. After 3 days, the supernatant was collected and centrifuged twice at 4,000 rpm for 15 minutes at 4°C to remove cell debris.

[0184] Antigens and antibodies Production of recombinant YFV antigens: The coding regions for prM and the entire soluble E (sE) region of the YFV Asibi strain (Uniprot ID: Q6DV88, residues 122–678 of the genome polyprotein) were cloned into pMT-puro, an insect expression vector encoding a C-terminal double Strep tag. Expression construct design was based on previously published structures of flavivirus antigens 61, 62, and 63. The YFV prM / E construct was used to generate an inducible, stable Drosophila S2 line. Protein expression was induced by the addition of copper sulfate and allowed to proceed for 5–7 days. The recombinant protein was affinity purified from the culture supernatant using a StrepTrap HP column (GE Healthcare). An additional purification step was performed using a size-exclusion chromatography step using an S200Increase column (GE Healthcare). The final protein preparation was stored in phosphate-buffered saline (pH 7.4) supplemented with an additional 150 mM NaCl. Small aliquots were stored at −70°C until use. Additional flavivirus antigens used in this study, i.e., DENV-2 E, DENV-4 E, WNV E, and ZIKV E, were expressed and purified essentially as described for YFV sE.

[0185] Flavivirus NS1 protein antigens: NS1 proteins from dengue virus (serotypes 1–4), JEV, TBEV, WNV, and YFV were purchased from Native Antigen Company (catalog numbers FLAVX4-NS1-100 and DENVX4-NS1-100). ZIKV NS1 was purchased from Meridian Life Sciences (catalog number R01636). Positive control antibodies reactive with the above NS1 proteins, i.e., anti-DENV NS1 (catalog number AbDENVNS1-DA034) and anti-ZIKV NS1 (catalog number AbZIKVNS1-B4-100), were obtained from Native Antigen Company. Anti-YFV NS1 protein antibody was purchased from Meridian Life Sciences (catalog number C01906M). Anti-WNV NS1 antibody (catalog no. HM484-X0632) and anti-TBEV NS1 antibody (catalog no. HM477-X1462) were purchased from East Coast Bio. Flavivirus cross-reactive serum was used to detect JEV NS1 protein.

[0186] YFV-17D DIII protein: The DIII region (aa 293-397) of the YFV-17D E protein (Uniprot ID: P03314) was produced in Drosophila S2 cells using a modified pT350 vector (Felix Rey, Institut Pasteur, France). Protein expression was induced with CdCl2, and the supernatant was harvested 5-7 days after induction. The recombinant protein was purified using size-exclusion chromatography using a Strep-Tactin column (IBA) and an S200Increase column (GE Healthcare) in 10 mM Tris (pH 8) / 150 mM NaCl buffer.

[0187] Single B cell sorting For plasmablast sorting, PBMCs were stained with anti-human CD38 (PE), CD27 (BV421), CD20 (PE-Cy7), CD3 (PerCP-Cy5.5), CD8 (PerCP-Cy5.5), CD14 (PerCP-Cy5.5), and CD16 (PerCP-Cy5.5). Plasmablasts were defined as CD19+CD3-CD20- / loCD27highCD38high cells. For MBC sorting, B cells were purified using a MACS B cell isolation kit (Miltenyi Biotec, catalog no. 130-091-151) and subsequently stained with anti-human CD19 (PE-Cy7), CD20 (PE-Cy7), CD3 (PerCP-Cy5.5), CD8 (PerCP-Cy5.5), CD14 (PerCP-Cy5.5), CD16 (PerCP-Cy5.5), IgD (BV421), IgM (AF-488), CD27 (BV510), CD21 (BV605), CD71 (APC-Cy7), and a mixture of double-labeled (APC and PE) YFV E tetramers (25 nM each). Tetramers were freshly prepared for each experiment, and B cells that showed reactivity to the YFV E tetramer were single-cell sorted. Single cells were analyzed using a BD FACS Aria. Using a 96-well PCR plate (BioRAD) containing 20 μL / well of lysis buffer (5 μL 5X first-strand cDNA buffer (Invitrogen), 0.625 μL NP-40 (New England Biolabs), 0.25 μL RNaseOUT (Invitrogen), 1.25 μL dithiothreitol (Invitrogen), and 12.6 μL dH2O), the plates were immediately stored at -80°C. Flow cytometry data were analyzed using FlowJo software.

[0188] Amplification and cloning of antibody variable genes Antibody variable genes (IgH, IgK, and IgL) were amplified by reverse transcription PCR and nested PCR using a cocktail of IgG- and IgM-specific primers, as previously described (Tiller et al., J Immunol 2008). The primers used in the second PCR contained 40 base pairs of 5' and 3' homology to the digested expression vector, which allowed cloning by homologous recombination into S. cerevisiae. The lithium acetate method for chemical conversion was used to clone the PCR products into S. cerevisiae (Gietz and Schiestl, Nat Protoc 2007). 10 μL of unpurified heavy and light chain PCR products and 200 ng of digested expression vector were used per transformation reaction. Following transformation, individual yeast colonies were picked for sequencing and characterization.

[0189] Expression and purification of IgG and Fab fragments IgG was expressed in S. cerevisiae cultures grown in 24-well plates as previously described (Bornholdt et al., Science 2016b). After 6 days, cultures were harvested by centrifugation, and IgG was purified by protein A affinity chromatography. Bound antibody was eluted with 200 mM acetic acid / 50 mM NaCl (pH 3.5) into 1 / 8 volume of 2 M Hepes (pH 8.0) and buffer-exchanged into PBS (pH 7.0).

[0190] Two YFV E-reactive control mAbs, 5A and 4G2, were produced within the human IgG1 constant region. The publicly available variable region sequences of the two control antibodies, 4G2 and 5A, were synthesized as gBlock fragments (IDT) with homologous overhangs for recombination cloning into S. cerevisiae. Subsequent production was performed as described above.

[0191] Fab fragments were generated by digesting IgG with papain for 2 hours at 30°C. The digestion was terminated by the addition of iodoacetamide, and the Fab and Fc mixture was passed over protein A agarose to remove the Fc fragment and undigested IgG. The flow-through from the protein A resin was then analyzed using CaptureSelect (商標) The IgG-CH1 affinity resin (ThermoFischer Scientific) was passed over and eluted with 200 mM acetic acid / 50 mM NaCl (pH 3.5) into 1 / 8 volume of 2 M Hepes (pH 8.0). The Fab fragments were then buffer exchanged into PBS, pH 7.0.

[0192] Measurement of binding kinetics Surface plasmon resonance kinetics (SPR) of IgG binding: A Biacore 8K system docked with a CAP sensor chip was used. The sample compartment was set to 10 °C, the flow cell temperature was set to 25 °C, and the data collection rate was set to 10 Hz. HBS-EP+ (10 mM HEPES (pH 7.3), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the running buffer. In each cycle, biotin CAPture reagent (GE Healthcare) diluted 1:20 in the running buffer was injected for 600 s at a flow rate of 5 μL / min on flow cells 1 and 2, followed by 900 s of capture (1 μL / min) of biotinylated YFV E antigen (25 nM in HBS-EP+) on flow cell 2, achieving a minimum capture level of 400 RU. Antibodies (36–288 nM in HBS-EP+) were then injected for 300 s (30 μL / min) over flow cells 1 and 2, dissociation was monitored for 300 s (30 μL / min), and the surface was regenerated at the oligonucleotide level with 6 M guanidine-HCl in 0.25 M NaOH for 120 s (10 μL / min). A minimum of two blank (HBS-EP+) injections were also performed under identical conditions as described above and used to evaluate and subtract system artifacts. Data were aligned, double-referenced, and fitted to a bivalent analyte binding model using Biacore 8K Evaluation Software version 1.0.

[0193] Surface plasmon resonance kinetics (SPR) of Fab binding: A Biacore 8K system docked with a CAP sensor chip was used. The sample compartment was set to 10 °C, the flow cell temperature was set to 25 °C, and the data collection rate was set to 10 Hz. HBS-EP+ (10 mM HEPES (pH 7.3), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the running buffer. In each cycle, biotin CAPture reagent (GE Healthcare) diluted 1:20 in the running buffer was injected for 600 s at a flow rate of 1 μL / min on flow cells 1 and 2, followed by 900 s of capture (1 μL / min) of biotinylated YFV E antigen (15 nM in HBS-EP+) on flow cell 2, achieving a minimum capture level of 275 RU. Fabs (A5: 27-1 nM in HBS-EP+, 4G2: 4-0.125 nM in HBS-EP+) were then injected for 300 s (30 μL / min) over flow cells 1 and 2, dissociation was monitored for 1200 s (30 μL / min), and the surface was regenerated at the oligonucleotide level with 6 M guanidine-HCl in 0.25 M NaOH for 185 s (10 μL / min). A minimum of two blank (HBS-EP+) injections were also performed under identical conditions as described above and used to evaluate and subtract system artifacts. Data were aligned, double-referenced, and fitted to a 1:1 binding model using Biacore 8K Evaluation Software version 1.0.

[0194] Biolayer Interferometry Kinetics (BLI): For monovalent apparent KD determination, IgG binding to recombinant YFV E antigen was measured by biolayer interferometry (BLI) using a ForteBio Octet HTX instrument (Molecular Devices). IgG was captured (1.5 nM) on an anti-human IgG capture (AHC) biosensor (Molecular Devices) and left in PBSF (PBS with 0.1% w / v BSA) for a minimum of 30 min. After a short (60 s) baseline step in PBSF, the IgG-loaded biosensor chip was exposed to YFV E antigen (100 nM in PBSF) for 180 s with orbital shaking at 1000 rpm and then immersed in PBSF to measure any dissociation of the antigen from the biosensor chip surface for 180 s with orbital shaking at 1000 rpm. Data for which the binding response was >0.1 n m were interstep corrected (for related steps) and fitted to a 1:1 binding model using ForteBio data analysis software version 11.1.

[0195] To determine the bivalent apparent KD, IgG binding to recombinant biotinylated YFV E antigen was measured by biolayer interferometry (BLI) using a ForteBio Octet HTX instrument (Molecular Devices). Recombinant biotinylated YFV E was immobilized on a streptavidin biosensor (Molecular Devices) and left in PBSF (PBS with 0.1% w / v BSA) for a minimum of 30 minutes. After a short (60-second) baseline step in PBSF, the antigen-loaded biosensor chip was exposed to IgG (100 nM in PBSF) (180 seconds, 1000 rpm orbital shaking) and then immersed in PBSF to measure any dissociation of IgG from the biosensor chip surface (180 seconds, 1000 rpm orbital shaking). Data with binding responses >0.1 nM were corrected between steps (for the relevant steps) and fitted to a 1:1 binding model using ForteBio data analysis software version 11.1.

[0196] High-throughput antibody epitope assignment Biolayer Interferometry (BLI) Epitope Binning: For epitope binning, control antibodies A5 and 4G2 (produced as human IgG1 chimeras) were captured on anti-human IgG capture biosensors (0.9 nm) (Molecular Devices). The biosensors were then blocked by exposing them to adalimumab (0.5 mg / mL, 20 min, 350 rpm orbital shaking). After a short (60 s) baseline step in PBSF, a cross-interaction check was performed between the sample IgG and the loaded biosensor (180 s, 1000 rpm orbital shaking). No cross-interactions were observed for this panel of IgGs. The loaded biosensor then underwent a second short (60 s) baseline step in PBSF, followed by an association step in 100 nM recombinant YFV E monomer (180 s, 1000 rpm orbital shaking). Finally, a binning step was performed on 100 nM sample IgG in PBS with 0.1% BSA (PBSF) (180 seconds, 1000 rpm orbital shaking). Data were analyzed using ForteBio data analysis software version 11.1. Sample IgGs with binning responses below 0.1 nM were determined to compete with the control antibody. Sample IgGs with binning responses above 0.1 nM were determined to be non-competitors to the control antibody.

[0197] High-Throughput Epitope Binning Using Carterra LSA (SPR) Binding kinetics and affinity. The kinetics and affinity constants of yellow fever antigen (45 kDa molecular weight, supplied by Adimab as purified recombinant monomer) binding to a library of 770+ADI mAbs (supplied as purified human IgG) were determined at 25 °C in a "capture kinetics" assay format using a Carterra high-throughput surface plasmon resonance (SPR) biosensor platform equipped with an HC-30M chip type. To prepare the surface for this experiment, the chip was coated as a "lawn" with the capture reagent, i.e., goat anti-human IgG Fc polyclonal (Southern Biotech, catalog number 2014-01) cross-adsorbed to serum proteins from multiple other species, using standard amine coupling in a running buffer of 10 mM Hepes (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20 (HBSET). Briefly, this involved priming a single flow cell (SFC) with HBSET running buffer, injecting a freshly prepared activation solution of 1:1:1 v / v / v 0.1 M N-hydroxysulfosuccinimide (Sulfo-NHS, Pierce) + 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Pierce) + 0.1 M MES (pH 5.5) (Carterra) for 10 min, binding 50 μg / ml goat anti-human IgG Fc diluted in 10 mM sodium acetate (pH 4.3) for 15 min, and quenching excess reactive ester with 1 M ethanolamine (pH 8.5) for 7 min. This resulted in an average final binding level of 6256 RU ± 4% variance (as judged by 384 reaction spots). A 96-channel printhead (96PH) was then primed in running buffer and used to capture ADI mAb as a ligand, which was diluted to 2 μg / ml in running buffer and batch printed 96 times at a time onto individual spots. Four serial docks of the 96PH were used to address all four print block locations, thus generating a 384-ligand array.The 96PH was returned to water for washing, and the SFC was docked onto the printed array and primed with assay running buffer of HBSET + 0.5 g / L BSA. Yellow fever monomer antigen analyte samples were prepared as an eight-member, four-fold dilution series spanning nominal concentrations of 0.02 to 367 nM and injected into the SFC at increasing concentrations after several buffer (blank) injections. Association and dissociation times were 5 and 20 minutes, respectively. Data were analyzed in Carterra's kinetics software as follows: Binding data on reaction spots were double-referenced by subtracting the response from a local reference spot (representing the bare capture reagent) and then subtracting the response from a buffer blank analyte. The double-referenced data were globally fit to a simple Langmuir model, allowing each spot its own association rate constant (ka), dissociation rate constant (kd), and Rmax value. The equilibrium dissociation (affinity) constant (KD) was calculated from the ratio of the reaction rate constants, KD = kd / ka).

[0198] Epitope binning experiments: A Carterra LSA was used to perform epitope binning assays in a classic sandwich assay format using six benchmark mAbs (ADI-49582, ADI-44112, ADI-45107, ADI-49147, 4G2, and 5A) as analytes to probe the epitope diversity of the 770+ ADI library as ligands. A HCX-30M (pre-activated) chip type was used, and the experiment was performed at 25°C. The SFC and 96PH were primed in a running buffer of 25 mM Mes (pH 5.5) + 0.01% Tween 20. ADI mAbs were diluted to 2 μg / ml in 10 mM sodium acetate (pH 4.5) (binding buffer) and bound via the 96PH using a 7-minute contact time at each print block location. After four sequential dockings of the 96PH to construct the 384-ligand array, the SFC was docked over the entire surface by injecting ethanolamine at pH 8.5 for 7 minutes to quench excess reactive esters. Final binding levels for each mAb ranged from 1,000 to 4,000 RU per spot. The 96PH was returned to water for washing, and the SFC was primed in an assay running buffer of HBSET + 0.5 g / L BSA. Each binning cycle involved simultaneous sample delivery, whereby antigen (50 nM yellow fever monomer) and antibody analyte (20 μg / mL mAb or buffer) samples were injected sequentially over the 384-ligand array with minimal dissociation time between them. Typical association times were 3 or 5 minutes, and the surface was regenerated with 75 mM phosphoric acid after each binning cycle. Binding data were analyzed using Carterra's Epitope software.

[0199] Microtiter Neutralization Assay Monoclonal antibodies were serially diluted in DMEM high glucose medium (Gibco) containing 10% heat-inactivated FBS (Gibco), 1% Gluta-MAX (Gibco), 1% P / S (Gibco), and 25 mM HEPES (Gibco) and incubated with YFV-17D or ZIKV for 1 hour at room temperature. YFV-17D or ZIKV was diluted to achieve 60% endpoint infection. The antibody-virus mixture was added to 5x10 cells seeded the day before. 3The cells were added in triplicate to a 96-well plate (Costar 3595) containing a monolayer of Huh 7.5.1 cells. The cells were incubated for 2 days at 37°C and 5% CO2. The cells were then fixed with 4% paraformaldehyde (Sigma) for 10 minutes and then washed three times with Tris buffer, pH 7.6 (50 mM Tris, 150 mM NaCl (all Fisher Scientific)). The fixed cells were incubated for 1 hour at room temperature (RT) with the pan-flavivirus mouse mAb 4G2 (ATCC) at 2 μg / ml in Tris buffer containing 3% nonfat dry milk (BioRad), 0.5% Triton X-100 (MP Biomedicals), and 0.05% Tween 20 (Fisher Scientific). The cells were then washed three times and stained with Alexa Fluor 978 (Alexa Fluor 978). Cells were incubated with secondary antibody conjugated to 488 goat anti-mouse (Invitrogen) at a 1:500 dilution for 1 hour at room temperature. Cells were washed again, and nuclei were stained with Hoechst-33342 (Invitrogen) at a 1:2,000 dilution in PBS. Viral infectivity was measured by automated enumeration of Alexa Fluor 488-positive cells from images captured using a Cytation-5 automated fluorescence microscope (BioTek) and analyzed using Gen5 data analysis software (BioTek). The half-maximal inhibitory concentrations (IC50s) of mAbs were calculated using nonlinear regression analysis with GraphPad Prism software. Virus neutralization data were subjected to nonlinear regression analysis (four-parameter, variable-slope sigmoidal dose-response equation, GraphPad Prism) to extract half-maximal inhibitory concentrations (IC50s).

[0200] Neutralization of donor plasma samples was performed exactly as described above for purified IgG. Serial dilutions of plasma were preincubated with YFV-17D infectious stock for 1 h before addition to cell monolayers.

[0201] Purified total human IgG from a non-immunized donor was used as a negative control in purified IgG neutralization assays against YFV-17D and ZIKV (catalog no. AB_2337042, Jackson Immuno Research).

[0202] FRNT assay Virus-specific mAbs were screened as previously described. Briefly, all purified mAbs were serially diluted in 199 medium (Thermo Scientific) containing 5% heat-inactivated fetal bovine serum (FBS) (Gibco-Invitrogen) and incubated with YFV-17DD at 37°C. After 1 hour of incubation, the Ab-virus mixture was added in duplicate to a 96-well plate containing an 80% confluent monolayer of Vero E6 cells. The plate was incubated at 37°C for 1.5 hours. The wells were then covered with 1% methylcellulose in supplemented OptiMEM GlutaMAX medium (Invitrogen) with 5% heat-inactivated FBS (Gibco-Invitrogen) and 1% amphotericin B and incubated at 37°C and 5% CO2 for 72 hours. The cells were then fixed and permeabilized with Perm / Wash buffer (BD Biosciences) for 30 minutes. After permeabilization, cells were washed with phosphate-buffered saline (PBS) and incubated for 2 hours with a 1:2000 dilution of anti-flavivirus antibody (MAB10216, EMD Millipore) in Perm / Wash buffer. After incubation, cells were washed with PBS and incubated for 2 hours with an anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (115035146, Jackson ImmunoResearch Laboratories). Plates were washed and developed with peroxidase substrate (KPL). The half-maximal inhibitory concentrations (IC50s) of mAbs were calculated using nonlinear regression analysis with GraphPad Prism software.

[0203] Serum and purified IgG ELISA For NS1 and E binding ELISAs, 96-well plates (Corning, catalog no. 3690) were coated with 5 μg / ml of NS1 or E protein diluted in PBS and incubated overnight at 4°C. The wells were washed and then blocked with 5% nonfat dry milk (NFDM) in PBS for 1 hour at 37°C. The wells were washed three times with PBS, and serial dilutions of human plasma in 5% NFDM-PBS were added and incubated for 1 hour at 37°C. The plates were then washed three times with PBS, and secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher Scientific, catalog no. 31413) or anti-human IgM (Sigma Aldrich, catalog no. AP114P) detection antibodies were added at a 1:8000 dilution in 5% NFDM-PBS for 1 hour at 37°C. After washing three times with PBS, detection reagent was added according to the manufacturer's recommendations (Thermo Scientific, catalog no. 34029), and absorbance was measured at 450 nM wavelength using a Spectramax microplate reader (Molecular Devices).

[0204] For virus-binding ELISA, 96-well ELISA plates were coated with 5 μg / ml 4G2 (Millipore MAB10216) diluted in PBS and incubated for 2 hours at 37°C. After washing three times with PBS, whole YFV-17D virus particles were diluted in PBS, pH 7.4, and incubated overnight at 4°C. The plates were then washed three times with PBS and blocked with 5% NFDM-PBS for 1 hour at 37°C. After removal of the blocking solution, test antibodies diluted in 5% NFDM-PBS were allowed to bind for 1 hour at 37°C. The plates were then washed three times with PBS, and secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher Scientific, catalog no. 31413) or anti-human IgM (Sigma Aldrich, catalog no. AP114P) detection antibodies were added at a 1:8000 dilution in 5% NFDM-PBS for 1 hour at 37°C. After washing three times with PBS, detection reagent was added according to the manufacturer's recommendations (Thermo Scientific, catalog no. 34029), and absorbance was measured at 450 nM wavelength using a Spectramax microplate reader (Molecular Devices).

[0205] Binding of purified IgG to viral particles was performed as described above. IgG was diluted in 5% NFDM-PBS and tested at 100 nM concentration for single-point reactivity testing of day 14 antibodies from plasmablasts and MBCs.

[0206] All references, patents, and patent publications cited herein are hereby incorporated by reference in their entirety for all that they teach. Further aspects of the present invention are described below: [Section 1] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a Yellow Fever Virus (YFV) protein, wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the antibody or the antigen-binding fragment thereof is at least 70% identical to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 3 of an antibody selected from Antibody No. 1 to Antibody No. 152 disclosed in Table 3, and the antibody or the antigen-binding fragment thereof also has the following characteristics: a) the antibody or antigen-binding fragment thereof cross-competes with the antibody or antigen-binding fragment thereof for binding to YFV; b) the antibody or antigen-binding fragment thereof exhibits neutralizing activity towards YFV in vitro; c) the antibody or antigen-binding fragment thereof has an in vitro neutralizing capacity (IC) of about 0.5 micrograms per milliliter (μg / ml) to about 5 μg / ml, about 0.05 μg / ml to about 0.5 μg / ml, or less than about 0.05 mg / ml 50 ), and d) The antibody or antigen-binding fragment thereof binds to the envelope protein of YFV. [Section 2] Item 1, wherein the antibody or antigen-binding fragment thereof comprises at least two of properties a) to d). [Section 3] The antibody or antigen-binding fragment thereof a) the CDRH3 amino acid sequence of any one of the antibodies designated as Antibody No. 1 to Antibody No. 152 disclosed in Table 3; b) the CDRH2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; c) the CDRH1 amino acid sequence of any one of the antibodies designated as Antibody No. 1 to Antibody No. 152 disclosed in Table 3; d) the CDRL3 amino acid sequence of any one of the antibodies designated as Antibody No. 1 to Antibody No. 152 disclosed in Table 3; e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; f) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; or g) An isolated antibody or antigen-binding fragment thereof according to paragraph 1, comprising any combination of two or more of a), b), c), d), e), and f). [Section 4] The antibody or antigen-binding fragment thereof a) the heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; and b) An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising at least one of the light chain (LC) amino acid sequences of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 in Table 3. [Section 5] The isolated antibody or antigen-binding fragment thereof according to item 1, wherein the antibody is selected from the group consisting of antibodies that are at least 70% identical to any one of the antibodies designated Antibody No. 1 to Antibody No. 152 in Table 3. [Section 6] Item 1. The isolated antibody or antigen-binding fragment thereof according to Item 1, wherein the antibody is selected from the group consisting of antibodies designated as Antibody No. 1 to Antibody No. 152 disclosed in Table 3. [Section 7] An isolated nucleic acid sequence encoding the antibody or antigen-binding fragment thereof of paragraph 1. [Section 8] Item 8. An expression vector comprising the isolated nucleic acid sequence of Item 7. [Section 9] A host cell transfected, transformed or transduced with a nucleic acid sequence according to paragraph 7. [Section 10] 1. A pharmaceutical composition comprising: One or more of the isolated antibodies or antigen-binding fragments thereof according to paragraph 1; and a pharmaceutically acceptable carrier and / or excipient. [Section 11] 1. A pharmaceutical composition comprising: One or more nucleic acid sequences according to paragraph 7; and a pharmaceutically acceptable carrier and / or excipient. [Section 12] A host cell comprising the nucleic acid sequence of paragraph 7. [Section 13] A method for treating or preventing a yellow fever virus (YFV) infection or at least one symptom associated with a YFV infection, comprising administering one or more antibodies or antigen-binding fragments thereof described in paragraph 1 to a patient in need of or suspected of needing treatment or prevention, such that the YFV infection is treated or prevented, or the at least one symptom associated with a YFV infection is treated, alleviated, or reduced in severity. [Section 14] 1. A method of treating or preventing either a yellow fever virus (YFV) infection or at least one symptom associated with said YFV infection, comprising: A method comprising administering one or more antibodies or antigen-binding fragments thereof described in paragraph 1 to a patient in need of or suspected of in need of treatment or prevention, so that the YFV infection is treated or prevented, or at least one symptom associated with YFV infection is treated, alleviated, or reduced in severity. [Section 15] Item 14. The method of paragraph 13, wherein the method further comprises administering a second therapeutic agent to the patient. [Section 16] Item 16. The method of item 15, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, a vaccine specific to YFV, an siRNA specific to a YFV antigen, and a secondary antibody specific to a YFV antigen. [Section 17] 11. The pharmaceutical composition of claim 10, for use in preventing Yellow Fever Virus (YFV) infection in a patient in need of or suspected of needing such prevention, or for treating a patient suffering from a YFV infection, or for ameliorating at least one symptom or complication associated with said infection, wherein such use results in either the prevention of said infection, or the prevention, amelioration, or reduction in severity and / or duration of at least one symptom or complication associated with said infection, and wherein said use comprises the step of administering the pharmaceutical composition to said patient. [Section 18] 11. The pharmaceutical composition of claim 10, for use in treating or preventing either a Yellow Fever Virus (YFV) infection or at least one symptom associated with said YFV infection in a patient in need of or suspected of being in need of treatment or prevention, wherein such use results in either prevention of said infection or prevention, or prevention, or prevention, or amelioration, or reduction in severity and / or duration of at least one symptom or complication associated with said infection, said use comprising the step of administering said pharmaceutical composition to said patient. [Section 19] 11. Use of the pharmaceutical composition of clause 10 in the manufacture of a medicament for preventing Yellow Fever Virus (YFV) infection in a patient in need thereof, or for treating a patient suffering from YFV infection, or for ameliorating at least one symptom or complication associated with said infection, wherein said infection is prevented, or at least one symptom or complication associated with said infection is either prevented, ameliorated, or reduced in severity and / or duration, said use comprising the step of administering said pharmaceutical composition to said patient. [Section 20] 11. Use of the pharmaceutical composition of clause 10 in the manufacture of a medicament for the prevention of either a Yellow Fever Virus (YFV) infection or at least one symptom associated with said YFV infection in a patient in need of or suspected of being in need of such prevention, wherein such use results in either the prevention of said infection or the prevention, amelioration, or reduction in severity and / or duration of at least one symptom or complication associated with said infection, said use comprising the step of administering said pharmaceutical composition to said patient. [Section 21] 1. A Yellow Fever Virus (YFV) antibody or antigen-binding fragment thereof, comprising the sequence: a) QQX1X2X3X4X5X6T, where X1 is Y, F, or A, X2 is N, H, or Y, X3 is R, S, T, or D, X4 is D, F, Y, W, or P, X5 is P or S, and X6 is Y, F, K, or W; b) QX1X2X3X4TX5X6T, where X1 is Q or H, X2 is A or S, X3 is S or Y, X4 is T or S, X5 is R or P, and X6 is Y, L, W, or R; or c) A YFV antibody or antigen-binding fragment thereof, comprising a CDRL3 containing the consensus motif GTWDX1SX2X3SAGX4V, where X1 is S or T, X2 is S or is not an amino acid, X3 is L or P, and X4 is K, G, or R. [Section 22] A yellow fever virus (YFV) antibody or antigen-binding fragment thereof, comprising a CDRH3 containing a consensus motif having the sequence AX1X2YDSX3X4YYX5X6X7X8, where X1 is K or R, X2 is Y, F, T, A, G, Y, or H, X3 is S, N, or R, X4 is A or G, X5 is W or Y, X6 is F, L, I, A, or E, X7 is D, E, or H, and X8 is Y, H, or S. [Section 23] 1. A Yellow Fever Virus (YFV) antibody or antigen-binding fragment thereof, comprising a YFV binding domain CDRL2, wherein the CDRL2 binding domain comprises a consensus motif having the sequence: a) X1X2X3X4RPS, where X1 is D or E, X2 is N, V, or D, X3 is K, N, D, or S, and X4 is K, E, or R; b) X1X2X3X4LX5X6, where X1 is A, G, or R, X2 is A or T, X3 is S or T, X4 is T, G, S, or I, X5 is Q or R, and X6 is S or R; c) X1X2SX3RAX4, where X1 is G, D, R, or A, X2 is A or S, X3 is S, T, or N, and X4 is T or A; d) X1VX2X3RPS, where X1 is D, E, or R, X2 is S, T, N, or A, and X3 is N, K, or Q; or e) A YFV antibody or antigen-binding fragment thereof, comprising X1ASX2LEX3, wherein X1 is R, Q, or K, X2 is T, S, G, R, or I, and X3 is T or S. [Section 24] 1. A Yellow Fever Virus (YFV) antibody or antigen-binding fragment thereof, comprising the sequence: a) X1 is D, E, or S, X2 is I or V, X3 is F or Y, X4 is X or T, X5 is G or E, X6 is S, G, or T, X7 is T or A, X8 is N, S, H, K, or T, X9 is N or S, and X 10 is S or F, and X 11 is L or V, and X 12 is K or E, X1X2X3HX4X5X6X7X8YX9PX 10 X 11 X 12 S, or b) X1 is V or L, X2 is I or M, X3 is S, W, or L, X4 is F or Y, X5 is E or G, X6 is S or T, X7 is K, N, or Y, X8 is F, W, or Y, X9 is Y or F, and X 10 is V or L, and X 11 is K or R, X1X2X3X4DX5X6X7KX8X9ADSX 10 X 11 A YFV antibody or antigen-binding fragment thereof, comprising a CDRH2 containing a consensus motif having G. [Section 25] 1. A Yellow Fever Virus (YFV) antibody or antigen-binding fragment thereof, comprising the sequence: a) RX1SX2X3X4X5X6X7X8X9, where X1 is A or T, X2 is Q or R, X3 is S or T, X4 is I or V, X5 is S or T, X6 is S, N, T, F, D, or G, X7 is N, Y, W, F, or K, X8 is L or V, and X9 is A or N; b) SGSX1SNX2GX3X4X5VX6, where X1 is N or S, X2 is I or F, X3 is S or N, X4 is N, Y, S, or D, X5 is Y, F, or D, and X6 is S or A; c) A YFV antibody or antigen-binding fragment thereof, comprising a CDRL1 containing the consensus motif X1GTX2X3DX4GX5X6X7X8VS, where X1 is A or T, X2 is S, G, or R, X3 is S or T, X4 is V, F, or I, X5 is G or A, X6 is Y, D, or F, X7 is K or N, and X8 is Y or F. [Section 26] 1. A Yellow Fever Virus (YFV) antibody or antigen-binding fragment thereof, comprising the sequence: a) X1X2FX3X4X5X6X7X8, where X1 is F, Y, or L, X2 is T, A, S, or N, X3 is S or T, X4 is S, T, or R, X5 is Y or L, X6 is G, A, T, W, S, or D, X7 is M, I, or L, and X8 is H, S, N, or T; b) XI SIX 2 X3 X4 X5 X6 WX7, where X1 is G or I, X2 is S or T, X3 is S, T, G or is not an amino acid, X4 is D, S, T or G, X5 is Y, N or D, X6 is W or Y and X7 is S or T; c) A YFV antibody or antigen-binding fragment thereof, comprising a CDRH1-binding domain containing the consensus motif FX1FSDX2YMX3, wherein X1 is I or T, X2 is H or Y, and X3 is A or D. [Section 27] Item 28. The YFV antibody or antigen-binding fragment thereof according to any one of Items 22 to 27, which binds to the E protein of YFV. [Section 28] 28. The YFV antibody or antigen-binding fragment thereof according to any one of items 22 to 27, which further binds to the E protein of DENV-2, DENV-4, WNV, and / or ZIKV. [Section 29] Antibodies that bind to YFV E protein. [Section 30] The antibody of claim 29, wherein the antibody binds to at least one of an epitope within the FL of domain II of the YFV E protein, a protein proximal to the FL of domain II of the YFV E protein, and a protein within domain III of YFV. [Section 31] The antibody has the following characteristics: a) the antibody or antigen-binding fragment thereof exhibits a clean or low polyreactivity profile; b) the antibody or antigen-binding fragment thereof has an in vitro neutralizing capacity (IC) of about 0.5 micrograms per milliliter (μg / ml) to about 5 μg / ml, about 0.05 μg / ml to about 0.5 μg / ml, or less than about 0.05 mg / ml.50 30. The antibody of claim 29, wherein the antibody exhibits one or more of the following characteristics: (a) the antibody or antigen-binding fragment thereof exhibits a specific binding affinity to YFV-17D particles; (b) the antibody or antigen-binding fragment thereof binds to YFV-17D particles; and (c) the antibody or antigen-binding fragment thereof binds to the envelope protein of YFV. [Section 32] The antibody is about 1×10 -6 M ~ approx. 1×10 -10 30. The antibody of paragraph 29, which exhibits an equilibrium dissociation constant of M. [Section 33] The antibody is a high affinity binder and has a binding affinity of at least 10 -9 The antibody of paragraph 29, which exhibits binding affinity for YFV of M. [Section 34] The antibody was 1×10 -3 s -1 The antibody of paragraph 29, having the following dissociation rate constant: [Section 35] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a Yellow Fever Virus (YFV) protein, wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the antibody or antigen-binding fragment thereof is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween, to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 3 of an antibody selected from Antibody No. 1 to Antibody No. 152 disclosed in Table 3, and the antibody or antigen-binding fragment thereof also has the following characteristics: a) the antibody or antigen-binding fragment thereof cross-competes with the antibody or antigen-binding fragment thereof for binding to YFV; b) the antibody or antigen-binding fragment thereof exhibits neutralizing activity towards YFV in vitro; c) the antibody or antigen-binding fragment thereof has an in vitro neutralizing capacity (IC) of about 0.5 micrograms per milliliter (μg / ml) to about 5 μg / ml, about 0.05 μg / ml to about 0.5 μg / ml, or less than about 0.05 mg / ml 50 ) or d) The antibody or antigen-binding fragment thereof binds to the envelope protein of YFV. [Section 36] Item 1, wherein the antibody or antigen-binding fragment thereof comprises at least two, at least three, or at least four of properties a) to d). [Section 37] The antibody or antigen-binding fragment thereof a) the CDRH3 amino acid sequence of any one of the antibodies designated as Antibody No. 1 to Antibody No. 152 disclosed in Table 3; b) the CDRH2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; c) the CDRH1 amino acid sequence of any one of the antibodies designated as Antibody No. 1 to Antibody No. 152 disclosed in Table 3; d) the CDRL3 amino acid sequence of any one of the antibodies designated as Antibody No. 1 to Antibody No. 152 disclosed in Table 3; e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; f) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3; or g) An isolated antibody or antigen-binding fragment thereof according to paragraph 35 or 36, comprising any combination of two or more of a), b), c), d), e), and f). [Section 38] The antibody or antigen-binding fragment thereof a) the heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3, and / or b) An isolated antibody or antigen-binding fragment thereof according to any one of items 35 to 37, comprising the light chain (LC) amino acid sequence of any one of the antibodies designated Antibody No. 1 to Antibody No. 152 as disclosed in Table 3. [Section 39] 39. The isolated antibody or antigen-binding fragment thereof according to any one of paragraphs 35 to 38, wherein the antibody is selected from the group consisting of antibodies that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages of identity therebetween, to at least one of the antibodies designated Antibody No. 1 to Antibody No. 152 disclosed in Table 3. [Section 40] 40. The isolated antibody or antigen-binding fragment thereof according to any one of items 35 to 39, wherein the antibody is selected from the group consisting of antibodies designated as antibody No. 1 to antibody No. 152 disclosed in Table 3. [Section 41] 41. An isolated nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to any one of items 35 to 40. [Section 42] 42. An expression vector comprising the isolated nucleic acid sequence of paragraph 41. [Section 43] A host cell transfected, transformed or transduced with the nucleic acid sequence of Paragraph 41 or the expression vector of Paragraph 42. [Section 44] A pharmaceutical composition comprising one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of items 35 to 40, and a pharmaceutically acceptable carrier and / or excipient. [Section 45] A pharmaceutical composition comprising one or more nucleic acid sequences according to paragraph 41 or one or more expression vectors according to paragraph 42, and a pharmaceutically acceptable carrier and / or excipient. [Section 46] A host cell comprising the nucleic acid sequence of Paragraph 41 or the expression vector of Paragraph 42. [Section 47] 1. A method for treating or preventing yellow fever virus (YFV) infection or at least one symptom associated with YFV infection, comprising administering to a patient in need of or suspected of being in need of treatment or prevention: a) one or more antibodies or antigen-binding fragments thereof according to any one of items 35 to 40; b) the nucleic acid sequence according to item 41, c) the expression vector according to item 42; d) a host cell according to paragraph 43, or e) administering the pharmaceutical composition according to item 44 or 45, Thus, the method wherein the YFV infection is treated or prevented, or at least one symptom associated with the YFV infection is treated, alleviated, or reduced in severity. [Section 48] 1. A method for treating or preventing either a yellow fever virus (YFV) infection or at least one symptom associated with said YFV infection, comprising administering to a patient in need of or suspected of being in need of treatment or prevention: a) one or more antibodies or antigen-binding fragments thereof according to any one of items 35 to 40; b) the nucleic acid sequence according to item 41, c) the expression vector according to item 42; d) a host cell according to paragraph 43, or e) administering the pharmaceutical composition according to item 44 or 45, Thus, the method wherein the YFV infection is treated or prevented, or at least one symptom associated with the YFV infection is treated, alleviated, or reduced in severity. [Section 49] Item 49. The method according to any one of Items 47 to 48, further comprising administering a second therapeutic agent to the patient. [Section 50] Item 50. The method of item 49, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, a vaccine specific to YFV, an siRNA specific to a YFV antigen, and a secondary antibody specific to a YFV antigen. [Section 51] A pharmaceutical composition comprising any one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of items 35 to 41, and a pharmaceutically acceptable carrier and / or excipient. [Section 52] 52. The pharmaceutical composition of clause 51 for use in preventing Yellow Fever Virus (YFV) infection in a patient in need thereof or suspected of being in need thereof, or for treating a patient suffering from a YFV infection, or for ameliorating at least one symptom or complication associated with said infection, wherein such use results in the infection being prevented, or at least one symptom or complication associated with said infection being prevented, ameliorated, or reduced in severity and / or duration. [Section 53] 52. The pharmaceutical composition of claim 51 for use in treating or preventing either a Yellow Fever Virus (YFV) infection or at least one symptom associated with said YFV infection in a patient in need of or suspected of needing treatment or prevention, wherein such use results in either the infection being prevented, or at least one symptom or complication associated with said infection being prevented, ameliorated, or reduced in severity and / or duration. [Section 54] 52. Use of the pharmaceutical composition of clause 51 in the manufacture of a medicament for preventing Yellow Fever Virus (YFV) infection in a patient in need thereof, or for treating a patient suffering from YFV infection, or for ameliorating at least one symptom or complication associated with said infection, wherein said infection is prevented, or at least one symptom or complication associated with said infection is either prevented, ameliorated, or reduced in severity and / or duration.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a Yellow Fever Virus (YFV) protein, wherein the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 308 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 460; b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 307 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 459; c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 311 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 463; d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 320 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 471; or e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 341 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 492 1. An isolated antibody or antigen-binding fragment thereof comprising:

2. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 307 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

459.

3. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 308 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

460.

4. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 311 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

463.

5. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 320 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

471.

6. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 341 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

492.

7. 7. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 6, having the following characteristics: a) exhibits neutralizing activity against YFV in vitro; b) an in vitro neutralizing capacity (IC) of about 0.5 micrograms per milliliter (μg / ml) to about 5 μg / ml, about 0.05 μg / ml to about 0.5 μg / ml, or less than about 0.05 mg / ml ５０ ) and / or c) Binds to the envelope protein of YFV.

8. In vitro neutralization capacity (IC) against YFV of about 0.5 micrograms / milliliter (μg / ml) to about 5 μg / ml ５０ 8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment exhibits the following sequence:

9. In vitro neutralization capacity (IC) against YFV of about 0.05 μg / ml to about 0.5 μg / ml ５０ 8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment exhibits the following sequence:

10. In vitro neutralization capacity (IC) against YFV of less than about 0.05 mg / ml ５０ 8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment exhibits the following sequence:

11. The antibody or antigen-binding fragment thereof is about 1 x 10 －６ M ~ approx. 1 x 10 －１０ 11. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 10, which exhibits an equilibrium dissociation constant of M.

12. 12. The isolated antibody or antigen-binding fragment thereof of any one of claims 7 to 11, wherein the antibody or antigen-binding fragment thereof comprises at least two of properties a), b) and / or c).

13. 12. The isolated antibody or antigen-binding fragment thereof of any one of claims 7 to 11, wherein the antibody or antigen-binding fragment thereof comprises properties a), b) and c).

14. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 13 and a pharmaceutically acceptable carrier and / or excipient.

15. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 13 and a pharmaceutically acceptable carrier and / or excipient for treating or preventing yellow fever virus (YFV) infection or at least one symptom caused by YFV infection.

16. A pharmaceutical composition for treating or preventing a yellow fever virus (YFV) infection or at least one symptom caused by a YFV infection, comprising an isolated nucleic acid sequence encoding the isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 13, and a pharmaceutically acceptable carrier and / or excipient.

17. 17. The pharmaceutical composition of claim 15 or 16, wherein at least one symptom resulting from YFV infection is treated, ameliorated, or reduced in severity.

18. The pharmaceutical composition of any one of claims 15 to 17, wherein at least one symptom caused by YFV infection is fever, chills, headache, back pain, muscle pain, loss of appetite, nausea, vomiting or fatigue.

19. Use of an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 13 in the manufacture of a medicament for treating or preventing yellow fever virus (YFV) infection or at least one symptom caused by YFV infection.

20. Use of an isolated nucleic acid in the manufacture of a medicament for treating or preventing a yellow fever virus (YFV) infection or at least one symptom resulting from a YFV infection, wherein the isolated nucleic acid encodes an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 13.

21. 21. The use of claim 19 or 20, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 308 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

460.

22. 22. The use according to any one of claims 19 to 21, wherein at least one symptom resulting from YFV infection is treated, ameliorated, or reduced in severity.

23. 22. The use according to any one of claims 19 to 21, wherein at least one symptom resulting from YFV infection is fever, chills, headache, back pain, muscle pain, loss of appetite, nausea, vomiting or fatigue.

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