METHODS FOR IDENTIFICATION OF ANTIGEN BINDING SPECIFICITY OF ANTIBODIES
20220315982 · 2022-10-06
Inventors
- Marion Francis Setliff (Nashville, TN, US)
- Ivelin Stefanov Georgiev (Brentwood, TN, US)
- Andrea Shiakolas (Nashville, TN, US)
Cpc classification
C12N15/1096
CHEMISTRY; METALLURGY
International classification
C12N15/10
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to a method for simultaneous detection of antigens and antigen specific antibodies. LIBRA-seq (Linking B Cell Receptor to Antigen specificity through sequencing) is developed to simultaneously recover both antigen specificity and paired heavy and light chain BCR sequence. LIBRA-seq is a next-generation sequencing-based readout for BCR-antigen binding interactions that utilizes oligonucleotides (oligos) conjugated to recombinant antigens.
Claims
1. A method for simultaneous detection of an antigen and an antibody that specifically binds said antigen, comprising: labeling a plurality of antigens with unique antigen barcodes; providing a plurality of barcode-labeled antigens to a population of B-cells; allowing the plurality of barcode-labeled antigens to bind to the population of B-cells; washing unbound antigens from the population of B-cells; separating the B-cells into single cell emulsions; introducing into each single cell emulsion a unique cell barcode-labeled bead; preparing a single cell cDNA library from the single cell emulsions; performing PCR amplification reactions to produce a plurality of amplicons, wherein the amplicons comprise: 1) the cell barcode and the antigen barcode, 2) the cell barcode and an antibody sequence, and 3) a unique molecular identifier (UMI); sequencing the plurality of amplicons; removing a sequence lacking the cell barcode, the UMI, or the antigen barcode; aligning the antibody sequence to a reference library of immunoglobulin V, D, J and C sequences; constructing a UMI count matrix comprising the cell barcode, the antigen barcode, and the antibody sequence; determining a LIBRA-seq score; and determining that the antibody specifically binds an antigen if the LIBRA-seq score of the antibody for the antigen is increased in comparison to a control sample.
2. The method of claim 1, wherein the barcode-labeled antigens are labeled with a first barcode comprising a DNA sequence or an RNA sequence.
3. The method of claim 1, wherein the cell barcode-labeled beads are labeled with a second barcode comprising a DNA sequence or an RNA sequence.
4. The method of claim 1, wherein the antibody sequence comprises an immunoglobulin heavy chain (VDJ) sequence, or an immunoglobulin light chain (VJ) sequence.
5. The method of claim 1, wherein the barcode-labeled antigens comprise an antigen from a pathogen or an animal.
6. The method of claim 5, wherein the antigen from a pathogen comprises an antigen from a virus.
7. The method of claim 6, wherein the antigen from a virus comprises an antigen from human immunodeficiency virus (HIV), an antigen from influenza virus, or an antigen from respiratory syncytial virus (RSV).
8. The method of claim 1, further comprising determining a level of somatic hypermutation of the antibody specifically binding to the antigen.
9. The method of claim 1, further comprising determining a length of a complementarity-determining region (CDR) of the antibody specifically binding to the antigen.
10. The method of claim 1, further comprising determining a motif of a CDR of the antibody specifically binding to the antigen.
11. The method of claim 9, wherein the CDR is selected from the group consisting of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.
12. A method of determining a broadly neutralizing antibody to a pathogen, said method comprising: labeling a plurality of antigens derived from the pathogen with unique antigen barcodes; providing a plurality of barcode-labeled antigens to a population of B-cells; allowing the plurality of barcode-labeled antigens to bind to the population of B-cells; washing unbound antigens from the population of B-cells; separating the B-cells into single cell emulsions; introducing into each single cell emulsion a unique cell barcode-labeled bead; preparing a single cell cDNA library from the single cell emulsions; performing PCR amplification reactions to produce a plurality of amplicons, wherein the amplicons comprise: 1) the cell barcode and the antigen barcode, 2) the cell barcode and an antibody sequence, and 3) a unique molecular identifier (UMI); sequencing the plurality of amplicons; removing a sequence lacking a cell barcode, unique molecular identifier (UMI), or an antigen barcode; aligning the antibody sequence to a reference library of immunoglobulin V, D, J and C sequences; constructing a UMI count matrix comprising the cell barcode, the antigen barcode, and the antibody sequence; determining a LIBRA-seq score; and determining that the antibody is a broadly neutralizing antibody if the LIBRA-seq scores of the antibody for two or more antigens are increased in comparison to a control.
13. The method of claim 12, wherein the barcode-labeled antigens are labeled with a first barcode comprising a DNA sequence or an RNA sequence.
14. The method of claim 12, wherein the cell barcode-labeled beads are labeled with a second barcode comprising a DNA sequence or an RNA sequence.
15. The method of claim 12, wherein the antibody sequence comprises an immunoglobulin heavy chain (VDJ) sequence, or an immunoglobulin light chain (VJ) sequence.
16. The method of claim 12, wherein the barcode-labeled antigens comprise an antigen from a pathogen or an animal.
17. The method of claim 16, wherein the antigen from a pathogen comprises an antigen from a virus.
18. The method of claim 17, wherein the antigen from a virus comprises an antigen from human immunodeficiency virus (HIV), an antigen from influenza virus, or an antigen from respiratory syncytial virus (RSV).
19. The method of claim 12, further comprising determining a level of somatic hypermutation of the antibody specifically binding to the antigen.
20. The method of claim 12, further comprising determining a length of a complementarity-determining region (CDR) of the antibody specifically binding to the antigen.
21. The method of claim 12, further comprising determining a motif of a CDR of the antibody specifically binding to the antigen.
22. The method of claim 20, wherein the CDR is selected from the group consisting of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.
Description
DESCRIPTION OF DRAWINGS
[0044] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate aspects described below.
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DETAILED DESCRIPTION
[0058] Recent advances in next-generation sequencing (NGS) enable high-throughput interrogation of antibody repertoires at the sequence level, including paired heavy and light chains (Busse et al., 2014; Dekosky et al., 2013; Tan et al., 2014). However, annotation of NGS antibody sequences for their cognate antigen partner(s) generally requires synthesis, production and characterization of individual recombinant monoclonal antibodies (DeFalco et al., 2018; Setliff et al., 2018). Recent efforts to develop new antibody screening technologies have sought to overcome throughput limitations while still uniting antibody sequence and functional information. For example, natively-paired human BCR heavy and light chain amplicons can be expressed and screened as Fab (Wang et at, 2018) or scFV (Adler et al., 2017b, 2017a) in a yeast display system. Although these various antibody discovery technologies have led to the identification of a number of potently neutralizing antibodies, they remain limited by the number of antigens against which single cells can simultaneously be screened efficiently.
[0059] LIBRA-seq (LInking B Cell Receptor to Antigen specificity through sequencing) is developed to simultaneously recover both antigen specificity and paired heavy and light chain BCR sequence. LIBRA-seq is a next-generation sequencing-based readout for BCR-antigen binding interactions that utilizes oligonucleotides (oligos) conjugated to recombinant antigens. Antigen barcodes are recovered during paired-chain BCR sequencing experiments and bioinformatically mapped to single cells. The LIBRA-seq method was applied to PBMC samples from two HIV-infected subjects, and from these, HIV- and influenza-specific antibodies were successfully identified, including both known and novel broadly neutralizing antibody (bNAb) lineages. LIBRA-seq is high-throughput, scalable, and applicable to many targets. This single, integrated assay enables the mapping of monoclonal antibody sequences to panels of diverse antigens theoretically unlimited in number and facilitates the rapid identification of cross-reactive antibodies that serves as therapeutics or vaccine templates.
[0060] Disclosed herein are systems and methods for simultaneous detection of antigens and antigen specific antibodies.
[0061] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.
[0063] The following definitions are provided for the full understanding of terms used in this specification.
Terminology
[0064] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
[0065] As used herein, the term “subject” or “host” can refer to living organisms such as mammals, including, but not limited to humans, livestock, dogs, cats, and other mammals. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a human
[0066] “Nucleotide,” “nucleoside,” “nucleotide residue,” and “nucleoside residue,” as used herein, can mean a deoxyribonucleotide or ribonucleotide residue, or other similar nucleoside analogue. A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3′-AMP (3′-adenosine monophosphate) or 5′-GMP (5′-guanosine monophosphate). There are many varieties of these types of molecules available in the art and available herein.
[0067] The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers.
[0068] The method and the system disclosed here including the use of primers, which are capable of interacting with the disclosed nucleic acids, such as the antigen barcode as disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions. Typically, the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner Typically, the disclosed primers hybridize with the disclosed nucleic acids or region of the nucleic acids or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids.
[0069] The term “amplification” refers to the production of one or more copies of a genetic fragment or target sequence, specifically the “amplicon”. As it refers to the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as “PCR product.”
[0070] The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.
[0071] As used herein, the term “antigen” refers to a molecule that is capable of stimulating an immune response such as by production of antibodies specific for the antigen. Antigens of the present invention can be, for example, an antigen from human immunodeficiency virus (HIV), an antigen from influenza virus, or an antigen from respiratory syncytial virus (RSV). Antigens of the present invention can also be, for example, a human antigen (e.g. an oncogene-encoded protein).
[0072] In the present invention, “specific for” and “specificity” means a condition where one of the molecules involved in selective binding. Accordingly, an antibody that is specific for one antigen selectively binds that antigen and not other antigens.
[0073] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to specifically interact with the HIV virus, such that the HIV viral infection is prevented, inhibited, reduced, or delayed. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
[0074] Each antibody molecule is made up of the protein products of two genes, heavy-chain gene and light-chain gene. The heavy-chain gene is constructed through somatic recombination of V, D, and J gene segments. In humans, there are 51 VH, 27 DH, 6 JH, 9 CH gene segments on human chromosome 14. The light-chain gene is constructed through somatic recombination of V and J gene segments. There are 40 Vκ, 31 Vλ, 5 Jκ, 4 Jλ gene segments on human chromosome 14 (80 VJ). The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The “light chains” of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
[0075] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
[0076] The disclosed monoclonal antibodies can be made using any procedure which produces monoclonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
[0077] The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Pat. No. 5,804,440 to Burton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.
[0078] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross linking antigen.
[0079] As used herein, the term “antibody or antigen binding fragment thereof” or “antibody or fragments thereof” encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab′).sub.2, Fab′, Fab, Fv, sFv, scFv and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain HIV virus binding activity are included within the meaning of the term “antibody or antigen binding fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
[0080] Also included within the meaning of “antibody or antigen binding fragment thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies). Also included within the meaning of “antibody or antigen binding fragment thereof” are immunoglobulin single variable domains, such as for example a nanobody.
[0081] The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin. Biotechnol. 3:348-354, 1992).
[0082] As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
[0083] “Pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
[0084] “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
[0085] As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, and improvement or remediation of damage.
[0086] “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as coughing relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
Methods
[0087] In some aspects, disclosed herein is a method for simultaneous detection of an antigen and an antibody that specifically binds said antigen, comprising: [0088] labeling a plurality of antigens with unique antigen barcodes; [0089] providing a plurality of barcode-labeled antigens to a population of B-cells; [0090] allowing the plurality of barcode-labeled antigens to bind to the population of B-cells; [0091] washing unbound antigens from the population of B-cells; [0092] separating the B-cells into single cell emulsions; [0093] introducing into each single cell emulsion a unique cell barcode-labeled bead; [0094] preparing a single cell cDNA library from the single cell emulsions; [0095] performing PCR amplification reactions to produce a plurality of amplicons, wherein the amplicons comprise: 1) the cell barcode and the antigen barcode, 2) the cell barcode and an antibody sequence, and 3) a unique molecular identifier (UMI); [0096] sequencing the plurality of amplicons; [0097] removing a sequence lacking the cell barcode, the UMI, or the antigen barcode; [0098] aligning the antibody sequence to a reference library of immunoglobulin V, D, J and C sequences; [0099] constructing a UMI count matrix comprising the cell barcode, the antigen barcode, and the antibody sequence; [0100] determining a LIBRA-seq score; and [0101] determining that the antibody specifically binds an antigen if the LIBRA-seq score of the antibody for the antigen is increased in comparison to a control sample.
[0102] Following a LIBRA-seq experiment, there are 2 resulting pairs of FASTQ files: (1) B cell receptor libraries (containing heavy and light chain contigs), and (2) antigen barcode libraries (containing antigen-identifying DNA barcode sequences from the antigen screening library). In some embodiments, it should be understood that the methods described herein are for uniting the information from these two sequencing libraries. Accordingly, in some embodiments, the above noted step of removing a sequence lacking the cell barcode, the UMI, or the antigen barcode is for removing a sequence from the antigen barcode library lacking the cell barcode, the UMI, or the antigen barcode. The general structure of the antigen barcode should be look like, for example,
[0103] Processing of antigen barcode reads and BCR sequence contigs. A pipeline shown herein takes paired-end fastq files of oligo libraries as input, processes and annotates reads for cell barcode, UMI, and antigen barcode, and generates a cell barcode—antigen barcode UMI count matrix. BCR contigs are processed using cellranger (10× Genomics) using GRCh38 as reference. For the antigen barcode libraries, initial quality and length filtering is carried out by fastp (Chen et al., 2018) using default parameters for filtering. This results in only high-quality reads being retained in the antigen barcode library (
[0104] (1) The reverse complement of the R2 sequence is determined (Skip step 1 if using R1).
[0105] (2) The sequence is screened for possessing an exact match to any of the valid 10× cell barcodes present in the filtered_contig.fasta file output by cell ranger during processing of BCR V(D)J fastq files. Sequences without a BCR-associated cell barcode are discarded.
[0106] (3) The 10 bases immediate 3′ to the cell barcode are annotated as the read's UMI.
[0107] (4) The remainder of the sequence 3′ to the UMI is screened for a 13 or 15 bp sequence with a hamming distance of 0, 1, or 2 to any of the antigen barcodes used in the screening library. Following this processing, only sequences around the expected lengths are retained (the lengths of sequences can be from more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases shorter to more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases longer than the expected lengths), thus allowing for a deletion, an insertion outside the cell barcode, or bases flanking the cell barcode.
[0108] This general process requires that sequences possess all elements needed for analysis (cell barcode, UMI, and antigen barcode), but is permissive to insertions or deletions in the TSO region between the UMI and antigen barcode. After processing each sequence one-by-one, cell barcode—UMI—antigen barcode collisions are screened. Any cell barcode—UMI combination (indicative of a unique oligo molecule) that has multiple antigen barcodes associated with it is removed. A cell barcode—antigen barcode UMI count matrix is then constructed, which served as the basis of subsequent analysis. Additionally, the BCR contigs are aligned (filtered_contigs.fasta file output by Cellranger, 10× Genomics) to IMGT reference genes using HighV-Quest (Alamyar et al., 2012). The output of HighV-Quest is parsed using ChangeO (Gupta et al., 2015), and merged with the UMI count matrix.
[0109] The above stated procedure can be summarized as the following steps:
[0110] 1) Remove low quality reads;
[0111] 2) Remove reads too long or too short to be a valid antigen barcode read containing a cell barcode, UMI, and antigen barcode;
[0112] 3) For each quality read, annotate: [0113] a. Cell barcode, [0114] b. UMI [0115] c. Antigen barcode, allowing for sequencing/PCR errors by using a hamming distance threshold.
[0116] Determination of LIBRA-seq Score. Starting with the UMI count matrix, all counts of more than one UMIs (for example, more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 UMIs) were set to 0, with the idea that these low counts can be attributed to noise. After this, the UMI count matrix was subset to contain only cells with a count of one or more UMIs than the minimum value in the above noted step of noise filtering for at least 1 antigen. The centered-log ratios (CLR) of each antigen UMI count for each cell were then calculated (Mimitou et al., 2019; Stoeckius et al., 2017, 2018). Because UMI counts were on different scales for each antigen, due to differential oligo loading during oligo-antigen conjugation, the CLRs UMI counts were rescaled using the StandardScaler method in scikit learn (Pedregosa and Varoquaux, 2011). Lastly, A correction procedure was performed to the z-score-normalized CLRs from UMI counts of 0, setting them to the minimum for each antigen for donor NIAID 45 and N90 experiments, and to −1 for the Ramos B cell line experiment. These CLR-transformed, Z-score-normalized, corrected values served as the final LIBRA-seq scores. LIBRA-seq scores were visualized using Cytobank (Kotecha et al., 2010).
[0117] Identification of sequence feature—antigen specificity associations. Following determination of LIBRA-seq scores (above), and because antibody sequence is united with antigen specificity (in the form of a LIBRA-seq score), sequence-specificity associations can be made.
[0118] Accordingly, in some embodiments, the method of any preceding aspect further comprises determining a level of somatic hypermutation of the antibody specifically binding to the antigen
[0119] In some embodiments, the method of any preceding aspect further comprises determining a length of a complementarity-determining region (CDR) of the antibody specifically binding to the antigen. The term “complementarity determining region (CDR)” used herein refers to an amino acid sequence of an antibody variable region of a heavy chain or light chain. CDRs are necessary for antigen binding and determine the specificity of an antibody. Each variable region typically has three CDRs identified as CDR1 (CDRH1 or CDRL1, where “H” indicates the heavy chain CDR1 and “L” indicates the light chain CDR1), CDR2 (CDRH2 or CDRL2), and CDR3 (CDRH3 or CDRL3). The CDRs may provide contact residues that play a major role in the binding of antibodies to antigens or epitopes. Four framework regions, which have more highly conserved amino acid sequences than the CDRs, separate the CDR regions in the VH or VL.
[0120] Accordingly, in some embodiments, the method of any preceding aspect further comprises determining a motif of a CDR of the antibody specifically binding to the antigen. In some embodiments, the CDR is selected from the group consisting of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.
[0121] In some embodiments, the method of any preceding aspect further comprises identification of IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, or IGLJ genes, or combinations thereof, associated with any particular combination of antigen specificities.
[0122] In some embodiments, the method of any preceding aspect further comprises identification of mutations in heavy or light FW1, FW2, FW3 or FW4 associated with any particular combination of antigen specificities.
[0123] In some embodiments, the method of any preceding aspect further comprises identification of overall gene expression profiles or select up- or down-regulated genes associated with any particular combination of antigen specificities.
[0124] In some embodiments, the method of any preceding aspect further comprises identification of surface markers, via, for example, fluorescence-activated cell sorting, or oligo-conjugated antibodies associated with any particular combination of antigen specificities
[0125] In some embodiments, the method of any preceding aspect further comprises identification of any combination of BCR sequence feature (for example, immunoglobulin gene, sequence motif, or CDR length), gene expression profile, or surface marker profile associated with any particular combination of antigen specificities.
[0126] In some embodiments, the method of any preceding aspect further comprises training a machine learning algorithm on sequence features, sequence motifs, or encoded sequence properties (such as via Kidera factors), associated with any particular combination of antigen specificities for subsequent application to sequenced antibodies lacking antigen specificity information due to not using LIBRA-seq or otherwise.
[0127] In some aspects, disclosed herein is a method for simultaneous detection of an antigen and an antibody that specifically binds said antigen, comprising: [0128] labeling a plurality of antigens with unique antigen barcodes; [0129] providing a plurality of barcode-labeled antigens to a population of B-cells; [0130] allowing the plurality of barcode-labeled antigens to bind to the population of B-cells; [0131] washing unbound antigens from the population of B-cells; [0132] separating the B-cells into single cell emulsions; [0133] introducing into each single cell emulsion a unique cell barcode-labeled bead; [0134] preparing a single cell cDNA library from the single cell emulsions; [0135] performing PCR amplification reactions to produce a plurality of amplicons, wherein the amplicons comprise: 1) the cell barcode and the antigen barcode, 2) the cell barcode and an antibody sequence, and 3) a unique molecular identifier (UMI); [0136] sequencing the plurality of amplicons; [0137] removing a sequence lacking the cell barcode, the UMI, or the antigen barcode; [0138] aligning the antibody sequence to a reference library of immunoglobulin V, D, J and C sequences; [0139] constructing a UMI count matrix comprising the cell barcode, the antigen barcode, and the antibody sequence; [0140] determining a LIBRA-seq score; and [0141] determining that the antibody specifically binds an antigen if the LIBRA-seq score of the antibody for the antigen is increased in comparison to a control sample.
[0142] In some embodiments, the barcode-labeled antigens are labeled with a first barcode comprising a DNA sequence or an RNA sequence. In some embodiments, the cell barcode-labeled beads are labeled with a second barcode comprising a DNA sequence or an RNA sequence.
[0143] It should be understood that the barcode described above is conjugated to the barcode-labeled antigen in a way that are known to one of ordinary skill in the art. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. An oligonucleotide barcode can also be conjugated to an antigen using the Solulink Protein-Oligonucleotide Conjugation Kit (TriLink cat no. S-9011) according to manufacturer's instructions. Briefly, the oligo and protein are desalted, and then the amino-oligo is modified with the 4FB crosslinker, and the biotinylated antigen protein is modified with S-HyNic. Then, the 4FB-oligo and the HyNic-antigen are mixed together. This causes a stable bond to form between the protein and the oligonucleotide. In some embodiments, the cell barcode-labeled beads are labeled with a second barcode comprising a DNA sequence or an RNA sequence. In some embodiments, the cell barcode-labeled beads are labeled with a second barcode comprising a DNA sequence. In some embodiments, the cell barcode-labeled beads are labeled with a second barcode comprising an RNA sequence. In some embodiments, the cell barcode-labeled beads are labeled with a barcode on the inside of the bead. In some embodiments, the cell barcode-labeled beads are labeled with a barcode encapsulated within the bead. In some embodiments, the cell barcode-labeled beads are labeled with a barcode on the outside of the bead.
[0144] As used herein, “beads” is not limited to a specific type of bead. Rather, a large number of beads are available and are known to one of ordinary skill in the art. A suitable bead may be selected on the basis of the desired end use and suitability for various protocols. In some embodiments, the bead is or comprises a particle or a bead. In some embodiments, the solid support bead is magnetic. Beads comprise particles have been described in the prior art in, for example, U.S. Pat. Nos. 5,084,169, 5,079,155, 473,231, and 8,110,351. The particle or bead size can be optimized for binding B cell in a single cell emulsion and optimized for the subsequent PCR reaction.
[0145] These oligos, which contain the cell barcode, both: (1) enable amplification of cellular mRNA transcripts through the template switch oligo that is part of the oligo containing the cell barcode, and (2) directly anneal to the antigen barcode-containing oligos from the antigen. In some embodiments, the oligos delivered from the beads have the general structure: P5_PCR_handle-Cell_barcode-UMI-Template_switch_oligo.
[0146] It is noted above that the antibody is determined as specifically binding an antigen if the LIBRA-seq score of the antibody for the antigen is increased in comparison to a control sample. It should be understood herein that, as taught by
[0147] In some embodiments, the antibody sequence comprises an immunoglobulin heavy chain (VDJ) sequence, or an immunoglobulin light chain (VJ) sequence. In some embodiments, the antibody sequence comprises an immunoglobulin heavy chain (VDJ) sequence. In some embodiments, the antibody sequence comprises an immunoglobulin light chain (VJ) sequence.
[0148] In some embodiments, the barcode-labeled antigens comprise an antigen from a pathogen or an animal In some embodiments, the barcode-labeled antigens comprise an antigen from a pathogen. In some embodiments, the barcode-labeled antigens comprise an antigen from an animal In some embodiments, the animal is a mammal, including, but not limited to, primates (e.g., humans and nonhuman primates), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.
[0149] In some embodiments, the antigen from a pathogen comprises an antigen from a virus. In some embodiments, the antigen from a virus comprises an antigen from human immunodeficiency virus (HIV), an antigen from influenza virus, or an antigen from respiratory syncytial virus (RSV).
[0150] In some embodiments, the antigen from a virus comprises an antigen from human immunodeficiency virus (HIV). In some embodiments, the antigen from a virus comprises an antigen from influenza virus. In some embodiments, the antigen from a virus comprises an antigen from respiratory syncytial virus (RSV).
[0151] In some embodiments, the antigen from HIV comprises an antigen from HIV-1. In some embodiments, the antigen from HIV comprises an antigen from HIV-2. In some embodiments, the antigen from HIV comprises HIV-1 Env. In some embodiments, the antigen from influenza virus comprises hemagglutinin (HA). In some embodiments, the antigen from RSV comprises an RSV F protein. In some embodiments, the antigen is selected from the antigens listed in Table 1.
TABLE-US-00001 TABLE 1 Antigen screening library for human B-cell sample analysis. For a set of pathogens, shown are selected protein targets, number of strains, and resulting total number of antigens in the screening library. Pathogen Protein targets # Strains # Antigens in library CMV g.sup.B 2 2 D ngue E, prM 6 10 Hepatitis B HBsAg 2 2 Hepatitis C E2, E1E2 2 4 HIV-1 gp140, gp120, MPER 3 9 HPV L1 3 3 HSV-1 g.sup.B 1 1 influenza HA
NA
12 Malaria PfCSP 1 1 Measles H, F 1 2 Mumps HN, NP 1 2 Norovirus P 10 10 Rhinovius VP1 5 5 Rotavirus VP7, VP4
8 RSV F
G 4 8 Rub
a E1 1 1 Staphylococcus aureus HtsA, SirA, IsdB, SstD 1 4 UPEC Hma, IutA, FyuA, IreA 1 4 Z
ka E
prM 1 2 *influenza: A (6 HA, 4 NA) and B (2 HA); {circumflex over ( )}rotavirus: 6 G, 2 P variants)
indicates data missing or illegible when filed
[0152] In some embodiments, the population of B-cells comprise a memory B-cell, a plasma cell, a naïve B cell, an activated B-cell, or a B-cell line. In some embodiments, the population of B-cells comprise a memory B-cell, a plasma cell, a naïve B cell, an activated B-cell, or a B-cell line. In some embodiments, the population of B-cells comprise a plasma cell. In some embodiments, the population of B-cells comprise a naïve B cell. In some embodiments, the population of B-cells comprise an activated B-cell. In some embodiments, the population of B-cells comprise a B-cell line.
[0153] In another aspect, disclosed herein is a method of determining a broadly neutralizing antibody to a pathogen, said method comprising: [0154] labeling a plurality of antigens derived from the pathogen with unique antigen barcodes; [0155] providing a plurality of barcode-labeled antigens to a population of B-cells; [0156] allowing the plurality of barcode-labeled antigens to bind to the population of B-cells; [0157] washing unbound antigens from the population of B-cells; [0158] separating the B-cells into single cell emulsions; [0159] introducing into each single cell emulsion a unique cell barcode-labeled bead; [0160] preparing a single cell cDNA library from the single cell emulsions; [0161] performing PCR amplification reactions to produce a plurality of amplicons, wherein the amplicons comprise: 1) the cell barcode and the antigen barcode, 2) the cell barcode and an antibody sequence, and 3) a unique molecular identifier (UMI); [0162] sequencing the plurality of amplicons; [0163] removing a sequence lacking a cell barcode, unique molecular identifier (UMI), or an antigen barcode; [0164] aligning the antibody sequence to a reference library of immunoglobulin V, D, J and C sequences; [0165] constructing a UMI count matrix comprising the cell barcode, the antigen barcode, and the antibody sequence; [0166] determining a LIBRA-seq score; and [0167] determining that the antibody is a broadly neutralizing antibody if the LIBRA-seq scores of the antibody for two or more antigens are increased in comparison to a control.
[0168] Polypeptides and Polynucleotides
[0169] In some aspects, disclosed herein is a polynucleotide comprising a sequence set forth in the specification.
[0170] In some aspects, disclosed herein is a polypeptide, wherein the polypeptide is encoded by a polynucleotide sequence set forth in the specification.
[0171] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein [0172] the VH comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 667-711; and/or [0173] the VL comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 802-845.
[0174] In some embodiments, the VH comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions) when compared to SEQ ID NOs: 667-711. In some embodiments, the VL comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions) when compared to SEQ ID NOs: 802-845.
[0175] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a light chain variable region (VL) that comprises a light chain complementarity determining region (CDRL)1, CDRL2, and CDRL3 and a heavy chain variable region (VH) that comprises a heavy chain complementarity determining region (CDRH)1, CDRH2, and CDRH3, wherein [0176] the CDRH1 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 712-740; and/or [0177] the CDRL1 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 846-876.
[0178] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a light chain variable region (VL) that comprises a light chain complementarity determining region (CDRL)1, CDRL2, and CDRL3 and a heavy chain variable region (VH) that comprises a heavy chain complementarity determining region (CDRH)1, CDRH2, and CDRH3, wherein [0179] the CDRH2 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 741-767; and/or [0180] the CDRL2 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 877-891.
[0181] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a light chain variable region (VL) that comprises a light chain complementarity determining region (CDRL)1, CDRL2, and CDRL3 and a heavy chain variable region (VH) that comprises a heavy chain complementarity determining region (CDRH)1, CDRH2, and CDRH3, wherein [0182] the CDRH3 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 768-801 or 917-936; and/or [0183] the CDRL3 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 892-916 or 937-938.
[0184] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a light chain variable region (VL) that comprises a light chain complementarity determining region (CDRL)1, CDRL2, and CDRL3 and a heavy chain variable region (VH) that comprises a heavy chain complementarity determining region (CDRH)1, CDRH2, and CDRH3, wherein [0185] the CDRH1 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 712-740; [0186] the CDRL1 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 846-876; [0187] the CDRH2 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 741-767; [0188] the CDRL2 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 877-891; [0189] the CDRH3 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 768-801 or 917-936; and/or [0190] the CDRL3 comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 892-916 or 937-938.
[0191] In some embodiments, the CDRH1 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NOs: 712-740. In some embodiments, the CDRH2 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NOs: 741-767. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID Nos: 768-801 or 917-936. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 770. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 771. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 917. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 918. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 919. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 920. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 921. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 922. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 923. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 924. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 925. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 926. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 927. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 928. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 929. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 930. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 931. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 932. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 933. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 934. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 935. In some embodiments, the CDRH3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 936. In some embodiments, the CDRH3 comprises a polypeptide sequence selected from SEQ ID NOs: 770-771 or 917-936.
[0192] In some embodiments, the CDRL1 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NOs: 846-876. In some embodiments, the CDRL2 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NOs: 877-891. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NOs: 892-916 or 937-938. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 894. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 895. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 896. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 897. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 902. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 903. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 904. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 905. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 906. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 907. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 908. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 911. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 915. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 937. In some embodiments, the CDRL3 comprises at least one amino acid substitution (including, for example, at least 1, 2, 3, 4, 5, or 6 substitutions) when compared to SEQ ID NO: 938. In some embodiments, the CDRL3 comprises a polypeptide sequence selected from the group consisting of SEQ ID NOs: 894-897, 902-908, 911, 915, 937, or 938.
[0193] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a heavy chain variable region (VH) that comprises a VDJ junction, wherein [0194] the VDJ junction comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 775 or 939-948.
[0195] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a light chain variable region (VL) that comprises a VJ junction, wherein [0196] the VJ junction comprises an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 892, 893, 899, 900, 909, 910, 912, 913, 914, or 916.
[0197] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VDJ junction comprising an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 775 or 939-948, and wherein the VL comprises a VJ junction comprising an amino acid sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 892, 893, 899, 900, 909, 910, 912, 913, 914, or 916.
[0198] In some aspects, disclosed herein is a polypeptide comprising a sequence set forth in
[0199] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a heavy chain variable region (VH) that is encoded by a polynucleotide at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 223-444.
[0200] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a light chain variable region (VL) that is encoded by a polynucleotide at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 445-666.
[0201] In some aspects, disclosed herein is a recombinant antibody, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is encoded by a polynucleotide at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 223-444, and wherein the VL is encoded by a polynucleotide at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 445-666.
[0202] In some aspects, disclosed herein is a therapeutic antibody comprising the polypeptide of any preceding aspect. The term “neutralizing antibody” is any antibody or antigen-binding fragment thereof that binds to a pathogen and interferes with the ability of the pathogen to infect a cell and/or cause disease in a subject. Typically, the neutralizing antibodies used in the method of the present disclosure bind to the surface of the pathogen and inhibit or reduce infection by the pathogen by at least 99 percent, at least 95 percent, at least 90 percent, at least 85 percent, at least 80 percent, at least 75 percent, at least 70 percent, at least 60 percent, at least 50 percent, at least 45 percent, at least 40 percent, at least 35 percent, at least 30 percent, at least 25 percent, at least 20 percent, or at least 10 percent relative to infection by the pathogen (e.g., HIV or influenza) in the absence of said antibody(ies) or in the presence of a negative control.
[0203] In some embodiments, the neutralizing antibody comprises a polypeptide sequence set forth in the specification. In some embodiments, the neutralizing antibody comprises 3602-870, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with the sequence of 3602-870, or a polypeptide comprising a portion of 3602-870. As used herein, “broadly neutralizing antibody” or “BNAb” is understood as an antibody obtained by any method that when delivered at an effective dose can be used as a therapeutic agent for the prevention or treatment of HIV or influenza infection or an infection-related disease against a broad array of different HIV or influenza strains (for example, more than 3 strains of HIV/influenza, preferably more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more strains of HIV/influenza). In some embodiments, the broadly neutralizing antibody comprises a polypeptide sequence set forth in the specification. In some embodiments, the neutralizing antibody comprises 3602-870, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with the sequence of 3602-870, or a polypeptide comprising a portion of 3602-870.
[0204] Accordingly, in some embodiments, the neutralizing antibody comprises a VH and a VL, wherein the VH comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 685, and wherein the VL comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 813. In some embodiments, the neutralizing antibody comprises a VH comprising a CDRH1, CDRH2, and CDRH3, wherein the CDRH1 comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 713, wherein the CDRH2 comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 749, and wherein the CDRH3 comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 773. In some embodiments, the neutralizing antibody comprises a VL comprising a CDRL1, CDRL2, and CDRL3, wherein the CDRL1 comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 851, wherein the CDRL2 comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 879, and wherein the CDRL3 comprises a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 893.
[0205] In some aspect, disclosed herein is a method of treating HIV infection in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant polypeptide and/or neutralizing antibody of any preceding aspect.
[0206] In some aspect, disclosed herein is a method of treating flu infection in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant polypeptide and/or neutralizing antibody of any preceding aspect.
EXAMPLES
[0207] The following examples are set forth below to illustrate the systems, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Example 1. LIBRA-seq Method
[0208] LIBRA-seq transforms antibody-antigen interactions into sequencing-detectable events by conjugating DNA-barcoded oligos to each antigen in a screening library. All antigens are labeled with the same fluorophore, which enables sorting of antigen-positive B cells by fluorescence-activated cell sorting (FACS) before encapsulation of single B cells via droplet microfluidics. Antigen barcodes and BCR transcripts are tagged with a common cell barcode from bead-delivered oligos, enabling direct mapping of BCR sequence to antigen specificity (
[0209] To investigate the ability of LIBRA-seq to accurately unite BCR sequence and antigen specificity, a mapping experiment was devised using two Ramos B-cell lines with differing BCR sequences and antigen specificities (Weaver et al., 2016). These engineered B-cell lines do not display endogenous BCR and instead express specific, user-defined surface IgM BCR sequences (Weaver et al., 2016). To that end, two well-characterized BCRs were selected: VRC01, a CD4-binding site-directed HIV-1 bNAb (Wu et al., 2010), and Fe53, a bNAb recognizing the stem of group 1 influenza hemagglutinins (HA) (Lingwood et al., 2012). These two populations of B -cell lines were mixed at a 1:1 ratio and incubated with three unique DNA-barcoded antigens: two variants of the trimeric HIV-1 Env protein from strains BG505 and CZA97 (Georgiev et al., 2015; van Gils et al., 2013; Ringe et al., 2017), and trimeric hemagglutinin from strain H1 A/New Caledonia/20/1999 (Whittle et al., 2014) (
[0210] 2321 cells with BCR sequence and antigen mapping information were recovered, highlighting the high throughput capacity of LIBRA-seq (
Example 2. Isolation of Antibodies from a Known HIV bNAb Lineage
[0211] LIBRA-seq was next used to analyze the antibody repertoire of donor NIAID 45, who had been living with HIV-1 without antiretroviral therapy for approximately 17 years at the time of sample collection. This sample was selected as an appropriate target for LIBRA-seq analysis because a large lineage of HIV-1 bNAbs had been identified previously from this donor (Bonsignori et al., 2018; Wu et al., 2010, 2015). This lineage consists of the prototypical bNAb VRC01, as well as multiple clades of clonally related bNAbs with diverse neutralization phenotypes (Wu et al., 2015). The same BG505, CZA97, and H1 A/New Caledonia/20/99 antigen screening library was used in the Ramos B-cell line experiments, recovering paired V.sub.H:V.sub.L antibody sequences with antigen mapping for 866 cells (
[0212] To further investigate LIBRA-seq in monoclonal antibody isolation, new members of the VRC01 antibody lineage were identified from the LIBRA-seq-identified antigen-specific B cells. 29 BCRs that were clonally related to previously-identified members of the VRC01 lineage (
Example 3. Identification of Additional Broadly-Reactive Anti-HIV and Anti-Influenza Antibodies
[0213] To further assess the ability of LIBRA-seq to accurately identify antigen-specific B cells, a number of putative HIV-specific and influenza-specific monoclonal antibodies were produced from donor 45 that did not belong to the VRC01 lineage. In particular, seven additional anti-HIV antibodies were recombinantly produced, three of which were clonally related (2723-2121, 2723-422, and 2723-2304) (
Example 4. Discovery of an HIV bNAb using a Nine-Antigen Screening Library
[0214] Having validated LIBRA-seq with three antigens on both Ramos B cell lines and primary B cells from a patient sample, experiment was performed to increase the number of antigens in the screening library. To that end, the B cell repertoire of NIAID donor N90 was screened against nine antigens (
[0215] To increase the number of antigens in the screening library, a panel consisted of five HIV-1 Env trimers from a variety of clades, BG505 (clade A), B41 (clade B), ZM106.9 (clade C), ZM197 (clade C) and KNH1144 (clade A) was utilized (van Gils et al., 2013; Harris et al., 2011; Joyce et al., 2017; Julien et al., 2015; Pugach et al., 2015; Ringe et al., 2017), along with four diverse hemagglutinin trimers (H1 A/New Caledonia/20/99, H1 A/Michigan/45/2015, H5 A/Indonesia/5/2005, and H7 A/Anhui/1/2013) (
[0216] The B cells with the highest LIBRA-seq scores in the N90 sample were analyzed, especially those cells that had LIBRA-seq scores for any antigen above one (901 cells) (
[0217] Cells that had high LIBRA-seq scores for each of multiple HIV-1 antigens were also observed, including 124 cells that had high scores for four or more SOSIPs (
Example 5. Discussion
[0218] Disclosed herein is a method to interrogate antibody-antigen interactions via a sequencing-based readout were disclosed. New members of two known HIV-specific bNAb lineages were identified from previously characterized human infection samples and a novel bNAb lineage. Additionally, many other broadly-reactive HIV-specific antibodies were identified and investigated regarding their specificity for a subset of them. Within both HIV-1 infection samples, influenza-specific antibodies were also isolated using hemagglutinin screening probes, highlighting LIBRA-seq for use in methods of simultaneously screening B cell repertoires against multiple, diverse antigen targets. The NGS-based coupling of antibody sequence and specificity enables screening of potentially millions of single B cells for reactivity to a larger repertoire of epitopes than purely fluorescence-based methods, since sequence space is not hindered by spectral overlap. Using LIBRA-seq therefore helps to maximize lead discovery per experiment, an important consideration when preserving limited sample.
[0219] Beyond LIBRA-seq's importance in antibody discovery, the high-throughput coupling of antibody sequence and specificity can enable high-resolution immune profiling. For example, in donor N90, the use of specific germline genes (e.g., IGHV1-69, IGHV4-39, and IGHV1-18) was enriched in B cells that exhibited broad, as opposed to strain-specific, HIV-1 antigen reactivity (
Example 6. Methods and Materials
[0220] Antigen expression and purification. For the different LIBRA-seq experiments, a total of six HIV-1 gp140 SOSIP variants from strains BG505 (clade A), CZA97 (clade C), B41 (clade B), ZM197 (clade C), ZM106.9 (clade C), KNH1144 (clade A) and four influenza hemagglutinin variants from strains A/New Caledonia/20/99 (H1N1) (GenBank ACF41878), A/Michigan/45/2015 (H1N1) (GenBank AMA11475), A/Indonesia/5/2005 (H5N1) (GenBank ABP51969), and A/Anhui/1/2013 (H7N9) (GISAID EPI439507) were expressed as recombinant soluble antigens.
[0221] The single-chain variants (Georgiev et al., 2015) of BG505, CZA97, B41, ZM197, ZM106.9, and KNH1144 each containing an Avi tag, were expressed in 293F mammalian cells using polyethylenimine (PEI) transfection reagent and cultured for 5-7 days. Next, cultures were centrifuged at 6000 rpm for 20 minutes. Supernatant was 0.45 μm filtered with Nalgene Rapid Flow Disposable Filter Units with PES membrane, and then run slowly over an affinity column of agarose bound Galanthus nivalis lectin (Vector Laboratories cat no. AL-1243-5) at 4° C. The column was washed with PBS, and proteins were eluted with 30 mL of 1 M methyl-α-D-mannopyranoside. The protein elution was buffer exchanged 3× into PBS and concentrated using 30 kDa Amicon Ultra centrifugal filter units. Concentrated protein was run on a Superdex 200 Increase 10/300 GL sizing column on the AKTA FPLC system, and fractions were collected on an F9-R fraction collector. Fractions corresponding to correctly folded antigen were analyzed by SDS-PAGE, and antigenicity by ELISA was characterized with known monoclonal antibodies specific for that antigen.
[0222] Recombinant HA proteins all contained the HA ectodomain with a point mutation at the sialic acid-binding site (Y98F), T4 fibritin foldon trimerization domain, Avi tag, and hexahistidine tag, and were expressed in Expi 293F mammalian cells using Expifectamine 293 transfection reagent (Thermo Fisher Scientific) cultured for 4-5 days. Culture supernatant was harvested and cleared as above, and then adjusted pH and NaCl concentration by adding 1M Tris-HCl (pH 7.5) and 5M NaCl to 50 mM and 500 mM, respectively. Ni Sepharose excel resin (GE Healthcare) was added to the supernatant to capture hexahistidine tag. Resin was separated on a column by gravity and captured HA protein was eluted by a Tris-NaCl (pH 7.5) buffer containing 300 mM imidazole. The eluate was further purified by a size exclusion chromatography with a HiLoad 16/60 Superdex 200 column (GE Healthcare). Fractions containing HA were concentrated, analyzed by SDS-PAGE and tested for antigenicity by ELISA with known antibodies. Proteins were frozen in LN2 and stored at −80C° until use.
[0223] All antigens included an AviTag modification at the C-terminus of their sequence, and after purification, each AviTag labeled antigen was biotinylated using the BirA-500: BirA biotin-protein ligase standard reaction kit (Avidity LLC, cat no. BirA500).
[0224] Oligonucleotide barcode design. Oligo used herein possess a 13-15 bp antigen barcode, a sequence capable of annealing to the template switch oligo that is part of the 10× bead-delivered oligos, and contain truncated TruSeq small RNA read 1 sequences in the following structure: 5′-CCTTGGCACCCGAGAATTCCANNNNNNNNNNNNNCCCATATAAGA*A*A-3′ (SEQ ID NO: 949), where Ns represent the antigen barcode. For the cell line and NIAID45 experiments, we used the following antigen barcodes: CATGATTGGCTCA (SEQ ID NO: 950) (BG505), TGTCCGGCAATAA (SEQ ID NO: 951) (CZA97), GATCGTAATACCA (SEQ ID NO: 952) (H1 A/New Caledonia/20/99). For the N90 experiment, we used longer antigen barcodes (15 bp), as follows: TCCTTTCCTGATAGG (SEQ ID NO: 953) (ZM106.9), TAACTCAGGGCCTAT (SEQ ID NO: 954) (KNH1144), GCTCCTTTACACGTA (SEQ ID NO: 955) (ZM197), GCAGCGTATAAGTCA (SEQ ID NO: 956) (B41), ATCGTCGAGAGCTAG (SEQ ID NO: 957) (BG505), CAGGTCCCTTATTTC (SEQ ID NO: 958) (A/Indonesia/5/2005), ACAATTTGTCTGCGA (SEQ ID NO: 959) (A/Anhui/1/2013), TGACCTTCCTCTCCT (SEQ ID NO: 960) (A/Michigan/45/2015), AATCACGGTCCTTGT (SEQ ID NO: 961) (A/New Caledonia/20/99). Oligos were ordered from Sigma-Aldrich and IDT with a 5′ amino modification and HPLC purified.
[0225] Conjugation of oligonucleotide barcodes to antigens. For each antigen, a unique DNA “barcode” was directly conjugated to the antigen itself. In particular, 5′ amino-oligonucleotides were conjugated directly to each antigen using the Solulink Protein-Oligonucleotide Conjugation Kit (TriLink cat no. S-9011) according to manufacturer's instructions. Briefly, the oligo and protein were desalted, and then the amino-oligo was modified with the 4FB crosslinker, and the biotinylated antigen protein was modified with S-HyNic. Then, the 4FB-oligo and the HyNic-antigen were mixed together. This causes a stable bond to form between the protein and the oligonucleotide. The concentration of the antigen-oligo conjugates was determined by a BCA assay, and the HyNic molar substitution ratio of the antigen-oligo conjugates was analyzed using the NanoDrop according to the Solulink protocol guidelines. AKTA FPLC was used to remove excess oligonucleotide from the protein-oligo conjugates. Additionally, the antigen-oligo conjugates were analyzed via SDS-PAGE with a silver stain.
[0226] Fluorescent labeling of antigens. After attaching DNA barcodes directly to a biotinylated antigen, the barcoded antigens were mixed with streptavidin labeled with fluorophore phycoerythrin (PE). The streptavidin-PE was mixed with biotinylated antigen at a 5× molar excess of antigen to streptavidin. 1/5 of the streptavidin-oligo conjugate was added to the antigen every 20 minutes with constant rotation at 4° C.
[0227] B cell lines production and identification by sequencing. B cell lines were engineered from a clone of Ramos Burkitt's lymphoma that do not display endogenous antibody, and they ectopically express specific surface IgM B cell receptor sequences. The B cell lines used expressed B cell receptor sequences for HIV-1 specific antibody VRC01 and influenza specific antibody Fe53. The cells are cultured at 37° C. with 5% CO2 saturation in complete RPMI, made up of RPMI supplemented with 15% fetal bovine serum, 1% L-Glutamine, and 1% Penicillin/Streptomycin. Although endogenous heavy chains are scrambled, endogenous light chain transcripts remain and are detectable by sequencing. We thus identified and classified single Ramos Burkitt's B cells as either VRC01 or FE53 based on their heavy chain sequences. These Ramos B cell lines were validated for binding to our antigen probes by FACS.
[0228] Donor PBMCs. Donor NIAID45 Peripheral blood mononuclear cells were collected from donor NIAID45 on July 12, 2007. Donor NIAID45, from whom antibodies VRC01, VRC02, VRC03, VRC06, VRC07, VRC08, NIH45-46, and others from the VRC01 bNAb lineage had been previously isolated, was enrolled in investigational review board approved clinical protocols at the National Institute of Allergy and Infectious Diseases and had been living with HIV without antiretroviral treatment for approximately 17 years at the time of sample collection. Donor N90 Peripheral blood mononuclear cells were collected from donor N90 on May 29, 2008. Donor N90, from whom antibody lineage VRC38 had been previously isolated, was enrolled in investigational review board approved clinical protocols at the National Institute of Allergy and Infectious Diseases and had been living with HIV without antiretroviral treatment through the timepoint of sample collection since diagnosis in 1985 (Wu et al., 2012).
[0229] Enrichment of antigen-specific IgG+B cells. For the given sample, cells were stained and mixed with fluorescently labeled DNA-barcoded antigens and other antibodies, and then sorted using fluorescence activated cell sorting (FACS). First, cells were counted and viability was assessed using Trypan Blue. Then, cells were washed with DPBS supplemented with 1% Bovine serum albumin (BSA) through centrifugation at 300 g for 7 minutes. Cells were resuspended in PBS-BSA and stained with a variety of cell markers. For donor NIAID 45 PBMCs, these markers included CD3-APCCy7, IgG-FITC, CD19-BV711, CD14-V500, and LiveDead-V500. Additionally, fluorescently labeled antigen-oligo conjugates (described above) were added to the stain, so antigen-specific sorting could occur. For donor N90 PBMCs, these markers included LiveDead-APCCy7, CD14-APCCy7, CD3-FITC, CD19-BV711, and IgG-PECy5. Additionally, fluorescently labeled antigen-oligo conjugates were added to the stain, so antigen-specific sorting could occur. After staining in the dark for 30 minutes at room temperature, cells were washed 3 times with PBS-BSA at 300 g for 7 minutes. Then, cells were resuspended in PBS-BSA and sorted on the cell sorter. Antigen positive cells were bulk sorted and then they were delivered to the Vanderbilt VANTAGE sequencing core at an appropriate target concentration for 10× Genomics library preparation and NGS analysis. FACS data were analyzed using Cytobank (Kotecha et al., 2010).
[0230] 10× single cell processing and next generation sequencing. Single-cell suspensions were loaded onto the Chromium microfluidics device (10× Genomics) and processed using the B-cell VDJ solution according to manufacturer's suggestions for a target capture of 10,000 B cells per 1/8 10× cassette for B cell lines, 9,000 cells for B cells from donor NIAID45, and 4,000 for donor N90, with minor modifications in order to intercept, amplify and purify the antigen barcode libraries. The library preparation follows the CITE-seq protocol (available at cite-seq.com), with the exception of an increase in the number of PCR cycles of the antigen barcodes. Briefly, following cDNA amplification using an additive primer (5′ -CCTTGGCACCCGAGAATT*C*C-3′) (SEQ ID NO: 962) to increase the yield of antigen barcode libraries (Stoeckius et al., 2017), SPRI separation was used to size separate antigen barcode libraries from cellular mRNA libraries, PCR amplified for 10-12 cycles, and purified using 1.6× purification. Sample preparation for the cellular mRNA library continued according to 10× Genomics-suggested protocols, resulting in Illumina-ready libraries. Following library construction, we sequenced both BCR and antigen barcode libraries on a NovaSeq 6000 at the VANTAGE sequencing core, dedicating ˜2.5% of a flow cell to each experiment, with a target 10% of this fraction dedicated to antigen barcode libraries. This resulted in ˜334 5 million reads for the cell line V(D)J libraries (˜96,500 reads/cell), ˜376.3 million reads for donor NIAID45 V(D)J libraries (˜79,300 reads/cell), and ˜272 4 million reads for the N90 V(D)J libraries (˜151,400 reads/cell). Additionally, this sequencing depth resulted in ˜46.7 million total reads for antigen barcode library of the cell lines, ˜39 6 million reads for the antigen barcode library of donor NIAID45, and ˜82 9 million reads for the antigen barcode library for N90.
[0231] Processing of antigen barcode reads and BCR sequence contigs. A pipeline shown herein takes paired-end fastq files of oligo libraries as input, processes and annotates reads for cell barcode, UMI, and antigen barcode, and generates a cell barcode—antigen barcode UMI count matrix. BCR contigs are processed using cellranger (10× Genomics) using GRCh38 as reference. For the antigen barcode libraries, initial quality and length filtering is carried out by fastp (Chen et al., 2018) using default parameters for filtering. This results in only high-quality reads being retained in the antigen barcode library (
[0232] Determination of LIBRA-seq Score. Starting with the UMI count matrix, all counts of 1, 2, or 3 UMIs were set to 0, with the idea that these low counts can be attributed to noise. After this, the UMI count matrix was subset to contain only cells with a count of at least 4 UMIs for at least 1 antigen. The centered-log ratios (CLR) of each antigen UMI count for each cell were then calculated (Mimitou et al., 2019; Stoeckius et al., 2017, 2018). Because UMI counts were on different scales for each antigen, due to differential oligo loading during oligo-antigen conjugation, the CLRs UMI counts were rescaled using the StandardScaler method in scikit learn (Pedregosa and Varoquaux, 2011). Lastly, A correction procedure was performed to the z-score-normalized CLRs from UMI counts of 0, setting them to the minimum for each antigen for donor NIAID 45 and N90 experiments, and to −1 for the Ramos B cell line experiment. These CLR-transformed, Z-score-normalized, corrected values served as the final LIBRA-seq scores. LIBRA-seq scores were visualized using Cytobank (Kotecha et al., 2010).
[0233] Phylogenetic trees. Phylogenetic trees of antibody heavy chain sequences were constructed in order to assess the relative relatedness of antibodies within a given lineage. For the VRC01 lineage, the 29 sequences identified by LIBRA-seq and 52 sequences identified from the literature were aligned using clustal within Geneious. We then used the PhyML maximum likelihood (Guindon et al., 2009) plugin in Geneious (available at www.geneious.com/plugins/phyml-plugin/) to infer a phylogenetic tree. The resulting tree was then rooted to the inferred unmutated common ancestor (Bonsignori et al., 2018) (accession MK032222). A similar process was used to build a phylogenetic tree for lineage 2121, with one exception. Rather than using an inferred germline precursor, the IGHV and IGHJ genes were germline-reverted and the CDRH3 nucleotide sequence of the lineage member was used with the least IGHV somatic mutation. Trees were annotated and visualized in iTol (Letunic and Bork, 2019).
[0234] Antibody expression and purification. For each antibody, variable genes were inserted into plasmids encoding the constant region for the heavy chain (pFUSE-CHIg, Invivogen) and light chain (pFUSE2-CLIg, Invivogen) and synthesized from GenScript. In cases where the IgBLAST-aligned sequence was missing any residues at the beginning of framework 1 or end of framework 4, sequences were completed with germline residues. mAbs were expressed in Expi 293F mammalian cells by co-transfecting heavy chain and light chain expressing plasmids using polyethylenimine (PEI) transfection reagent and cultured for 5-7 days. Next, cultures were centrifuged at 6000 rpm for 20 minutes. Supernatant was 0.45 μm filtered with Nalgene Rapid Flow Disposable Filter Units with PES membrane. Filtered supernatant was run over a column containing Protein A agarose resin that had been equilibrated with PBS. The column was washed with PBS, and then antibodies were eluted with 100 mM Glycine HCl at pH 2.7 directly into a 1:10 volume of 1 M Tris-HCL pH 8. Eluted antibodies were buffer exchanged into PBS 3 times using 10 kDa Amicon Ultra centrifugal filter units.
[0235] Enzyme linked immunosorbent assay (ELISA). For ELISAs, soluble hemagglutinin protein was plated at 2 μg/ml overnight at 4° C. The next day, plates were washed three times with PBS supplemented with 0.05% Tween20 (PBS-T) and coated with 5% milk powder in PBS-T. Plates were incubated for one hour at room temperature and then washed three times with PBS-T. Primary antibodies were diluted in 1% milk in PBS-T, starting at 10 μg/ml with a serial 1:5 dilution and then added to the plate. The plates were incubated at room temperature for one hour and then washed three times in PBS-T. The secondary antibody, goat anti-human IgG conjugated to peroxidase, was added at 1:20,000 dilution in 1% milk in PBS-T to the plates, which were incubated for one hour at room temperature. Plates were washed three times with PBS-T and then developed by adding TMB substrate to each well. The plates were incubated at room temperature for ten minutes, and then 1 N sulfuric acid was added to stop the reaction. Plates were read at 450 nm.
[0236] For recombinant trimer capture for single-chain SOSIPs, 2 μg/ml of a mouse anti-AviTag antibody (GenScript) was coated overnight at 4 C in phosphate-buffered saline (PBS) (pH 7.5). The next day plates were washed three times with PBS-T, and blocked with 5% milk in PBS-T. After an hour incubation at room temperature and three washes with PBS-T, 2 μg/ml of recombinant trimer proteins diluted in 1% milk PBS-T were added to the plate and incubated for one hour at room temperature. Primary and secondary antibodies, along with substrate and sulfuric acid, were added as described above. ELISAs were performed in at least two experimental replicates and data were graphed using GraphPad Prism 8.0.0. Data shown is representative of one replicate, with error bars representing standard error of the mean for technical duplicates within that experiment. The area under the curve (AUC) was calculated using GraphPad Prism 8.0.0.
[0237] TZM-bl Neutralization Assays. Antibody neutralization was assessed using the TZM-bl assay as described (Sarzotti-Kelsoe et al., 2014). This standardized assay measures antibody-mediated inhibition of infection of JC53BL-13 cells (also known as TZM-bl cells) by molecularly cloned Env-pseudoviruses. Viruses that are highly sensitive to neutralization (Tier 1) and those representing circulating strains that are moderately sensitive (Tier 2) were included. Antibodies were tested against a variety of Tier 1 viruses and the Tier 2 Global panel plus additional viruses, including a subset of the antigens used for LIBRA-seq. Murine leukemia virus (MLV) was included as an HIV-specificity control and VRC01 was used as a positive control. Results are presented as the concentration of monoclonal antibody (in μg/ml) required to inhibit 50% of virus infection (IC.sub.50).
[0238] Surface Plasmon Resonance and Fab competition. The binding of antibody 2723-2121 to BG505 DS-SOSIP (Do Kwon et al., 2015) was assessed by surface plasmon resonance on Biacore T-200 (GE-Healthcare) at 25° C. with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P-20) as the running buffer. Antibodies VRC01 and PGT145 were tested as positive control, and antibody 17b was tested as negative control to confirm that the trimer was in the closed conformation. Antibody 2723-2121 was captured on a flow cell of CM5 chip immobilized with ˜7500 RU of anti-human Fc antibody, and binding was measured by flowing over a 200 nM solution BG505-DS SOSIP in running buffer. Similar runs were performed with VRC01, PGT145 and 17b IgGs. To determine the epitope of antibody 2723-2121, we captured 2723-2121 IgG on a single flow cell of CM5 chip immobilized with ˜7500 RU of anti-human Fc antibody. Next 200 nM BG505 DS-SOSIP, either alone or with different concentrations of antigen binding fragments (Fab) of VRC01 or PGT145 or VRC34 was flowed over the captured 2723-2121 flow cell for 60 s at a rate of 10 μl/min. The surface was regenerated between injections by flowing over 3M MgCl.sub.2 solution for 10 s with flow rate of 100 μl/min. Blank sensorgrams were obtained by injection of same volume of HBS-EP+ buffer in place of trimer with Fabs solutions. Sensorgrams of the concentration series were corrected with corresponding blank curves. The binding of antibody 3602-870 to BG505 DS-SOSIP was assessed by surface plasmon resonance in the same way as described for 2723-2121. For 3602-870, competition experiments were performed with PGT145 Fab, PGT122 Fab, and VRC01 Fab.
[0239] ADCP, ADCD, Trogocytosis, ADCC Assays. Antibody-dependent cellular phagocytosis (ADCP) was performed using gp120 ConC coated neutravidin beads as previously described (Ackerman et at, 2011). Phagocytosis score was determined as the percentage of cells that took up beads multiplied by the fluorescent intensity of the beads. Antibody-dependent complement deposition (ADCD) was performed as in (Richardson et al., 2018a) where CEM.NKR.CCRS gp120 ConC coated target cells were opsonized with mAb and incubated with complement from a healthy donor. C3b deposition was then determined by flow cytometry with complement deposition score determined as the percentage of C3b positive cells multiplied by the fluorescence intensity. Antibody dependent cellular trogocytosis (ADCT) was measured as the percentage transfer of PKH26 dye of the surface of CEM.NKR.CCRS target cells to CSFE stained monocytic cell line THP-1 cells in the presence of HIV specific mAbs as described elsewhere (Richardson et al., 2018b). Antibody-dependent cellular cytotoxicity (ADCC) was done using a GranToxiLux based assay (Pollara et al., 2011) with gp120 ConC coated CEM.NKR.CCRS target cells and PBMCs from a healthy donor. The percentage of granzyme B present in target cells was measured by flow cytometry.
[0240] Statistics. ELISA error bars (standard error) were calculated using GraphPad Prism version 8.0.0. The Pearson's r value comparing BG505 and CZA97 LIBRA-seq scores for Ramos B-cell lines was calculated using Cytobank. Spearman correlations and associated p values were calculated using SciPy in Python.
TABLE-US-00002 TABLE 1 Nucleic acid sequences encoding heavy and light chains of antibodies and the cell barcodes thereof. SEQ ID NO for SEQ ID NO for SEQ ID NO for Heavy Chain Light Chain Donor Index Cell Barcode Contig Contig Selection logic N90 585 1 223 445 Cross-reactive HIV N90 1758 2 224 446 Cross-reactive HIV N90 3086 3 225 447 Cross-reactive HIV N90 2163 4 226 448 Cross-reactive HIV N90 627 5 227 449 Cross-reactive HIV N90 3218 6 228 450 Cross-reactive HIV N90 490 7 229 451 Cross-reactive HIV N90 84 8 230 452 Cross-reactive HIV N90 3023 9 231 453 Cross-reactive HIV N90 370 10 232 454 Cross-reactive HIV N90 2064 11 233 455 Cross-reactive HIV N90 2673 12 234 456 Cross-reactive HIV N90 3279 13 235 457 Cross-reactive HIV N90 2394 14 236 458 Cross-reactive HIV N90 2429 15 237 459 Cross-reactive HIV N90 1582 16 238 460 Cross-reactive HIV N90 2808 17 239 461 Cross-reactive HIV N90 2320 18 240 462 Cross-reactive HIV N90 2052 19 241 463 Cross-reactive HIV N90 1057 20 242 464 Cross-reactive HIV N90 1140 21 243 465 Cross-reactive HIV N90 2538 22 244 466 Cross-reactive HIV N90 2212 23 245 467 Cross-reactive HIV N90 1925 24 246 468 Cross-reactive HIV N90 528 25 247 469 Cross-reactive HIV N90 3353 26 248 470 Cross-reactive HIV N90 2302 27 249 471 Cross-reactive HIV N90 318 28 250 472 Cross-reactive HIV N90 3258 29 251 473 Cross-reactive HIV N90 2664 30 252 474 Cross-reactive HIV N90 2548 31 253 475 Cross-reactive HIV N90 1762 32 254 476 Cross-reactive HIV N90 1062 33 255 477 Cross-reactive HIV N90 1284 34 256 478 Cross-reactive HIV N90 592 35 257 479 Cross-reactive HIV N90 2876 36 258 480 Cross-reactive HIV N90 1887 37 259 481 Cross-reactive HIV N90 1178 38 260 482 Cross-reactive HIV N90 2507 39 261 483 Cross-reactive HIV N90 957 40 262 484 Cross-reactive HIV N90 3359 41 263 485 Cross-reactive HIV N90 1904 42 264 486 Cross-reactive HIV N90 1692 43 265 487 Cross-reactive HIV N90 1661 44 266 488 Cross-reactive HIV N90 1407 45 267 489 Cross-reactive HIV N90 1042 46 268 490 Cross-reactive HIV N90 1954 47 269 491 Cross-reactive HIV N90 1442 48 270 492 Cross-reactive HIV N90 2211 49 271 493 Cross-reactive HIV N90 451 50 272 494 Cross-reactive HIV N90 3544 51 273 495 Cross-reactive HIV N90 3232 52 274 496 Cross-reactive HIV N90 3226 53 275 497 Cross-reactive HIV N90 2985 54 276 498 Cross-reactive HIV N90 180 55 277 499 Cross-reactive HIV N90 2427 56 278 500 Cross-reactive HIV N90 1433 57 279 501 Cross-reactive HIV N90 979 58 280 502 Cross-reactive HIV N90 889 59 281 503 Cross-reactive HIV N90 442 60 282 504 Cross-reactive HIV N90 389 61 283 505 Cross-reactive HIV N90 3494 62 284 506 Cross-reactive HIV N90 3093 63 285 507 Cross-reactive HIV N90 2420 64 286 508 Cross-reactive HIV N90 2232 65 287 509 Cross-reactive HIV N90 1884 66 288 510 Cross-reactive HIV N90 463 67 289 511 Cross-reactive HIV N90 334 68 290 512 Cross-reactive HIV N90 223 69 291 513 Cross-reactive HIV N90 3415 70 292 514 Cross-reactive HIV N90 1992 71 293 515 Cross-reactive HIV N90 1987 72 294 516 Cross-reactive HIV N90 1977 73 295 517 Cross-reactive HIV N90 1848 74 296 518 Cross-reactive HIV N90 1728 75 297 519 Cross-reactive HIV N90 1567 76 298 520 Cross-reactive HIV N90 1506 77 299 521 Cross-reactive HIV N90 1416 78 300 522 Cross-reactive HIV N90 1027 79 301 523 Cross-reactive HIV N90 934 80 302 524 Cross-reactive HIV N90 652 81 303 525 Cross-reactive HIV N90 624 82 304 526 Cross-reactive HIV N90 431 83 305 527 Cross-reactive HIV N90 350 84 306 528 Cross-reactive HIV N90 3345 85 307 529 Cross-reactive HIV N90 2504 86 308 530 Cross-reactive HIV N90 1753 87 309 531 Cross-reactive HIV N90 1690 88 310 532 Cross-reactive HIV N90 1324 89 311 533 Cross-reactive HIV N90 1314 90 312 534 Cross-reactive HIV N90 155 91 313 535 Cross-reactive HIV N90 1866 92 314 536 Cross-reactive HIV N90 654 93 315 537 Cross-reactive HIV N90 1487 94 316 538 Cross-reactive HIV N90 842 95 317 539 Cross-reactive HIV N90 523 96 318 540 Cross-reactive HIV N90 284 97 319 541 Cross-reactive HIV N90 208 98 320 542 Cross-reactive HIV N90 1149 99 321 543 Cross-reactive HIV N90 1882 100 322 544 Cross-reactive HIV N90 1662 101 323 545 Cross-reactive HIV N90 1572 102 324 546 Cross-reactive HIV N90 404 103 325 547 Cross-reactive HIV N90 2978 104 326 548 Cross-reactive HIV N90 1261 105 327 549 Cross-reactive HIV N90 845 106 328 550 Cross-reactive HIV N90 1125 107 329 551 Cross-reactive HIV N90 3035 108 330 552 Cross-reactive HIV N90 3272 109 331 553 Cross-reactive HIV N90 2759 110 332 554 Cross-reactive HIV N90 2638 111 333 555 Cross-reactive HIV N90 2014 112 334 556 Cross-reactive HIV N90 1824 113 335 557 Cross-reactive HIV N90 1612 114 336 558 Cross-reactive HIV N90 1478 115 337 559 Cross-reactive HIV N90 1422 116 338 560 Cross-reactive HIV N90 942 117 339 561 Cross-reactive HIV N90 818 118 340 562 Cross-reactive HIV N90 445 119 341 563 Cross-reactive HIV N90 183 120 342 564 Cross-reactive HIV N90 30 121 343 565 Cross-reactive HIV N90 29 122 344 566 Cross-reactive HIV N90 3477 123 345 567 Cross-reactive HIV N90 2845 124 346 568 Cross-reactive HIV N90 587 125 347 569 Cross-reactive HIV N90 3330 126 348 570 Cross-reactive HIV N90 3047 127 349 571 Cross-reactive HIV N90 2612 128 350 572 Cross-reactive HIV N90 2148 129 351 573 Cross-reactive HIV N90 1657 130 352 574 Cross-reactive HIV N90 1016 131 353 575 Cross-reactive HIV N90 968 132 354 576 Cross-reactive HIV N90 277 133 355 577 Cross-reactive HIV N90 2309 134 356 578 Cross-reactive HIV N90 3140 135 357 579 Cross-reactive HIV N90 2790 136 358 580 Cross-reactive HIV N90 2726 137 359 581 Cross-reactive HIV N90 1308 138 360 582 Cross-reactive HIV N90 991 139 361 583 Cross-reactive HIV N90 406 140 362 584 Cross-reactive HIV N90 137 141 363 585 Cross-reactive HIV N90 3005 142 364 586 Cross-reactive HIV N90 2745 143 365 587 Cross-reactive HIV N90 3439 144 366 588 Cross-reactive HIV N90 3400 145 367 589 Cross-reactive HIV N90 1921 146 368 590 Cross-reactive HIV N90 1126 147 369 591 Cross-reactive HIV N90 256 148 370 592 Cross-reactive HIV N90 3109 149 371 593 Cross-reactive HIV N90 2967 150 372 594 Cross-reactive HIV N90 2337 151 373 595 Cross-reactive HIV N90 1705 152 374 596 Cross-reactive HIV N90 492 153 375 597 Cross-reactive HIV N90 1479 154 376 598 Cross-reactive HIV N90 2002 155 377 599 Cross-reactive HIV N90 1813 156 378 600 Cross-reactive HIV N90 1048 157 379 601 Cross-reactive HIV N90 931 158 380 602 Cross-reactive HIV N90 460 159 381 603 Cross-reactive HIV N90 245 160 382 604 Cross-reactive HIV N90 3543 161 383 605 Cross-reactive HIV N90 2495 162 384 606 Cross-reactive HIV N90 2294 163 385 607 Cross-reactive HIV N90 91 164 386 608 Cross-reactive HIV N90 2379 165 387 609 Cross-reactive HIV N90 1851 166 388 610 Cross-reactive HIV N90 1357 167 389 611 Cross-reactive HIV N90 129 168 390 612 Cross-reactive HIV N90 48 169 391 613 Cross-reactive HIV N90 1287 170 392 614 Cross-reactive HIV N90 505 171 393 615 Cross-reactive HIV N90 3434 172 394 616 Cross-reactive HIV N90 3260 173 395 617 Cross-reactive HIV N90 51 174 396 618 Cross-reactive HIV N90 3441 175 397 619 Cross-reactive HIV N90 2535 176 398 620 Cross-reactive HIV N90 510 177 399 621 Cross-reactive HIV N90 328 178 400 622 Cross-reactive HIV N90 3497 179 401 623 Cross-reactive HIV N90 1549 180 402 624 Cross-reactive HIV N90 884 181 403 625 Cross-reactive HIV N90 2943 182 404 626 Cross-reactive HIV N90 2487 183 405 627 Cross-reactive HIV N90 1733 184 406 628 Cross-reactive HIV N90 3333 185 407 629 Cross-reactive HIV N90 3087 186 408 630 Cross-reactive Flu N90 1282 187 409 631 Cross-reactive Flu N90 2363 188 410 632 Cross-reactive Flu N90 251 189 411 633 Cross-reactive Flu N90 1849 190 412 634 Cross-reactive Flu N90 3139 191 413 635 Cross-reactive Flu N90 3455 192 414 636 Cross-reactive Flu N90 3180 193 415 637 Cross-reactive Flu N90 1993 194 416 638 Cross-reactive Flu N90 206 195 417 639 Cross-reactive Flu N90 2361 196 418 640 Cross-reactive Flu N90 218 197 419 641 Cross-reactive Flu N90 833 198 420 642 Cross-reactive Flu N90 2976 199 421 643 Cross-reactive Flu N90 2883 200 422 644 Cross-reactive Flu N90 1910 201 423 645 Cross-reactive Flu N90 1724 202 424 646 Cross-reactive Flu N90 377 203 425 647 Cross-reactive Flu N90 1757 204 426 648 Cross-reactive Flu N90 3326 205 427 649 Cross-reactive Flu N90 1864 206 428 650 Cross-reactive Flu N90 2822 207 429 651 Cross-reactive Flu N90 1373 208 430 652 Cross-reactive Flu N90 2709 209 431 653 Cross-reactive Flu N90 2496 210 432 654 Cross-reactive Flu N90 2018 211 433 655 Cross-reactive Flu N90 3505 212 434 656 Cross-reactive Flu N90 2115 213 435 657 Cross-reactive Flu N90 2724 214 436 658 Cross-reactive Flu N90 3436 215 437 659 Cross-reactive Flu N90 2678 216 438 660 Cross-reactive Flu N90 645 217 439 661 Cross-reactive Flu N90 3007 218 440 662 Cross-reactive Flu N90 2539 219 441 663 Cross-reactive Flu N90 1900 220 442 664 Cross-reactive Flu N90 1499 221 443 665 Cross-reactive Flu N90 1367 222 444 666 Cross-reactive Flu
TABLE-US-00003 TABLE 2 Amino acid sequences for heavy and light chains and the CDRs thereof. SEQ ID SEQ ID NO for NO for Heavy SEQ ID SEQ ID SEQ ID Light SEQ ID SEQ ID SEQ ID mAb chain NO for NO for NO for chain NO for NO for NO for name aa CDRH1 CDRH2 CDRH3 aa CDRL1 CDRL2 CDRL3 Specificity 2723-4872 667 734 761 796 844 852 878 903 HIV 3602-2648 668 721 746 784 830 863 888 897 HIV 3602-3278 668 721 746 784 830 863 888 897 HIV 3602-520 668 721 746 784 830 863 888 897 HIV 2723-432 669 720 766 774 810 864 882 899 HIV 3602-1483 670 714 744 794 829 862 891 897 HIV 3602-1075 671 719 745 776 815 872 889 898 HIV 3602-2137 672 719 745 776 816 869 889 898 HIV 3602-2199 673 719 745 776 814 866 889 901 HIV 3602-3420 674 722 742 793 831 867 889 894 HIV 3602-1337 675 717 743 793 812 865 889 896 HIV 3602-1494 675 717 743 793 812 865 889 896 HIV 3602-1735 675 717 743 793 812 865 889 896 HIV 3602-2848 675 717 743 793 812 865 889 896 HIV 3602-392 675 717 743 793 812 865 889 896 HIV 3602-964 675 717 743 793 812 865 889 896 HIV 3602-1544 676 717 743 791 811 865 889 895 HIV 3602-1841 676 715 743 791 811 865 889 895 HIV 3602-1737 677 718 743 793 811 865 889 895 HIV 3602-819 677 718 743 785 811 865 889 895 HIV 2723-3862 678 738 751 798 832 855 877 906 HIV 2723-5847 678 738 751 798 833 855 877 906 HIV 2723-483 679 736 747 783 827 848 881 908 HIV 2723-7033 680 736 747 783 828 847 880 908 HIV 2723-6307 681 736 747 783 828 847 880 908 HIV 2723-4196 682 736 747 782 825 848 880 908 HIV 2723-1241 683 736 747 783 826 848 881 908 HIV 2723-4559 684 735 748 800 822 850 880 904 HIV 3602-870 685 713 749 773 813 851 879 893 HIV 3602-1707 686 723 752 768 809 868 889 900 flu 2723-2304 687 725 762 778 818 859 885 913 HIV 2723-422 688 726 763 780 817 859 885 913 HIV 2723-3415 689 739 753 777 819 860 878 909 flu 2723-2120 690 727 741 775 834 873 884 916 HIV 2723-2121 691 728 767 779 821 871 883 912 HIV 2723-1952 692 740 756 781 808 870 887 892 HIV 2723-3196 693 716 764 790 807 857 879 914 HIV 2723-2859 694 724 757 799 820 861 883 910 flu 2723-5469 695 730 758 787 839 876 890 911 HIV 2723-293 696 731 760 788 835 874 890 911 HIV 2723-4186 696 731 760 788 840 858 890 911 HIV 2723-2540 697 733 765 786 838 876 890 911 HIV 2723-3244 698 732 758 788 837 875 890 911 HIV 2723-6220 699 732 758 789 837 875 890 911 HIV 2723-5655 700 732 758 788 837 875 890 911 HIV 2723-6684 701 731 760 788 836 874 890 911 HIV 2723-2624 702 729 750 792 841 853 886 915 HIV 2723-5479 703 729 750 792 842 853 886 915 HIV 2723-3069 704 737 759 801 824 849 880 905 HIV 2723-4975 704 737 759 801 823 846 880 905 HIV 2723-6609 704 737 759 801 823 846 880 905 HIV 2723-3055 705 729 761 795 843 853 886 902 HIV 2723-3131 706 712 754 772 806 856 883 907 HIV 2723-4886 707 712 754 769 802 856 883 907 HIV 2723-4509 708 712 755 770 804 856 883 907 HIV 2723-1879 709 712 755 771 803 856 883 907 HIV 2723-229 710 712 755 770 805 856 883 907 HIV 2723-6245 711 734 761 797 845 854 885 903 HIV
TABLE-US-00004 TABLE 3 Sequences in FIG. 2. SEQ ID SEQ ID NO CDRH3 NO CDRL3 770 AMRDYCRDDNCNKWDLRH 907 QHRET 771 AMRDYCRDDNCNRWDLRH 907 QHRET 917 AMRDYCRDDSCNIWDLRH 907 QHRET 918 AMRDYCRDDNCNIWDLRH 907 QHRET 919 VRTAYCERDPCKGWVFPH 906 QFLEN 920 VRRFVCDHCSDYTFGH 904 QDQEF 921 VRRGHCDHCYEWTLQH 905 QDRQS 922 VRRGSCDYCGDFPWQY 908 QQFEF 923 VRRGSCGYCGDFPWQY 908 QQFEF 924 VRGSSCCGGRRHCNGADCFNWDFQY 903 QCLEA 925 VRGRSCCGGRRHCNGADCFNWDFQY 903 QCLEA 926 VRGKSCCGGRRYCNGADCFNWDFEH 915 QSFEG 927 VRGRSCCDGRRYCNGADCFNWDFEH 902 QCFEG 928 TRGKYCTARDYYNWDFEH 911 QQYEF 929 TRGKYCTARDYYNWDFEY 911 QQYEF 930 TRGKNCDDNWDFEH 911 QQYEF 931 TRGKNCNYNWDFEH 911 QQYEF
TABLE-US-00005 TABLE 4 Additional sequences in FIG. 2. SEQ SEQ ID ID VJ NO VDJ Junction NO junction 939 ARHRADYDFWNGNNLRGYFDP 912 QQYGSSPTT 940 ARHRANYDFWGGSNLRGYFDP 913 QQYGTSPTT 941 ARHRADYDFWGGSNLRGYFDP 913 QQYGTSPTT 942 ARDEVLRGSASWFLGPNEVRHYGMDV 899 MQSLQLRS 943 VGRQKYISGNVGDFDF 914 QQYTNLPPALN 944 ATGRIAASGFYFQH 892 HHYNSFSHT 775 AREHTMIFGVAEGFWFDP 916 SSRDTDDISVI 945 VTMSGYHVSNTYLDA 910 QQYANSPLT 946 ARGRVYSDY 909 QQSGTSPPWT
TABLE-US-00006 TABLE 5 Sequences in FIG. 3. SEQ ID NO CDRH3 SEQ ID NO CDRL3 932 VRGPSSGWWYHEYSGLDV 897 MQARQTPRLS 933 IRGPESGWFYHYYFGLGV 897 MQARQTPRLS 934 ARGPSSGWHLHYYFGMGL 937 MQSLETPRLS 934 ARGPSSGWHLHYYFGMGL 938 MQSLQTPRLS 935 VRGPSSGWHLHYYFGMDL 894 MEALQTPRLT 935 VRGPSSGWHLHYYFGMDL 896 METLQTPRLT 935 VRGPSSGWHLHYYFGMDL 895 MESLQTPRLT 936 VRGASSGWHLHYYFGMDL 895 MESLQTPRLT
TABLE-US-00007 TABLE 6 Additional sequences in FIG. 3. SEQ ID SEQ ID NO VDJ Junction NO VJ junction 947 ARDAGERGLRGYSVGFFDS 893 HQYGTTPYT 948 AKVVAGGQLRYFDWQEGHYYGMDV 900 MQSLQTPHS
[0241] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
[0242] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.