US20210139565A1 - Camelidae single-domain antibodies against yersinia pestis and methods of use - Google Patents

Camelidae single-domain antibodies against yersinia pestis and methods of use Download PDF

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US20210139565A1
US20210139565A1 US17/141,501 US202117141501A US2021139565A1 US 20210139565 A1 US20210139565 A1 US 20210139565A1 US 202117141501 A US202117141501 A US 202117141501A US 2021139565 A1 US2021139565 A1 US 2021139565A1
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sabs
pestis
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Camilla A. Mauzy
Serge Victor Marie Muyldermans
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US Air Force
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This invention relates generally to the field of single-domain antibodies. More particularly, it relates to single-domain antibodies and polypeptides against Yersinia pestis, nucleic acid sequences encoding the single-domain antibodies, and methods of using the same.
  • a diagnostic tool must be sensitive and specific, as well as able to withstand the extreme conditions often encountered in the field.
  • the value of a therapeutic tool is largely determined by parameters such as toxicity, immunogenicity, and efficacy after administration.
  • the therapeutic tool may be required to treat large number of people in the event of a bioterrorism attack. All of these requirements highlight the importance of a long shelf life and the production costs of biological weapon-related diagnostics and therapeutics.
  • VHH The N-terminal domain of these heavy chain-only antibodies, which is referred to as VHH, is variable in sequence, and it is the sole domain that interacts with the cognate antigen. Because of their small size (12-15kDa, 2.2nm diameter, and 4 nm height), VHHs are also known as single-domain antibodies (SAbs), which are commercially-available as NANOBODIES (NANOBODY and NANOBODIES are registered trademarks of Ablynx N.V., Belgium).
  • SAbs single-domain antibodies
  • SAbs make attractive as tools for biological weapon detection due to their high affinity and specificity for their respective targets and their high stability and solubility. Their small size gives SAbs the unique ability to recognize and bind to areas of an antigen that are often not normally accessible to full-size antibodies due to steric hindrance and other size constraints. In addition, SAbs may be economically produced in large quantities, and their sequences are relatively easy to tailor to a specific application. These properties, as well as their low immunogenicity, make SAbs uniquely suited for detection, diagnostics, and immunotherapeutics.
  • the present invention includes a composition comprising at least one single-domain antibody against one or more Yersinia pestis ( Y. pestis ) surface proteins, in which the one or more Y. pestis surface proteins are selected from the group consisting of YscF, F1, and LcrV, with each single-domain antibody comprising four framing regions (FRs) and three complementarity determining regions (CDRs), in which the at least one single-domain antibody is selected from the group consisting of: (1) at least one single-domain antibody comprising one CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7, one CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33, and one CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60; (2) at least one single-domain antibody comprising one CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19, one CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47, and one C
  • the at least one single-domain antibody is selected from the group consisting of SEQ ID NOs:154-160, 168-185, and 204-217. In a further embodiment, the at least one single-domain antibody further comprises at least one of a protein tag, a protein domain tag, or a chemical tag.
  • the composition comprises a plurality of single-domain antibodies against a single Y. pestis surface protein. In another embodiment, at least a portion of the plurality of single-domain antibodies is against different epitopes on the single Y. pestis surface protein. In another embodiment, the composition comprises a plurality of single-domain antibodies against at least two Y. pestis surface proteins.
  • the composition comprises a plurality of single-domain antibodies further comprising a polypeptide.
  • the plurality of single-domain antibodies comprising the polypeptide are against a single Y. pestis surface protein.
  • at least a portion of the plurality of single-domain antibodies comprising the polypeptide are against different epitopes on the single Y. pestis surface protein.
  • the plurality of single-domain antibodies comprising the polypeptide are against at least two Y. pestis surface proteins.
  • the polypeptide comprises a fusion protein.
  • the polypeptide comprises a multivalent protein complex, with the single-domain antibodies being joined together with at least one linker molecule.
  • at least one of the plurality of single-domain antibodies comprising the polypeptide further comprises at least one of a protein tag, a protein domain tag, or a chemical tag.
  • the present invention further includes at least one isolated nucleotide sequence encoding the at least one single-domain antibody, wherein the at least one isolated nucleotide sequence is selected from the group consisting of SEQ ID NOs:164-170, 189-206, and 221-234.
  • FIG. 1 is a graph of the binding response of IgG isolated from an immune alpaca to Y. pestis YscF (ELISA).
  • FIG. 2 is a graph of the binding response of IgG isolated from an immune alpaca to Y. pestis F1 (ELISA).
  • FIG. 3 is a graph of the binding response of IgG isolated from an immune alpaca to Y. pestis LcrV (ELISA).
  • FIG. 4 is a graph of polyclonal phage ELISA testing after each round of panning to isolate YscF-specific phages.
  • FIG. 5 is a graph of the ELISA for the presence of YscF-specific SAbs in the periplasmic extract of positive colonies.
  • FIG. 6 is a graph of polyclonal phage ELISA testing after each round of panning to isolate F1-specific phages.
  • FIGS. 7A-B are graphs of the ELISA for the presence of F1-specific SAbs in the periplasmic extract of positive colonies.
  • FIG. 8 is a graph of polyclonal phage ELISA testing after each round of panning to isolate LcrV-specific phages.
  • FIGS. 9A-B are graphs graph of the ELISA for the presence of LcrV-specific SAbs in the periplasmic extract of positive colonies.
  • FIG. 10 is a protein sequence alignment of seven exemplary YscF SAbs according to the present invention.
  • FIGS. 11A-B are protein sequence alignments of eighteen exemplary F1 SAbs according to the present invention.
  • FIGS. 12A-B are the protein sequence alignment of fourteen exemplary LcrV SAbs according to the present invention.
  • FIGS. 13A-B are the double-referenced sensorgrams obtained on the BIACORE T200 sensor instrument for selected LcrV SAbs.
  • FIGS. 14A-B are the double-referenced sensorgrams obtained on the BIACORE T200 sensor instrument for the two LcrV SAbs demonstrating the best binding capabilities.
  • FIGS. 15A-C are sequence alignments of nucleic acid sequences encoding the exemplary YscF SAbs according to the present invention.
  • FIGS. 16A-H are sequence alignments of nucleic acid sequences encoding the exemplary F1 SAbs according to the present invention.
  • FIGS. 17A-F are sequence alignments of nucleic acid sequences encoding the exemplary LcrV SAbs according to the present invention.
  • the present invention includes single-domain antibodies (SAbs) against three Yersinia pestis ( Y. pestis ) surface proteins (LcrV, YscF, and F1), the nucleic acids encoding the SAbs, and polypeptides comprising two or more SAbs capable of recognizing one or more Y. pestis surface proteins or epitopes.
  • SAbs single-domain antibodies against three Yersinia pestis ( Y. pestis ) surface proteins (LcrV, YscF, and F1)
  • the nucleic acids encoding the SAbs and polypeptides comprising two or more SAbs capable of recognizing one or more Y. pestis surface proteins or epitopes.
  • the present invention further includes methods for preventing or treating Y. pestis infections in a patient; methods for detecting and/or diagnosing Y. pestis infections; and devices and methods for identifying and/or detecting Y. pestis on a surface and/or in an environment.
  • Y. pestis the gram-negative bacillus that causes plague, is considered a Class A biological weapon.
  • Y. pestis infections occur in three different ways: infection of the lymph nodes (bubonic), the lungs (pneumonic), or the blood (septicemic).
  • the most serious, contagious, and often fatal mode of plague is pneumonic plague, which may be caused by inhalation of contaminated respiratory droplets from another infected person or from intentional release of aerosolized plague pathogen. While Y. pestis infections are treatable with antibiotics, diagnosis and treatment are often delayed.
  • LcrV is a 37 kDa virulence factor that is secreted and expressed on the Y. pestis cell surface prior to bacterial interaction with host cells, making it an excellent antigenic protein for antibody capture. It has been shown that anti-LcrV antibodies can block the delivery of Yops, a set of virulence proteins exported into the host cell upon contact. Additionally, it has been shown that a single sensitive, specific antibody could be used to capture LcrV from Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica.
  • LcrV The functional determination of LcrV provides a possible reason for the success of anti-LcrV Ab immunotherapeutics as it is hypothesized that the anti-LcrV/Ab complex prevents the formation and function of the tip complex, thus interfering with the translocation of virulent Yops critical to infection.
  • YscF has also been implicated as one of the “needle” proteins involved in T3SS injection of the virulent Yops proteins across eukaryotic membranes upon cell contact. Recent work using purified YscF to initiate an active immune response indicates that YscF-vaccinated mice have significant protection to a Y. pestis challenge. As with LcrV, these data indicate that YscF is an excellent antigen target for immunotherapeutic uses.
  • F1 protein which is a Y. pestis capsule protein, has likewise been identified as a potential therapeutic target and is one of the principal immunogens in currently available plague vaccines. Among other roles, F1 is thought to be involved in preventing Y. pestis uptake by macrophages.
  • SAbs in general including the presently disclosed Y. pestis SAbs, comprise four framework regions (FRs) interrupted by three complementarity determining regions (CDRs) to yield the following general structure:
  • sequence identity relates to the number of positions with identical nucleotides or amino acids divided by the total number of nucleotides or amino acids. The number of identical nucleotides or amino acids is determined by comparing corresponding positions of a designated first sequence (usually a reference sequence) with a second sequence.
  • sequence identity relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the designated or reference sequence. Any addition, deletion, insertion, or substitution of a nucleotide or amino acid is considered a difference when calculating the sequence identity.
  • the degree of sequence identity may also be determined using computer algorithms, such algorithms may include, for example, commercially-available Basic Local Alignment Search Tool, also known as BLAST (U.S. National Library of Medicine, Bethesda, Md.).
  • Y. pestis SAbs may be used as components of in vivo and in vitro assays and may also be used diagnostic testing and imaging.
  • the generally low toxicity and immunogenicity of SAbs further makes the present Y. pestis SAbs promising active and passive immunotherapeutic tools, particularly for self-administered fieldable therapeutics.
  • a self-administered treatment could provide sufficient temporary immunity and sufficiently slow the onset and progress of the disease to allow a person exposed to Y. pestis to reach a hospital for diagnosis and treatment.
  • the SAbs may be introduced by any suitable method including intravenous and subcutaneous injection, oral ingestion, inhalation, and topical administration.
  • the SAbs may bind to extracellular epitopes and antigens and may also bind to intracellular targets after introduction into the host cell by phagocytosis or other mechanisms.
  • the Y. pestis SAbs may be useful for decontamination and as field-stable capture elements for real-time biological weapon detection and quantitation.
  • Y. pestis SAbs demonstrate full functionality and high affinity for their respective antigen targets, which is likely due to the ability of SAbs to bind to protein clefts that are often inaccessible to larger, conventional antibodies. This ability to access areas located in interior pockets may allow therapeutic and detection tools based on the present Y. pestis SAbs to detect multiple strains of the pathogen, as well as related organisms in the Yersinia genus. SAb-based tools and techniques may also be less susceptible to genetic engineering of pathogen surface proteins and epitopes designed to elude current detectors and to circumvent immunity conferred by conventional vaccination.
  • the Y. pestis SAbs according to the present invention may be quickly, easily, and inexpensively produced in large quantities in a bacterial expression system such as E. coli with little or no loss of protein activity and little or no need for post-translational modification.
  • the SAbs are stable within a wide range of temperature, humidity, and pH. This stability may allow for stockpiling and long-term storage of the SAbs and SAb-based detection, diagnostic, and therapeutic tools in preparation for Y. pestis outbreaks and/or a bioterrorism attack, all without the need for costly climate control and/or monitoring.
  • the stability of SAbs in extreme environments may further allow for reusable sensors and detection devices.
  • FR and CDR regions Exemplary combinations of individual FR and CDR regions are shown in Table 2, and complete SAb protein sequences isolated according to the following Examples are listed in Tables 3, 5, and 7.
  • Unique sequences are each assigned a SEQ ID NO; sequences comprising less than four amino acids are not assigned a SEQ ID NO.
  • some SAbs share 100% sequence identity in one or more CDRs and/or FRs because the SAbs are either from clonally-related B-cells or from the same B-cell with diversification due to PCR error during library construction.
  • FIGS. 1-3 are graphs of the immune response to YscF, F1, and LcrV, respectively, in both conventional (IgG1) and heavy chain (IgG2 & IgG3) antibodies.
  • IgG1 and heavy chain (IgG2 & IgG3) antibodies As seen in FIGS. 1-3 , the IgG isolated from the immune animal exhibited a strong response toward all three antigens in both types of antibody.
  • VHH library was then constructed and screened for the presence of SAbs specific to YscF, F1, and LcrV.
  • Total RNA was extracted from peripheral blood lymphocytes isolated from the immune alpaca and used as a template for first strand cDNA synthesis with oligo(dT) primer.
  • VHH encoding sequences were amplified by PCR and cloned into the phagemid vector pHEN4.
  • pHEN4 vectors containing the amplified VHH sequences were transformed into electrocompetent cells to obtain a VHH library of about 1-2 ⁇ 10 8 independent transformants. About 75-93% of transformants harbored vectors with the correct insert sizes.
  • Antigen-specific SAbs were then selected from a phage display library.
  • the VHH library was subjected to four consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 700 ⁇ g/ml, 30 ⁇ g/well, in 25 mM Tris (pH not tested), 150 mM NaCl, 0.05% Tween-20, and 1 mM EDTA).
  • the enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from negative control (only blocked) wells. The enrichment was also evaluated by polyclonal phage ELISA, which is shown in FIG. 4 .
  • FIG. 10 is a protein sequence alignment of the seven exemplary YscF SAbs listed in Table 3, and FIGS. 15A-C are sequence alignments of the nucleic acid sequences listed in Table 4. Gaps are introduced in the sequences contained in FIGS. 10 and 15A -C as needed in order to align the respective protein and nucleic acid sequences with one another. Referring to FIG. 10 , the three CDRs are underlined in each sequence. The CDRs are defined according to the Kabat numbering system [Kabat, E. A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, Md.].
  • the library was subjected to four consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 200 ⁇ g/ml, 20 ⁇ g/well, in the presence of 0.005% Tween-20).
  • the enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from negative control (blocked only) wells.
  • the enrichment was also evaluated by polyclonal phage ELISA, which is shown in FIG. 6 .
  • FIGS. 11A-B are protein sequence alignments of the 18 exemplary F1 SAbs listed in Table 5, and FIGS. 16A-H are sequence alignments of the nucleic acid sequences listed in Table 6. Gaps are introduced in the sequences in FIGS. 11A-B and 16 A-H as needed in order to align the protein and nucleic acid sequences with one another. Referring to FIGS. 11A-B , the three CDRs are underlined in each sequence. The CDRs are defined according to the Kabat numbering system. The differences in the four FRs of each SAb (if any), as compared with 3F55 (SEQ ID NO:168), are in bold; any differences between the three CDRs of each SAb are not otherwise indicated.
  • the 18 exemplary Fl SAbs shown in FIGS. 11A-B and listed in Table 5 represent 10 different groups, which are listed in Table 9.
  • SAbs belonging to the same group are very similar, especially in the CDR3 region, and their amino acid sequences suggest that they are either from clonally-related B-cells resulting from somatic hypermutation or from the same B-cell with diversification due to PCR error during library construction.
  • the library was subjected to three consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 200 ⁇ g/ml, 20 ⁇ g/well).
  • the enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from negative control (blocked only) wells.
  • the enrichment was also evaluated by polyclonal phage ELISA, which is shown in FIG. 8 .
  • FIGS. 12A-B are the protein sequence alignment of the 14 exemplary LcrV SAbs listed in Table 7, and FIGS. 17A-F are sequence alignments of the nucleic acid sequences listed in Table 8. Gaps are introduced in the sequences in FIGS. 12A-B and 17A-F as needed in order to align the protein and nucleic acid sequences with one another.
  • the three CDRs are underlined in each sequence. The CDRs are defined according to the Kabat numbering system.
  • the differences in the four FRs of each SAb (if any), as compared with 1LCRV32 (SEQ ID NO:204), are shown in bold; any differences between the three CDRs of each SAb are not otherwise indicated.
  • the 14 exemplary LcrV SAbs shown in FIGS. 12A-B and listed in Table 7 represent six different groups, which are listed in Table 10. SAbs belonging to the same group are very similar, and their amino acid sequences suggest that they are from clonally-related B-cells resulting from somatic hypermutation or from the same B-cell with diversification due to PCR error during library construction.
  • Binding kinetics studies were conducted on selected LcrV and Fl SAbs.
  • LcrV and F1 protein was immobilized on the surface of a BIACORE CM5 chip (GE Healthcare Biosciences), and each SAb was allowed to associate/dissociate with the appropriate antigen.
  • the results of the binding kinetics study are shown in Table 11. Binding generally ranged from nM to pM, with the best two SAbs (LcrV-reactive SAbs SEQ ID NOs:209, 214) binding to the target in the mid-fM range.
  • the binding constants of the seven LcrV SAbs from Table 11 (SEQ ID NOs:204, 209, 211, 214-217) are shown in Table 12.
  • FIGS. 13A-B are the resulting double-referenced sensorgrams (colored by SAb concentration) obtained using a BIACORE T200 sensor instrument (General Electric Healthcare, United Kingdom) for six of the seven LcrV SAbs from Tables 11 and 12 (SEQ ID NOs:204, 209, 211, 214-217). A dissociation phase of 500 seconds was used for all concentrations of SAb. The overlaying curve fits are depicted in black, and the sensorgrams are based on a 1:1 binding model.
  • 14A-B are the double-referenced sensorgrams (colored by SAb concentration) obtained on the BIACORE T200 sensor instrument with overlaying curve fits (black), based on a 1:1 binding model. These data indicate that the SAb sequences of SEQ ID NOs:209 and 214 bind to the Y. pestis LcrV protein extremely and unusually tightly. Due to the nature of these two SAbs, both could bind to the Y. pestis bacteria in a manner that may make infection and/or replication difficult or impossible.
  • the present invention includes SAbs against at least one Y. pestis surface protein or antigen and the nucleotide sequences that encode the SAbs.
  • the Y. pestis surface protein may include YscF, F1, and/or LcrV.
  • the present invention includes a composition comprising a single SAb or a mixture of two or more different SAbs.
  • all of the SAbs may be against a single Y. pestis surface protein (single-antigen), or the SAbs may be against different epitopes on the same Y. pestis surface protein (single-antigen, multi-epitope).
  • the mixture of two or more different SAbs may further comprise SAbs against two or more Y. pestis surface proteins (multi-antigen).
  • SAbs against at least one Y. pestis YscF epitope may comprise one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60.
  • pestis YscF epitope may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146; a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60; and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153.
  • SAbs against at least one Y may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120;
  • pestis YscF epitope may comprise the specific arrangement of FRs and CDRs embodied in SEQ ID NOs:154-160.
  • the present invention further includes isolated nucleotide sequences selected from the group consisting of SEQ ID NOs:161-167 that encode the SAbs comprising SEQ ID NOs:154-160.
  • SAbs against at least one Y. pestis F1 epitope may comprise one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19; a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY.
  • pestis F1 epitope may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19; an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120; a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47; an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146; a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY; and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153.
  • SAbs against at least one Y. pestis Fl epitope comprise the specific arrangement of FRs and CDRs embodied in SEQ ID NOs:168-185.
  • the present invention further includes isolated nucleotide sequences selected from the group consisting of SEQ ID NOs:186-203 that encode the SAbs comprising SEQ ID NOs:168-185.
  • SAbs against at least one Y. pestis LcrV epitope may comprise one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26; a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI.
  • pestis LcrV epitope may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26; an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120; a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53; an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146; a CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI; and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153.
  • SAbs against at least one Y may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26; an FR2 sequence selected from the group consisting of SEQ ID NOs:
  • pestis LcrV epitope may comprise the specific arrangement of FRs and CDRs embodied in SEQ ID NOs:204-217.
  • the present invention further includes isolated nucleotide sequences selected from the group consisting of SEQ ID NOs:218-231 that encode the SAbs comprising SEQ ID NOs:204-217.
  • the present invention includes one or more SAbs against Y. pestis YscF, with each SAb comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence respectively having at least 15% sequence identity with a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60, in which the SAbs retain sufficient affinity for at least one of a Y. pestis YscF antigen or a Y. pestis YscF epitope.
  • the present invention further includes one or more SAbs against Y.
  • pestis YscF having at least 15% sequence identity with SEQ ID NOs:154-160, in which the SAbs retain sufficient affinity for at least one of a Y. pestis YscF antigen or a Y. pestis YscF epitope.
  • the present invention further includes one or more SAbs against Y. pestis Fl, with each SAb comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence respectively having at least 15% sequence identity with at least one of a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19, a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47, and a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY, in which the SAbs retain sufficient affinity for at least one of a Y. pestis F1 antigen or a Y. pestis F1 epitope.
  • the present invention further includes one or more SAbs against Y. pestis Fl having at least 15% sequence identity with SEQ ID NOs: 168-185, in which the SAbs retain sufficient affinity for at least one of a Y. pestis Fl antigen or a Y. pestis F1 epitope
  • the present invention further includes one or more SAbs against Y. pestis LcrV, with each SAb comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence respectively having at least 15% sequence identity with at least one of a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26, a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53, and a CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI, in which the SAbs retain sufficient affinity for at least one of a Y. pestis LcrV antigen or a Y. pestis LcrV epitope.
  • the present invention further includes one or more SAbs against Y.
  • pestis LcrV having at least 15% sequence identity with SEQ ID NOs: 204-217, in which the SAbs retain sufficient affinity for at least one of a Y. pestis LcrV antigen or a Y. pestis LcrV epitope.
  • the present invention further includes a polypeptide, which is used herein to refer to a structure comprising two or more of any of the above-described SAbs against Y. pestis YscF, F1, and/or LcrV in which the two or more SAbs are joined together.
  • the polypeptide may comprise a fusion protein that is created by joining together two or more SAbs at the genetic level. Two or more nucleic acid sequences encoding for two or more SAbs may be spliced together, and translation of the spliced nucleic acid sequence creates a longer, multi-antigen and/or multi-epitope fusion protein.
  • the fusion protein may contain up to four SAbs joined end-to-end in a substantially linear fashion, similar to beads on a string.
  • the fusion protein comprises SAbs that are all against a single Y. pestis surface protein or antigen i.e. a single-antigen fusion protein against either YscF, F1, or LcrV.
  • this single-antigen fusion protein further comprises SAbs that bind to two or more different epitopes (multi-epitope, single-antigen) on the single antigen.
  • the fusion protein may comprise SAbs against two or more different Y. pestis surface proteins i.e. a multi-antigen fusion protein.
  • the multi-antigen fusion protein may also comprise SAbs that bind to two or more different epitopes (multi-epitope, multi-antigen) on the same antigen(s).
  • each individual fusion protein molecule may bind to one Y. pestis surface protein molecule, or the individual fusion protein molecule may be bound to two or more separate Y. pestis surface protein molecules.
  • Use of a multi-antigen and/or multi-epitope fusion protein may increase avidity in enzyme immunosorbent assays.
  • the polypeptide may be created by joining two or more SAbs together with a protein or chemical linker to create a multivalent protein complex.
  • a linker molecule such as the verotoxin 1B-subunit may be used to create high avidity, pentavalent SAb complexes similar to keys on a key ring.
  • the multivalent protein complex may contain SAbs that are all against a single Y. pestis surface protein or antigen i.e. a single-antigen multivalent protein complex.
  • This single-antigen multivalent protein complex may further comprise SAbs that bind to two or more different epitopes (multi-epitope, single-antigen) on the single antigen.
  • the multivalent protein complex may comprise SAbs against two or more different Y. pestis surface proteins i.e. a multi-antigen multivalent protein complex.
  • the multi-antigen multivalent protein complex may further comprise SAbs that bind to two or more different epitopes (multi-epitope, multi-antigen) on the same antigen.
  • each multivalent protein complex may bind to one Y. pestis surface protein molecule, or the multivalent protein complex may be bound to two or more separate Y. pestis surface protein molecules.
  • These multi-antigen and/or multi-epitope multivalent protein complexes may generally demonstrate increased affinity for their respective epitope and/or antigen target(s) and may have numerous applications for biomarker assays or proteomics.
  • polypeptides as described herein comprise at least two SAbs, with the SAbs being selected from the following groups: (1) SAbs comprising one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60; (2) SAbs comprising one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19; a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY; and (3) SAbs comprising one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26; a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53
  • the polypeptides comprise at least two SAbs selected from the group consisting of: (1) SAbs comprising one each of a CDR1 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 27-33; and a CDR3 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs:54-60; (2) SAbs comprising one each of a CDR1 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 8-19; a CDR2 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 34-47; and a CDR3 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 61-71; and (3) SAbs comprising one each of a CDR1 sequence selected from the group consisting of sequence
  • the polypeptides may comprise at least two SAbs, with the SAbs being selected from the following groups: (1) SAbs comprising one set of CDR1, CDR2, and CDR3 sequences (as described above with respect to polypeptides according to the present invention) and one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102, an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120, an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146, and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153; and (2) SAbs selected from the group consisting of SEQ ID NOs:154-160, 168-185, and 204-217 and sequences having at least 15% sequence identity with SEQ ID NOs:154-160, 168-185, and 204-217.
  • SAbs selected from the following groups: (1) SAbs comprising one set of CDR1, CDR2, and CDR3 sequences (
  • any of the SAbs or polypeptides according to the present invention may further comprise a protein tag, a protein domain tag, or a chemical tag.
  • These tags generally comprise one or more additional amino acids or chemical molecules or residues that may be placed using known methods on the C- or N-terminus of the SAb or polypeptide without altering the activity or functionality of the SAb or polypeptide.
  • the tag may facilitate purification of the SAb or polypeptide, direct absorption and/or excretion in the body, and/or facilitate use in a variety of applications such as detecting and monitoring Y pestis.
  • the tag may include, but is not limited to, a histidine tag (HIS tag) and a poly-lysine tag.
  • the present invention further includes a method of preventing or treating a Y. pestis infection in a patient.
  • Y. pestis infections are frequently difficult to properly diagnose, which can result in delayed treatment, and a low toxicity treatment such as the presently disclosed SAbs may provide a valuable tool for cases of suspected Y. pestis exposure and/or infection and/or for patients presenting with ambiguous symptoms.
  • the method comprises identifying a patient who is suspected of having been exposed to and/or infected with Y. pestis, and administering to the patient a pharmaceutically active amount of one or more of the SAbs and/or polypeptides according to the present invention.
  • a “pharmaceutically active amount” refers generally to an amount that upon administration to the patient, is capable of providing directly or indirectly, one or more of the effects or activities disclosed herein.
  • the SAb(s) and/or polypeptide(s) may be administered as a form of passive immunotherapy in which the SAb(s) and/or polypeptide(s) are administered to the patient prior to at least one of exposure to or infection with Y. pestis.
  • the SAb(s) and/or polypeptide(s) may be administered after the patient is exposed to or infected with Y. pestis.
  • the SAb(s) and/or polypeptide(s) may be capable of being self-administered and may be administered to the patient using known techniques including, but not limited to, intravenous and subcutaneous injection, oral ingestion, inhalation, and topical administration.
  • the ability to self-administer the SAb(s) and/or polypeptide(s) may be particularly useful in the case of an outbreak or attack where access to medical personnel and treatment may be limited.
  • the present invention further includes a method of detecting and/or diagnosing a Y. pestis infection using one of more of the SAbs and/or polypeptides herein described.
  • the method may include detection of Y. pestis and diagnosis of the infection using known in vivo and/or in vitro assays such as enzyme linked immunosorbent assays (ELISAs), dot blot assays, and other suitable immunoassays.
  • ELISAs enzyme linked immunosorbent assays
  • the Y. pestis SAb(s) and/or polypeptide(s) may, for example, be used as a primary antibody or a capture antibody in an ELISA for the detection/diagnosis of a Y. pestis infection.
  • the SAb(s) and/or polypeptide(s) according to the present invention may further be coupled to one or more enzymes or markers for use in imaging.
  • the present invention further includes devices and methods for the identification and detection of Y. pestis on a surface and/or in an environment
  • a device for the environmental detection and/or quantification of Y. pestis may comprise one or more of the SAbs or polypeptides according to the present invention, with the SAb(s) and/or polypeptide(s) being used as a capture element.
  • a method of identifying and detecting Y. pestis using the device comprises contacting one or more of the SAbs or polypeptides with an unknown target and detecting binding between the SAbs or polypeptides and the unknown target to identify the unknown target as Y. pestis.
  • the method may further comprise use of the device to quantify an amount of Y. pestis on the surface and/or in the environment.
  • pestis F1 SAb DNA Sequences SEQ ID NO Name Sequence 186 3F55 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCT GTGCAGTTTCTGGAATGATGTACATTAGGGAGGCTATACGCTGGTACCGCCAGGCTCCAGGGAA GCAGCGCGAGTGGGTCGCCTTTGTAAGTAGTACTGGTAATCCACGCTATACAGACTCCGTGAAG GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGA CACCTGAGGACACGGCCGTCTATTACTGTAATACATACTTGGGCTCGAGGGACTACTGGGGCCA GGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCC GAGCATAG 187 3F85 CAGGTGCAGCTGCAGGAGTCTGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGGGGGGTC
  • pestis LcrV SAb Protein Sequences SEQ ID NO Name Sequence 204 1LCRV32 QVQLQESGGGMVEPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLERVSAINSDGDKTSYADSVKG RFTISRDNARNTLYLQMSNLKPEDTAVYYCADRDLYCSGSMCKDVLGGARYDFRGQGTQVTVSS 205 2LCRV4 QVQLQESGGGLVEPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLERVSAINSDGDKTSYADSVKG RFTISRDNARNTLYLQMSNLKPEDTAVYYCADRDLYCSGSMCKDVLGGARYDFRGQGTQVTVSS 206 2LCRV3 QVQLQESGGGMVEPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLERVSAINSDGDKTSYADSVKG RFTISRDNARNTLYLQMNNLKPEDTAVYYCADRDLYCSGSMCKDV
  • LcrV SAb Group Name SEQ ID NO 1 1LCRV32, 2LCRV4, 204-207 2LCRV3, 1LCRV52 2 1CLRV4, 1LCRV13, 208-213 2LCRV1, 1LCRV81, 1LCRV27, 1LCRV34 3 1LCRV31 214 4 1LCRV28 215 5 2LCRV11 216 6 1LCRV47 217

Abstract

Single-domain antibodies (SAbs) against three Yersinia pestis surface proteins (LcrV, YscF, and F1), nucleic acid sequences encoding the SAbs, and polypeptides comprising two or more SAbs capable of recognizing two or more epitopes and/or antigens. The present invention further includes methods for preventing or treating Y. pestis infections in a patient; methods for detecting and/or diagnosing Y. pestis infections; and devices and methods for identifying and/or detecting Y. pestis on a surface and/or in an environment.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of co-pending U.S. application Ser. No. 16/023,723, filed Jun. 29, 2018, which was a continuation of U.S. application Ser. No. 13/906,386, filed May 31, 2013, which claimed the benefit of and priority to U.S. Provisional Application No. 61/653,488, filed on May 31, 2012. The disclosure of each application is incorporated herein by reference, in its entirety.
    • RIGHTS OF THE GOVERNMENT
  • The invention described herein may be manufactured and used by or for the Government of the United States for all governmental purposes without the payment of any royalty.
    • FIELD OF THE INVENTION
  • This invention relates generally to the field of single-domain antibodies. More particularly, it relates to single-domain antibodies and polypeptides against Yersinia pestis, nucleic acid sequences encoding the single-domain antibodies, and methods of using the same.
    • BACKGROUND OF THE INVENTION Description of the Related Art
  • Increasing threats of bioterrorism have led to the development of new diagnostic and therapeutic tools for pathogens that can potentially be used as biological weapons. Many of these pathogens, such as the causative agents of plague, anthrax, and tularemia, are relatively easy to manipulate via genetic engineering and may be designed to evade detection by sensor devices. Many of these biological weapons candidates also display resistance to current medical treatments. To be useful, a diagnostic tool must be sensitive and specific, as well as able to withstand the extreme conditions often encountered in the field. The value of a therapeutic tool is largely determined by parameters such as toxicity, immunogenicity, and efficacy after administration. In addition, the therapeutic tool may be required to treat large number of people in the event of a bioterrorism attack. All of these requirements highlight the importance of a long shelf life and the production costs of biological weapon-related diagnostics and therapeutics.
  • Members of the family Camelidae, which includes alpacas, camels, and llamas, produce conventional antibodies, as well as antibodies consisting only of a dimer of heavy-chain polypeptides. The N-terminal domain of these heavy chain-only antibodies, which is referred to as VHH, is variable in sequence, and it is the sole domain that interacts with the cognate antigen. Because of their small size (12-15kDa, 2.2nm diameter, and 4 nm height), VHHs are also known as single-domain antibodies (SAbs), which are commercially-available as NANOBODIES (NANOBODY and NANOBODIES are registered trademarks of Ablynx N.V., Belgium).
  • SAbs make attractive as tools for biological weapon detection due to their high affinity and specificity for their respective targets and their high stability and solubility. Their small size gives SAbs the unique ability to recognize and bind to areas of an antigen that are often not normally accessible to full-size antibodies due to steric hindrance and other size constraints. In addition, SAbs may be economically produced in large quantities, and their sequences are relatively easy to tailor to a specific application. These properties, as well as their low immunogenicity, make SAbs uniquely suited for detection, diagnostics, and immunotherapeutics.
    • SUMMARY OF THE INVENTION
  • The present invention includes a composition comprising at least one single-domain antibody against one or more Yersinia pestis (Y. pestis) surface proteins, in which the one or more Y. pestis surface proteins are selected from the group consisting of YscF, F1, and LcrV, with each single-domain antibody comprising four framing regions (FRs) and three complementarity determining regions (CDRs), in which the at least one single-domain antibody is selected from the group consisting of: (1) at least one single-domain antibody comprising one CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7, one CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33, and one CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60; (2) at least one single-domain antibody comprising one CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19, one CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47, and one CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY; and (3) at least one single-domain antibody comprising one CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26, one CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53, and one CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI, with the four framing regions of each single-domain antibody comprising one FR1 sequence selected from the group consisting of SEQ ID NOs:79-102, one FR2 sequence selected from the group consisting of SEQ ID NOs:103-120, one FR3 sequence selected from the group consisting of SEQ ID NOs:121-146, and one FR4 sequence selected from the group consisting of SEQ ID NOs:147-153.
  • In one embodiment, the at least one single-domain antibody is selected from the group consisting of SEQ ID NOs:154-160, 168-185, and 204-217. In a further embodiment, the at least one single-domain antibody further comprises at least one of a protein tag, a protein domain tag, or a chemical tag.
  • In one embodiment, the composition comprises a plurality of single-domain antibodies against a single Y. pestis surface protein. In another embodiment, at least a portion of the plurality of single-domain antibodies is against different epitopes on the single Y. pestis surface protein. In another embodiment, the composition comprises a plurality of single-domain antibodies against at least two Y. pestis surface proteins.
  • In an alternative embodiment, the composition comprises a plurality of single-domain antibodies further comprising a polypeptide. In one embodiment, the plurality of single-domain antibodies comprising the polypeptide are against a single Y. pestis surface protein. In another embodiment, at least a portion of the plurality of single-domain antibodies comprising the polypeptide are against different epitopes on the single Y. pestis surface protein. In another embodiment, the plurality of single-domain antibodies comprising the polypeptide are against at least two Y. pestis surface proteins.
  • In a further embodiment, the polypeptide comprises a fusion protein. In another embodiment, the polypeptide comprises a multivalent protein complex, with the single-domain antibodies being joined together with at least one linker molecule. In a further embodiment, at least one of the plurality of single-domain antibodies comprising the polypeptide further comprises at least one of a protein tag, a protein domain tag, or a chemical tag.
  • The present invention further includes at least one isolated nucleotide sequence encoding the at least one single-domain antibody, wherein the at least one isolated nucleotide sequence is selected from the group consisting of SEQ ID NOs:164-170, 189-206, and 221-234.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a graph of the binding response of IgG isolated from an immune alpaca to Y. pestis YscF (ELISA).
  • FIG. 2 is a graph of the binding response of IgG isolated from an immune alpaca to Y. pestis F1 (ELISA).
  • FIG. 3 is a graph of the binding response of IgG isolated from an immune alpaca to Y. pestis LcrV (ELISA).
  • FIG. 4 is a graph of polyclonal phage ELISA testing after each round of panning to isolate YscF-specific phages.
  • FIG. 5 is a graph of the ELISA for the presence of YscF-specific SAbs in the periplasmic extract of positive colonies.
  • FIG. 6 is a graph of polyclonal phage ELISA testing after each round of panning to isolate F1-specific phages.
  • FIGS. 7A-B are graphs of the ELISA for the presence of F1-specific SAbs in the periplasmic extract of positive colonies.
  • FIG. 8 is a graph of polyclonal phage ELISA testing after each round of panning to isolate LcrV-specific phages.
  • FIGS. 9A-B are graphs graph of the ELISA for the presence of LcrV-specific SAbs in the periplasmic extract of positive colonies.
  • FIG. 10 is a protein sequence alignment of seven exemplary YscF SAbs according to the present invention.
  • FIGS. 11A-B are protein sequence alignments of eighteen exemplary F1 SAbs according to the present invention.
  • FIGS. 12A-B are the protein sequence alignment of fourteen exemplary LcrV SAbs according to the present invention.
  • FIGS. 13A-B are the double-referenced sensorgrams obtained on the BIACORE T200 sensor instrument for selected LcrV SAbs.
  • FIGS. 14A-B are the double-referenced sensorgrams obtained on the BIACORE T200 sensor instrument for the two LcrV SAbs demonstrating the best binding capabilities.
  • FIGS. 15A-C are sequence alignments of nucleic acid sequences encoding the exemplary YscF SAbs according to the present invention.
  • FIGS. 16A-H are sequence alignments of nucleic acid sequences encoding the exemplary F1 SAbs according to the present invention.
  • FIGS. 17A-F are sequence alignments of nucleic acid sequences encoding the exemplary LcrV SAbs according to the present invention.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The present invention includes single-domain antibodies (SAbs) against three Yersinia pestis (Y. pestis) surface proteins (LcrV, YscF, and F1), the nucleic acids encoding the SAbs, and polypeptides comprising two or more SAbs capable of recognizing one or more Y. pestis surface proteins or epitopes. The present invention further includes methods for preventing or treating Y. pestis infections in a patient; methods for detecting and/or diagnosing Y. pestis infections; and devices and methods for identifying and/or detecting Y. pestis on a surface and/or in an environment.
  • Y. pestis, the gram-negative bacillus that causes plague, is considered a Class A biological weapon. Y. pestis infections occur in three different ways: infection of the lymph nodes (bubonic), the lungs (pneumonic), or the blood (septicemic). The most serious, contagious, and often fatal mode of plague is pneumonic plague, which may be caused by inhalation of contaminated respiratory droplets from another infected person or from intentional release of aerosolized plague pathogen. While Y. pestis infections are treatable with antibiotics, diagnosis and treatment are often delayed. In the case of pneumonic plague, the early symptoms such as fever, headache, and nausea may easily be mistaken for more common illnesses, delaying proper diagnosis and treatment during the early stages of the disease and greatly increasing the chances of death. Untreated pneumonic plague has a mortality rate of almost 100%. In the case of battlefield personnel and persons stationed or living in rural areas, access to proper health care may be further limited by distance and availability.
  • Of particular interest for detection and treatment are three Y. pestis surface proteins, LcrV, YscF, and F1. LcrV is a 37 kDa virulence factor that is secreted and expressed on the Y. pestis cell surface prior to bacterial interaction with host cells, making it an excellent antigenic protein for antibody capture. It has been shown that anti-LcrV antibodies can block the delivery of Yops, a set of virulence proteins exported into the host cell upon contact. Additionally, it has been shown that a single sensitive, specific antibody could be used to capture LcrV from Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. The functional determination of LcrV provides a possible reason for the success of anti-LcrV Ab immunotherapeutics as it is hypothesized that the anti-LcrV/Ab complex prevents the formation and function of the tip complex, thus interfering with the translocation of virulent Yops critical to infection. YscF has also been implicated as one of the “needle” proteins involved in T3SS injection of the virulent Yops proteins across eukaryotic membranes upon cell contact. Recent work using purified YscF to initiate an active immune response indicates that YscF-vaccinated mice have significant protection to a Y. pestis challenge. As with LcrV, these data indicate that YscF is an excellent antigen target for immunotherapeutic uses. F1 protein, which is a Y. pestis capsule protein, has likewise been identified as a potential therapeutic target and is one of the principal immunogens in currently available plague vaccines. Among other roles, F1 is thought to be involved in preventing Y. pestis uptake by macrophages.
  • SAbs in general, including the presently disclosed Y. pestis SAbs, comprise four framework regions (FRs) interrupted by three complementarity determining regions (CDRs) to yield the following general structure:
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Like many SAbs, the CDR3 sequence of the presently disclosed Y. pestis SAbs is generally the most crucial in determining antigen specificity. SAbs directed against a particular antigen generally demonstrate some degree of homology or sequence identity between each FR and CDR. Where two nucleotide or amino acid sequences are the same length when aligned, the term “sequence identity” as used herein relates to the number of positions with identical nucleotides or amino acids divided by the total number of nucleotides or amino acids. The number of identical nucleotides or amino acids is determined by comparing corresponding positions of a designated first sequence (usually a reference sequence) with a second sequence. Where two nucleotide or amino acid sequences are of different length when aligned, the term “sequence identity” as used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the designated or reference sequence. Any addition, deletion, insertion, or substitution of a nucleotide or amino acid is considered a difference when calculating the sequence identity. The degree of sequence identity may also be determined using computer algorithms, such algorithms may include, for example, commercially-available Basic Local Alignment Search Tool, also known as BLAST (U.S. National Library of Medicine, Bethesda, Md.).
  • Y. pestis SAbs according to the present invention may be used as components of in vivo and in vitro assays and may also be used diagnostic testing and imaging. The generally low toxicity and immunogenicity of SAbs further makes the present Y. pestis SAbs promising active and passive immunotherapeutic tools, particularly for self-administered fieldable therapeutics. In the case of an outbreak or a biological weapon attack, a self-administered treatment could provide sufficient temporary immunity and sufficiently slow the onset and progress of the disease to allow a person exposed to Y. pestis to reach a hospital for diagnosis and treatment. The SAbs may be introduced by any suitable method including intravenous and subcutaneous injection, oral ingestion, inhalation, and topical administration. The SAbs may bind to extracellular epitopes and antigens and may also bind to intracellular targets after introduction into the host cell by phagocytosis or other mechanisms. In addition, the Y. pestis SAbs may be useful for decontamination and as field-stable capture elements for real-time biological weapon detection and quantitation.
  • Many of the presently disclosed Y. pestis SAbs demonstrate full functionality and high affinity for their respective antigen targets, which is likely due to the ability of SAbs to bind to protein clefts that are often inaccessible to larger, conventional antibodies. This ability to access areas located in interior pockets may allow therapeutic and detection tools based on the present Y. pestis SAbs to detect multiple strains of the pathogen, as well as related organisms in the Yersinia genus. SAb-based tools and techniques may also be less susceptible to genetic engineering of pathogen surface proteins and epitopes designed to elude current detectors and to circumvent immunity conferred by conventional vaccination.
  • The Y. pestis SAbs according to the present invention may be quickly, easily, and inexpensively produced in large quantities in a bacterial expression system such as E. coli with little or no loss of protein activity and little or no need for post-translational modification. In addition, the SAbs are stable within a wide range of temperature, humidity, and pH. This stability may allow for stockpiling and long-term storage of the SAbs and SAb-based detection, diagnostic, and therapeutic tools in preparation for Y. pestis outbreaks and/or a bioterrorism attack, all without the need for costly climate control and/or monitoring. The stability of SAbs in extreme environments may further allow for reusable sensors and detection devices.
  • The following examples and methods are presented as illustrative of the present invention or methods of carrying out the invention, and are not restrictive or limiting of the scope of the invention in any manner. Amino acid residues will be according to the standard three-letter or one-letter amino acid code as set out in Table 1. The materials and methods used in Examples 1-4 are described, for example, in Antibody Engineering, Eds. R. Kontermann & S. Dithel, Springer-Verlag, Berlin Heidelberg (2010) Isolation of antigen-specific Nanobodies, Hassanzadeh Ghassabeh Gh., et al., Vol. 2, Chapter 20, pp. 251-266. Exemplary combinations of individual FR and CDR regions are shown in Table 2, and complete SAb protein sequences isolated according to the following Examples are listed in Tables 3, 5, and 7. Unique sequences (individual CDRs and FRs and complete SAb sequences) are each assigned a SEQ ID NO; sequences comprising less than four amino acids are not assigned a SEQ ID NO. As seen in FIGS. 10-12, some SAbs share 100% sequence identity in one or more CDRs and/or FRs because the SAbs are either from clonally-related B-cells or from the same B-cell with diversification due to PCR error during library construction.
  • Example 1: Antibody Development and Construction of a VHH Library
  • All SAbs were developed using proteins (antigen) expressed from genes isolated from Y. pestis KIM5 (avirulentpgm-), which is similar in sequence to the same protein set in Y. pestis virulent strains (pgm+). An alpaca was injected subcutaneously on days 0, 7, 14, 21, 28 and 35, each time with about 165 μg YscF antigen, about 160 μg F1 antigen, and about 160 μg LcrV antigen. The same animal may be used for all experiments, but multiple animals may also be used. On day 39, anticoagulated blood was collected from the alpaca for the preparation of plasma and peripheral blood lymphocytes. Using plasma from the immune animal, IgG subclasses were obtained by successive affinity chromatography on protein A and protein G columns and were tested by ELISA to assess the immune response to YscF, F1, and LcrV antigens. FIGS. 1-3 are graphs of the immune response to YscF, F1, and LcrV, respectively, in both conventional (IgG1) and heavy chain (IgG2 & IgG3) antibodies. As seen in FIGS. 1-3, the IgG isolated from the immune animal exhibited a strong response toward all three antigens in both types of antibody.
  • A VHH library was then constructed and screened for the presence of SAbs specific to YscF, F1, and LcrV. Total RNA was extracted from peripheral blood lymphocytes isolated from the immune alpaca and used as a template for first strand cDNA synthesis with oligo(dT) primer. Using this cDNA, the VHH encoding sequences were amplified by PCR and cloned into the phagemid vector pHEN4. pHEN4 vectors containing the amplified VHH sequences were transformed into electrocompetent cells to obtain a VHH library of about 1-2×108 independent transformants. About 75-93% of transformants harbored vectors with the correct insert sizes. Antigen-specific SAbs were then selected from a phage display library.
  • Example 2: Isolation of YscF SAbs
  • For the YscF antigen, the VHH library was subjected to four consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 700 μg/ml, 30 μg/well, in 25 mM Tris (pH not tested), 150 mM NaCl, 0.05% Tween-20, and 1 mM EDTA). The enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from negative control (only blocked) wells. The enrichment was also evaluated by polyclonal phage ELISA, which is shown in FIG. 4. These experiments suggested that the phage population was enriched for antigen-specific phages only after the third round of panning. In total, 385 individual colonies (95, 143, and 47 from second, third, and fourth rounds, respectively) were randomly selected and analyzed by ELISA for the presence of YscF-specific SAbs in their periplasmic extracts. Out of these 385 colonies, 19 colonies (all from the third round) scored positive. Sequencing of positive colonies identified seven different SAbs, and the ELISA results for these seven SAbs are shown in FIG. 5. The protein sequences of the seven exemplary YscF SAbs according to the present invention are shown in Table 3, and the nucleic acid sequences encoding the seven exemplary YscF SAbs are shown in Table 4.
  • FIG. 10 is a protein sequence alignment of the seven exemplary YscF SAbs listed in Table 3, and FIGS. 15A-C are sequence alignments of the nucleic acid sequences listed in Table 4. Gaps are introduced in the sequences contained in FIGS. 10 and 15A-C as needed in order to align the respective protein and nucleic acid sequences with one another. Referring to FIG. 10, the three CDRs are underlined in each sequence. The CDRs are defined according to the Kabat numbering system [Kabat, E. A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, Md.]. The differences in the four FRs of each SAb (if any), as compared with 3YscF57 (SEQ ID NO:154), are in bold; any differences between the three CDRs of each SAb are not otherwise indicated. The seven exemplary YscF SAb sequences depicted in FIG. 10 and listed in Table 3 represent seven different groups i.e. they originate from seven clonally-unrelated B-cells.
  • Example 3: Isolation of F1 SAbs
  • For the F1 antigen, the library was subjected to four consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 200 μg/ml, 20 μg/well, in the presence of 0.005% Tween-20). The enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from negative control (blocked only) wells. The enrichment was also evaluated by polyclonal phage ELISA, which is shown in FIG. 6. These experiments suggested that the phage population was enriched for antigen-specific phages only after the third and fourth rounds of panning. In total, 285 individual colonies from second, third, and fourth rounds of panning (95 from each round) were randomly selected and analyzed by ELISA for the presence of F1-specific SAbs in their periplasmic extracts. Out of these 285 colonies, 55 scored positive (0, 29, and 26 from second, third, and fourth rounds, respectively). Sequencing of these 55 positive colonies identified 18 different SAbs, and the ELISA results for these 19 SAbs are shown in FIGS. 7A-B. The protein sequences of 18 exemplary F1 SAbs according to the present invention are shown in Table 5, and the nucleic acid sequences encoding the 18 Fl SAbs are shown in Table 6.
  • FIGS. 11A-B are protein sequence alignments of the 18 exemplary F1 SAbs listed in Table 5, and FIGS. 16A-H are sequence alignments of the nucleic acid sequences listed in Table 6. Gaps are introduced in the sequences in FIGS. 11A-B and 16A-H as needed in order to align the protein and nucleic acid sequences with one another. Referring to FIGS. 11A-B, the three CDRs are underlined in each sequence. The CDRs are defined according to the Kabat numbering system. The differences in the four FRs of each SAb (if any), as compared with 3F55 (SEQ ID NO:168), are in bold; any differences between the three CDRs of each SAb are not otherwise indicated. The 18 exemplary Fl SAbs shown in FIGS. 11A-B and listed in Table 5 represent 10 different groups, which are listed in Table 9. SAbs belonging to the same group are very similar, especially in the CDR3 region, and their amino acid sequences suggest that they are either from clonally-related B-cells resulting from somatic hypermutation or from the same B-cell with diversification due to PCR error during library construction.
  • Example 4: Isolation of LcrV SAbs
  • For the LcrV antigen, the library was subjected to three consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 200 μg/ml, 20 μg/well). The enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from negative control (blocked only) wells. The enrichment was also evaluated by polyclonal phage ELISA, which is shown in FIG. 8. These experiments suggested that the phage population was enriched for antigen-specific phages after the first, second, and third rounds of panning. 95 individual colonies from the second round of panning were randomly selected and analyzed by ELISA for the presence of LcrV-specific SAbs in their periplasmic extracts (not shown). Out of these 95 colonies, 85 scored positive. The VHHs from the 85 positive colonies were subjected to restriction fragment length polymorphism (RFLP) analysis using HinfI enzyme (not shown). Based on RFLP analysis, 40 colonies (several from each RFLP group) were selected for sequencing. Sequence analysis identified four different SAbs.
  • The high redundancy of the LcrV positive colonies identified after the second round of panning, together with the fact that the enrichment for antigen-specific phages was already good after the first round of panning, suggested that additional rounds of panning may have led to a loss of library diversity. To address this possibility and to identify additional unique sequences, 95 colonies from first round of panning were randomly selected and analyzed by ELISA for the presence of LcrV-specific SAbs in their periplasmic extracts, which is shown in FIGS. 9A-B. Out of these 95 colonies from the first round, 35 colonies were positive. These 35 colonies represented the four previously identified SAbs, as well as 10 novel sequences. The protein sequences of 14 exemplary LcrV SAbs according to the present invention are shown in Table 7, and the nucleic acid sequences encoding the 14 LcrV SAbs are shown in Table 8.
  • FIGS. 12A-B are the protein sequence alignment of the 14 exemplary LcrV SAbs listed in Table 7, and FIGS. 17A-F are sequence alignments of the nucleic acid sequences listed in Table 8. Gaps are introduced in the sequences in FIGS. 12A-B and 17A-F as needed in order to align the protein and nucleic acid sequences with one another. Referring to FIGS. 12A-B, the three CDRs are underlined in each sequence. The CDRs are defined according to the Kabat numbering system. The differences in the four FRs of each SAb (if any), as compared with 1LCRV32 (SEQ ID NO:204), are shown in bold; any differences between the three CDRs of each SAb are not otherwise indicated. The 14 exemplary LcrV SAbs shown in FIGS. 12A-B and listed in Table 7 represent six different groups, which are listed in Table 10. SAbs belonging to the same group are very similar, and their amino acid sequences suggest that they are from clonally-related B-cells resulting from somatic hypermutation or from the same B-cell with diversification due to PCR error during library construction.
  • Example: 5 Binding Kinetics of LcrV and F1 SAbs
  • Binding kinetics studies were conducted on selected LcrV and Fl SAbs. LcrV and F1 protein was immobilized on the surface of a BIACORE CM5 chip (GE Healthcare Biosciences), and each SAb was allowed to associate/dissociate with the appropriate antigen. The results of the binding kinetics study are shown in Table 11. Binding generally ranged from nM to pM, with the best two SAbs (LcrV-reactive SAbs SEQ ID NOs:209, 214) binding to the target in the mid-fM range. The binding constants of the seven LcrV SAbs from Table 11 (SEQ ID NOs:204, 209, 211, 214-217) are shown in Table 12. The KD is calculated as kd/ka (“n.b.” =no binding).
  • FIGS. 13A-B are the resulting double-referenced sensorgrams (colored by SAb concentration) obtained using a BIACORE T200 sensor instrument (General Electric Healthcare, United Kingdom) for six of the seven LcrV SAbs from Tables 11 and 12 (SEQ ID NOs:204, 209, 211, 214-217). A dissociation phase of 500 seconds was used for all concentrations of SAb. The overlaying curve fits are depicted in black, and the sensorgrams are based on a 1:1 binding model.
  • Of the described SAb sets, two SAbs (SEQ ID NOs:209, 214) demonstrate no discernible off rate (kd) within the limits of THE BIACORE instrument analyses (see Tables 11 and 12). In a second test, LcrV was immobilized on the surface of a BIACORE CM5 chip, and LcrV-reactive SAbs SEQ ID NOs:209 and 214 were allowed to associate/dissociate. A dissociation phase of 120 seconds was used for all concentrations of SAb except the highest concentration, for which a 3600 second dissociation was used. FIGS. 14A-B are the double-referenced sensorgrams (colored by SAb concentration) obtained on the BIACORE T200 sensor instrument with overlaying curve fits (black), based on a 1:1 binding model. These data indicate that the SAb sequences of SEQ ID NOs:209 and 214 bind to the Y. pestis LcrV protein extremely and unusually tightly. Due to the nature of these two SAbs, both could bind to the Y. pestis bacteria in a manner that may make infection and/or replication difficult or impossible.
  • The present invention includes SAbs against at least one Y. pestis surface protein or antigen and the nucleotide sequences that encode the SAbs. The Y. pestis surface protein may include YscF, F1, and/or LcrV. The present invention includes a composition comprising a single SAb or a mixture of two or more different SAbs. For compositions comprising a mixture of two or more different SAbs, all of the SAbs may be against a single Y. pestis surface protein (single-antigen), or the SAbs may be against different epitopes on the same Y. pestis surface protein (single-antigen, multi-epitope). The mixture of two or more different SAbs may further comprise SAbs against two or more Y. pestis surface proteins (multi-antigen).
  • In one embodiment of the present invention, SAbs against at least one Y. pestis YscF epitope may comprise one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60. In another embodiment, SAbs against at least one Y. pestis YscF epitope may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146; a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60; and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153. In a further embodiment, SAbs against at least one Y. pestis YscF epitope may comprise the specific arrangement of FRs and CDRs embodied in SEQ ID NOs:154-160. The present invention further includes isolated nucleotide sequences selected from the group consisting of SEQ ID NOs:161-167 that encode the SAbs comprising SEQ ID NOs:154-160.
  • In another embodiment, SAbs against at least one Y. pestis F1 epitope may comprise one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19; a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY. In another embodiment, SAbs against at least one Y. pestis F1 epitope may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19; an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120; a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47; an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146; a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY; and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153. In a further embodiment, SAbs against at least one Y. pestis Fl epitope comprise the specific arrangement of FRs and CDRs embodied in SEQ ID NOs:168-185. The present invention further includes isolated nucleotide sequences selected from the group consisting of SEQ ID NOs:186-203 that encode the SAbs comprising SEQ ID NOs:168-185.
  • In a further embodiment, SAbs against at least one Y. pestis LcrV epitope may comprise one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26; a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI. In another embodiment, SAbs against at least one Y. pestis LcrV epitope may comprise one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102; a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26; an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120; a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53; an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146; a CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI; and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153. In a further embodiment, SAbs against at least one Y. pestis LcrV epitope may comprise the specific arrangement of FRs and CDRs embodied in SEQ ID NOs:204-217. The present invention further includes isolated nucleotide sequences selected from the group consisting of SEQ ID NOs:218-231 that encode the SAbs comprising SEQ ID NOs:204-217.
  • In an alternative embodiment, the present invention includes one or more SAbs against Y. pestis YscF, with each SAb comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence respectively having at least 15% sequence identity with a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60, in which the SAbs retain sufficient affinity for at least one of a Y. pestis YscF antigen or a Y. pestis YscF epitope. The present invention further includes one or more SAbs against Y. pestis YscF having at least 15% sequence identity with SEQ ID NOs:154-160, in which the SAbs retain sufficient affinity for at least one of a Y. pestis YscF antigen or a Y. pestis YscF epitope.
  • The present invention further includes one or more SAbs against Y. pestis Fl, with each SAb comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence respectively having at least 15% sequence identity with at least one of a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19, a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47, and a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY, in which the SAbs retain sufficient affinity for at least one of a Y. pestis F1 antigen or a Y. pestis F1 epitope. The present invention further includes one or more SAbs against Y. pestis Fl having at least 15% sequence identity with SEQ ID NOs: 168-185, in which the SAbs retain sufficient affinity for at least one of a Y. pestis Fl antigen or a Y. pestis F1 epitope
  • The present invention further includes one or more SAbs against Y. pestis LcrV, with each SAb comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence respectively having at least 15% sequence identity with at least one of a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26, a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53, and a CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI, in which the SAbs retain sufficient affinity for at least one of a Y. pestis LcrV antigen or a Y. pestis LcrV epitope. The present invention further includes one or more SAbs against Y. pestis LcrV having at least 15% sequence identity with SEQ ID NOs: 204-217, in which the SAbs retain sufficient affinity for at least one of a Y. pestis LcrV antigen or a Y. pestis LcrV epitope.
  • In an another embodiment, the present invention further includes a polypeptide, which is used herein to refer to a structure comprising two or more of any of the above-described SAbs against Y. pestis YscF, F1, and/or LcrV in which the two or more SAbs are joined together. In one embodiment, the polypeptide may comprise a fusion protein that is created by joining together two or more SAbs at the genetic level. Two or more nucleic acid sequences encoding for two or more SAbs may be spliced together, and translation of the spliced nucleic acid sequence creates a longer, multi-antigen and/or multi-epitope fusion protein. The fusion protein may contain up to four SAbs joined end-to-end in a substantially linear fashion, similar to beads on a string.
  • In one embodiment, the fusion protein comprises SAbs that are all against a single Y. pestis surface protein or antigen i.e. a single-antigen fusion protein against either YscF, F1, or LcrV. In a further embodiment, this single-antigen fusion protein further comprises SAbs that bind to two or more different epitopes (multi-epitope, single-antigen) on the single antigen. In another embodiment, the fusion protein may comprise SAbs against two or more different Y. pestis surface proteins i.e. a multi-antigen fusion protein. The multi-antigen fusion protein may also comprise SAbs that bind to two or more different epitopes (multi-epitope, multi-antigen) on the same antigen(s). In use, each individual fusion protein molecule may bind to one Y. pestis surface protein molecule, or the individual fusion protein molecule may be bound to two or more separate Y. pestis surface protein molecules. Use of a multi-antigen and/or multi-epitope fusion protein may increase avidity in enzyme immunosorbent assays.
  • In another embodiment, the polypeptide may be created by joining two or more SAbs together with a protein or chemical linker to create a multivalent protein complex. For example, a linker molecule such as the verotoxin 1B-subunit may be used to create high avidity, pentavalent SAb complexes similar to keys on a key ring. In one embodiment, the multivalent protein complex may contain SAbs that are all against a single Y. pestis surface protein or antigen i.e. a single-antigen multivalent protein complex. This single-antigen multivalent protein complex may further comprise SAbs that bind to two or more different epitopes (multi-epitope, single-antigen) on the single antigen. In another embodiment, the multivalent protein complex may comprise SAbs against two or more different Y. pestis surface proteins i.e. a multi-antigen multivalent protein complex. The multi-antigen multivalent protein complex may further comprise SAbs that bind to two or more different epitopes (multi-epitope, multi-antigen) on the same antigen. In use, each multivalent protein complex may bind to one Y. pestis surface protein molecule, or the multivalent protein complex may be bound to two or more separate Y. pestis surface protein molecules. These multi-antigen and/or multi-epitope multivalent protein complexes may generally demonstrate increased affinity for their respective epitope and/or antigen target(s) and may have numerous applications for biomarker assays or proteomics.
  • In one embodiment of the present invention, polypeptides as described herein comprise at least two SAbs, with the SAbs being selected from the following groups: (1) SAbs comprising one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of SEQ ID NOs:27-33; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:54-60; (2) SAbs comprising one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:8-19; a CDR2 sequence selected from the group consisting of SEQ ID NOs:34-47; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:61-71, AEY, and PGY; and (3) SAbs comprising one each of a CDR1 sequence selected from the group consisting of SEQ ID NOs:20-26; a CDR2 sequence selected from the group consisting of SEQ ID NOs:48-53; and a CDR3 sequence selected from the group consisting of SEQ ID NOs:72-78 and GNI.
  • In a further embodiment, the polypeptides comprise at least two SAbs selected from the group consisting of: (1) SAbs comprising one each of a CDR1 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs:1-7; a CDR2 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 27-33; and a CDR3 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs:54-60; (2) SAbs comprising one each of a CDR1 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 8-19; a CDR2 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 34-47; and a CDR3 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 61-71; and (3) SAbs comprising one each of a CDR1 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 20-26; a CDR2 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 48-53; and a CDR3 sequence selected from the group consisting of sequences having at least 15% sequence identity with SEQ ID NOs: 72-78.
  • In another embodiment, the polypeptides may comprise at least two SAbs, with the SAbs being selected from the following groups: (1) SAbs comprising one set of CDR1, CDR2, and CDR3 sequences (as described above with respect to polypeptides according to the present invention) and one each of an FR1 sequence selected from the group consisting of SEQ ID NOs:79-102, an FR2 sequence selected from the group consisting of SEQ ID NOs:103-120, an FR3 sequence selected from the group consisting of SEQ ID NOs:121-146, and an FR4 sequence selected from the group consisting of SEQ ID NOs:147-153; and (2) SAbs selected from the group consisting of SEQ ID NOs:154-160, 168-185, and 204-217 and sequences having at least 15% sequence identity with SEQ ID NOs:154-160, 168-185, and 204-217.
  • In another embodiment, any of the SAbs or polypeptides according to the present invention may further comprise a protein tag, a protein domain tag, or a chemical tag. These tags generally comprise one or more additional amino acids or chemical molecules or residues that may be placed using known methods on the C- or N-terminus of the SAb or polypeptide without altering the activity or functionality of the SAb or polypeptide. The tag may facilitate purification of the SAb or polypeptide, direct absorption and/or excretion in the body, and/or facilitate use in a variety of applications such as detecting and monitoring Y pestis. The tag may include, but is not limited to, a histidine tag (HIS tag) and a poly-lysine tag.
  • The present invention further includes a method of preventing or treating a Y. pestis infection in a patient. Y. pestis infections are frequently difficult to properly diagnose, which can result in delayed treatment, and a low toxicity treatment such as the presently disclosed SAbs may provide a valuable tool for cases of suspected Y. pestis exposure and/or infection and/or for patients presenting with ambiguous symptoms. The method comprises identifying a patient who is suspected of having been exposed to and/or infected with Y. pestis, and administering to the patient a pharmaceutically active amount of one or more of the SAbs and/or polypeptides according to the present invention. As used throughout, a “pharmaceutically active amount” refers generally to an amount that upon administration to the patient, is capable of providing directly or indirectly, one or more of the effects or activities disclosed herein. In one embodiment, the SAb(s) and/or polypeptide(s) may be administered as a form of passive immunotherapy in which the SAb(s) and/or polypeptide(s) are administered to the patient prior to at least one of exposure to or infection with Y. pestis. In another embodiment, the SAb(s) and/or polypeptide(s) may be administered after the patient is exposed to or infected with Y. pestis. The SAb(s) and/or polypeptide(s). In all embodiments of the methods, the SAb(s) and/or polypeptide(s) may be capable of being self-administered and may be administered to the patient using known techniques including, but not limited to, intravenous and subcutaneous injection, oral ingestion, inhalation, and topical administration. The ability to self-administer the SAb(s) and/or polypeptide(s) may be particularly useful in the case of an outbreak or attack where access to medical personnel and treatment may be limited.
  • The present invention further includes a method of detecting and/or diagnosing a Y. pestis infection using one of more of the SAbs and/or polypeptides herein described. The method may include detection of Y. pestis and diagnosis of the infection using known in vivo and/or in vitro assays such as enzyme linked immunosorbent assays (ELISAs), dot blot assays, and other suitable immunoassays. The Y. pestis SAb(s) and/or polypeptide(s) may, for example, be used as a primary antibody or a capture antibody in an ELISA for the detection/diagnosis of a Y. pestis infection. The SAb(s) and/or polypeptide(s) according to the present invention may further be coupled to one or more enzymes or markers for use in imaging.
  • The present invention further includes devices and methods for the identification and detection of Y. pestis on a surface and/or in an environment A device for the environmental detection and/or quantification of Y. pestis may comprise one or more of the SAbs or polypeptides according to the present invention, with the SAb(s) and/or polypeptide(s) being used as a capture element. A method of identifying and detecting Y. pestis using the device comprises contacting one or more of the SAbs or polypeptides with an unknown target and detecting binding between the SAbs or polypeptides and the unknown target to identify the unknown target as Y. pestis. The method may further comprise use of the device to quantify an amount of Y. pestis on the surface and/or in the environment.
  • TABLE 1
    Amino Acid Code
    Alanine Ala A Methionine Met M
    Cysteine Cys C Asparagine Asn N
    Aspartic Acid Asp D Proline Pro P
    Glutamic Acid Glu E Glutamine Gln Q
    Phenylalanine Phe F Arginine Arg R
    Glycine Gly G Serine Ser S
    Histidine His H Threonine Thr T
    Isoleucine Ile I Valine Val V
    Lysine Lys K Tryptophan Trp W
    Leucine Leu L Tyrosine Tyr Y
  • TABLE 2
    Exemplary Combinations of FR and CDR Sequences
    ID ID ID ID ID ID ID
    # FR1 # CDR1 # FR2 # CDR2 # FR3 # CDR3 # FR4
    YscF SAb Sequences
     79 QVQLQESGG  1 GRTWR 103 WFRQ 27 VMSRSG 121 RFTISRDNAKN 54 GGGMY 147 WGKGTQ
    GLVQAGGSL AYYMG APGKE GTTSYA TVYLQMNNLA GPDLYG VTVSS
    RLSCAAS REFVA DSVKG PEDTATYYCK MTY
    A
     80 QVQLQESGG  2 GRAFS 103 WFRQ 28 ANWRSG 122 RFTISRDDAKN 55 GGGSRW 148 WGQGTQ
    GLVQAGGSL NYAMA APGKE GLTDYA TVYLQMNSLK YGRTTA VTVSS
    RLSCVAS REFVA DSVKG PEDTAVYYCA SWYDY
    A
     81 QVQLQESGG  3 GRTFSR 103 WFRQ 29 AISWSGS 123 RFTISRDHAKN 56 PAYGLR 149 RGQGTQ
    GLVQAGGSL YAMG APGKE STYYAD VMYLQMNGL PPYNY VTVSS
    RLSCAVS REFVA SVKG KPEDTGVYVC
    AR
     82 QVQLQESGG  4 QRTFSR 104 WFRQ 30 ATTWSG 124 RFTISRDNAKN 57 GRSSWF 150 WGRGTQ
    GLVQAGGSL YSLG APGEE ISSDYAD TGYLQMNNLK APWLTP VTVSS
    KLSCTAS RVFVA SVKG PEDTGVYYCA YEYDY
    A
     79 QVQLQESGG  5 GRTFSS 105 WFRQ 31 AIRWNG 125 RFTISRDLAKN 58 GVYDY 148 WGQGTQ
    GLVQAGGSL HAMA GPGEE DNIHYS TLYLQMNSLK VTVSS
    RLSCAAS RQFLA DSAKG PEDTAVYYCA
    R
     83 QVQLQESGG  6 GRTFG 106 WFRRA 32 GITRSGN 126 RFTISRDNAKN 59 DWGWR 148 WGQGTQ
    GLVQAGDSR RPFRYT PGKER NIYYSDS TVYLQMNSLK NY VTVSS
    ILSCTAS MG EFVG VKG PEDTAVYYCN
    A
     84 QVQLQESGG  7 GETVD 107 WFRQA 33 CISGSDG 127 RFTISRDNVKN 60 EIYDRR 148 WGQGTQ
    GLVQAGGSL DLAIG PGKER STYYAD TVYLQMNSLK WYRND VTVSS
    RLACAAS EEIS SLSG LEDTAVYYCY Y
    A
    F1 SAb Sequences
     81 QVQLQESGG  8 GMMYI 108 WYRQA 34 FVSSTGN 128 RFTISRDNAKN 61 YLGSRD 148 WGQGTQ
    GLVQAGGSL REAIR PGKQR PRYTDS TVYLQMNSLTP Y VTVSS
    RLSCAVS EWVA VKG EDTAVYYCNT
     85 QVQLQESGG  9 GMMYI 108 WYRQA 35 VVSSTG 128 RFTISRDNAKN 61 YLGSRD 148 WGQGTQ
    GLVQPGGSL RYTMR PGKQR NPHYAD TVYLQMNSLTP Y VTVSS
    RLSCAVS EWVA SVKG EDTAVYYCNT
     86 QVQLQESGG 10 GRAVN 109 WYRQA 36 FISVGGT 129 RFTVSRDNAKN * AEY 148 WGQGTQ
    GLVRPGGSL RYHIMH PGKQR TNYAGS TLYLQMNSLKP VTVSS
    RLSCAVS EWVT VKG EDTAVYYCNS
     87 QVQLQESGG 11 GIIFSD 108 WYRQA 37 QITRSQN 130 RFTVSRDNAKN 62 YDGRRP 148 WGQGTQ
    GSVQPGGSL YALT PGKQR INYTGSV TVHLQMNSLK PY VTVSS
    SLSCSAS EWVA KG PEDTAVYYCH
    A
     87 QVQLQESGG 11 GIIFSD 108 WYRQA 37 QITRSQN 130 RFTVSRDNAKN 63 YDGRRR 148 WGQGTQ
    GSVQPGGSL YALT PGKQR INYTGSV TVHLQMNSLK TY VTVSS
    SLSCSAS EWVA KG PEDTAVYYCH
    A
     88 QVQLQESGG 11 GIIFSD 108 WYRQA 37 QITRSQN 130 RFTVSRDNAKN 62 YDGRRP 148 WGQGTQ
    GLVQPGGSL YALT PGKQR INYTGSV TVHLQMNSLK PY VTVSS
    SLSCSAS EWVA KG PEDTAVYYCH
    A
     88 QVQLQESGG 11 GIIFSD 108 WYRQA 38 QITRRQ 130 RFTVSRDNAKN 64 YDGRRS 148 WGQGTQ
    GLVQPGGSL YALT PGKQR NINYTG TVHLQMNSLK PY VTVSS
    SLSCSAS EWVA SVKG PEDTAVYYCH
    A
     89 QVQLQESGG 11 GIIFSD 108 WYRQA 37 QITRSQN 131 RFTVSRDNAKN 62 YDGRRP 148 WGQGTQ
    GLVQPGGSL YALT PGKQR INYTGSV TVHLQMNSLKP PY VTVSS
    RLSCSAS EWVA KG EDAAVYYCHA
     90 QVQLQESGG 12 ARIFSI 108 WYRQA 39 AITTGGT 126 RFTISRDNAKN * PGY 148 WGQGTQ
    GLVQPGGSL YAMV PGKQR TNYADS TVYLQMNSLK VTVSS
    RLSCAAS EWVA VKG PEDTAVYYCN
    A
     90 QVQLQESGG 13 GVIASI 110 WYRQT 40 IITSGGN 132 RFTTSRDNARN 65 LVGAKD 148 WGQGTQ
    GLVQPGGSL SVLR PGKTR TRYADS TVYLQMNSLKP Y VTVSS
    RLSCAAS DWVA VKG EDTAVYYCNT
     91 QVQLQESGG 14 GTTFRS 111 WYRQA 41 FISSPGD 133 RFTISRDNAKN 66 NGIY 147 WGKGTQ
    GLVRPGGSL LVMK PGKER RTRYTE ALYLQMNGLK VTVSS
    RLSCEAS EWVA AVKG PEDTAVYYCN
    A
     92 QVQLQESGG 15 GFTFSN 112 WVRQA 42 TINSGG 134 RFTISRDNAKN 67 TASHIP 151 LSQGTQ
    GLVQSGDSL YAMS PGKGL GSTSYA TLYLQMNSLKP VTVSS
    RLSCAAS EWVS YSVKG EDTAVYYCAK
     90 QVQLQESGG 15 GFTFSN 112 WVRQA 43 TINIGGG 134 RFTISRDNAKN 67 TASHIP 151 LSQGTQ
    GLVQPGGSL YAMS PGKGL STSYAD TLYLQMNSLKP VTVSS
    RLSCAAS EWVS SVKG EDTAVYYCAK
     90 QVQLQESGG 16 GFTFRN 112 WVRQA 44 TINGGG 135 RFTISRDNAKN 68 TARDSR 149 RGQGTQ
    GLVQPGGSL YAMS PGKGL GITSYAD TMYLQMNSLK DS VTVSS
    RLSCAAS EWVS SVKG PEDTAVYYCA
    Q
     90 QVQLQESGG 17 GFTFSS 113 WVRLA 45 TINIAGG 134 RFTISRDNAKN 69 TAANWS 149 RGQGTQ
    GLVQPGGSL YAMS PGKGL ITSYADS TLYLQMNSLKP AQ VTVSS
    RLSCAAS EWVS VKG EDTAVYYCAK
     90 QVQLQESGG 17 GFTFSS 112 WVRQA 46 TINMGG 136 RFTISRHNAKN 70 TAGNWS 149 RGQGTQ
    GLVQPGGSL YAMS PGKGL GTTSYA  TLYLQMNSLKP AQ VTVSS
    RLSCAAS EWVS DSVKG EDTAVYYCAK
     90 QVQLQESGG 18 GFTFST 114 WIRQPP 47 TITSAGG 137 RFTISRDNAKN 71 LVNLAQ 152 TGQGTQ
    GLVQPGGSL SAMS GKARE SISYVNS TLYLQMNMLK VTVSS
    RLSCAAS VVA VKG PEDTAVYYCAR
     90 QVQLQESGG 19 GFTFST 114 WIRQPP 47 TITSAGG 137 RFTISRDNAKN 71 LVNLAQ 152 TGQGTQ
    GLVQPGGSL NAMS GKARE SISYVNS TLYLQMNMLK VTVSS
    RLSCAAS VVA VKG PEDTAVYYCAR
    LcrV SAb Sequences
     93 QVQLQESGG 20 GFRFSS 115 WVRQA 48 AINSDG 138 RFTISRDNARN 72 RDLYCS 149 RGQGTQ
    GMVEPGGSL YAMS PGKGL DKTSYA TLYLQMSNLKP GSMCKD VTVSS
    RLSCAAS ERVS DSVKG EDTAVYYCAD VLGGAR
    YDF
     94 QVQLQESGG 20 GFRFSS 115 WVRQA 48 AINSDG 138 RFTISRDNARN 72 RDLYCS 149 RGQGTQ
    GLVEPGGSL YAMS PGKGL DKTSYA TLYLQMSNLKP GSMCKD VTVSS
    RLSCAAS ERVS DSVKG EDTAVYYCAD VLGGAR
    YDF
     93 QVQLQESGG 20 GFRFSS 115 WVRQA 48 AINSDG 139 RFTISRDNARN 72 RDLYCS 149 RGQGTQ
    GMVEPGGSL YAMS PGKGL DKTSYA TLYLQMNNLK GSMCKD VTVSS
    RLSCAAS ERVS DSVKG PEDTAVYYCA VLGGAR
    D YDF
     95 QVQLQESGG 21 GLRFSS 115 WVRQA 48 AINSDG 138 RFTISRDNARN 72 RDLYCS 149 RGQGTQ
    GLVQSGESL YAMS PGKGL DKTSYA TLYLQMSNLKP GSMCKD VTVSS
    RLSCAAS ERVS DSVKG EDTAVYYCAD VLGGAR
    YDF
     96 QVQLQESGG 22 GFTFN 116 WYRQV 49 TITGASG 140 RFTISRDNAKN 73 YLTYDS 148 WGQGTQ
    GLVQPGGSL WYTM PGEER DTKYAD TVTLQMNSLKP GSVKGV VTVSS
    KLSCAAS A KMVA SVKG GDAAVYYCHA NY
     97 QVQLQESGG 22 GFTFN 116 WYRQV 49 TITGASG 141 RFTISRDNAKN 73 YLTYDS 148 WGQGTQ
    GLVRPGGSL WYTM PGEER DTKYAD TVTLQMNSLKP GSVKGV VTVSS
    KLSCAAS A KMVA SVKG GDTAVYYCHA NY
     98 QVQLQESGG 22 GFTFN 116 WYRQV 49 TITGASG 141 RFTISRDNAKN 73 YLTYDS 148 WGQGTQ
    GSVQPGGSL WYTM PGEER DTKYAD TVTLQMNSLKP GSVKGV VTVSS
    KLSCAAS A KMVA SVKG GDTAVYYCHA NY
     98 QVQLQESGG 22 GFTFN 116 WYRQV 49 TITGASG 141 RFTISRDNAKN 74 CLTYDS 148 WGQGTQ
    GSVQPGGSL WYTM PGEER DTKYAD TVTLQMNSLKP GSVKGV VTVSS
    KLSCAAS A KMVA SVKG GDTAVYYCHA NY
     99 QVQLQESGG 22 GFTFN 116 WYRQV 49 TITGASG 141 RFTISRDNAKN 73 YLTYDS 148 WGQGTQ
    GFVQPGGSL WYTM PGEER DTKYAD TVTLQMNSLKP GSVKGV VTVSS
    KLSCAAS A KMVA SVKG GDTAVYYCHA NY
     96 QVQLQESGG 22 GFTFN 116 WYRQV 49 TITGASG 141 RFTISRDNAKN 75 YLTYDS 148 WGQGTQ
    GLVQPGGSL WYTM PGEER DTKYAD TVTLQMNSLKP GSAKGV VTVSS
    KLSCAAS A KMVA SVKG GDTAVYYCHA NY
    100 QVQLQESGG 23 GSLLNI 117 WYRQA 50 TVTSSG 143 RFTISRDNAKN 76 HLRYGD 148 WGQGTQ
    GLVQPGGSL YAMG PGRQR TAEYAD TVYLQMNSLRP YVRGPP VTVSS
    GLSCAAS ELVA SVKG EDTGVYYCNA EYNY
    90 QVQLQESGG 24 GGTLG 118 WFRQA 51 CITSSDT 144 RFTISRDNAKN 77 GYYFRD 147 WGKGTQ
    GLVQPGGSL YYAIG PGKER SAYYAD TMYLQMNNLK YSDSYY VTVSS
    RLSCAAS EAVS SAKG PEDTAVYYCA YTGTGM
    A KV
    101 QVQLQESGG 25 GFTLDI 119 WFRQA 52 WIVGND 145 RFTISRDNAKN 78 YSGRPP 148 WGQGTQ
    GLVQPGGST YAIG PGKEH GRTYYI TVYLEMNSLKP VGRDGY VTVSS
    RLSCAAS EGVS DSVKG EDTAVYYCAA DY
    102 QVQLQESGG 26 GASLR 120 WSRQG 53 VMAPDY 146 RVAVRGDVVK * GNI 153 RGLGTQ
    GLVQPGGSL DRRVT PGKSLE GVHYFG NTVYLQVNAL VTVSS
    ILSCTIS IIA KPEDTAIYWCS
    M
    *These sequences have fewer than the required minimum of four amino acids and are not assigned a SEQ. NO.? 
  • TABLE 3
    Y. pestis YscF SAb Protein Sequences
    SEQ ID
    NO Name Sequence
    154 3yscf57 QVQLQESGGGLVQAGGSLRLSCAASGRTWRAYYMGWFRQAPGKEREFVAVMSRSGGTTSYADSVK
    GRFTISRDNAKNTVYLQMNNLAPEDTATYYCKAGGGMYGPDLYGMTYWGKGTQVTVSS
    155 3yscf124 QVQLQESGGGLVQAGGSLRLSCVASGRAFSNYAMAWFRQAPGKEREFVAANWRSGGLTDYADSVK
    GRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAAGGGSRWYGRTTASWYDYWGQGTQVTVSS
    156 3yscf15 QVQLQESGGGLVQAGGSLRLSCAVSGRTFSRYAMGWFRQAPGKEREFVAAISWSGSSTYYADSVKG
    RFTISRDHAKNVMYLQMNGLKPEDTGVYVCARPAYGLRPPYNYRGQGTQVTVSS
    157 3yscf24 QVQLQESGGGLVQAGGSLKLSCTASQRTFSRYSLGWFRQAPGEERVFVAATTWSGISSDYADSVKG
    RFTISRDNAKNTGYLQMNNLKPEDTGVYYCAAGRSSWFAPWLTPYEYDYWGRGTQVTVSS
    158 3yscf142 QVQLQESGGGLVQAGGSLRLSCAASGRTFSSHAMAWFRQGPGEERQFLAAIRWNGDNIHYSDSAKG
    RFTISRDLAKNTLYLQMNSLKPEDTAVYYCARGVYDYWGQGTQVTVSS
    159 3yscf75 QVQLQESGGGLVQAGDSRILSCTASGRTFGRPFRYTMGWFRRAPGKEREFVGGITRSGNNIYYSDSV
    KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADWGWRNYWGQGTQVTVSS
    160 3yscf140 QVQLQESGGGLVQAGGSLRLACAASGETVDDLAIGWFRQAPGKEREEISCISGSDGSTYYADSLSGRF
    TISRDNVKNTVYLQMNSLKLEDTAVYYCYAEIYDRRWYRNDYWGQGTQVTVSS
  • TABLE 4
    Y. pestis YscF SAb DNA Sequences
    SEQ ID
    NO Name Sequence
    161 3yscf57 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGACGCACCTGGAGAGCCTATTACATGGGCTGGTTCCGCCAGGCTCCAGGGAA
    GGAGCGTGAGTTTGTAGCAGTTATGAGTCGGAGCGGTGGCACCACATCCTATGCGGACTCCGTG
    AAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTACAAATGAACAACC
    TGGCACCTGAGGACACGGCCACGTATTATTGTAAGGCGGGGGGCGGAATGTACGGGCCGGACCT
    GTATGGTATGACATACTGGGGCAAAGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTAC
    GACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    162 3yscf124 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTACAGGCTGGGGGCTCTCTGAGACTCTCCT
    GTGTAGCCTCTGGACGCGCCTTCAGTAATTATGCGATGGCCTGGTTCCGCCAGGCTCCAGGGAAG
    GAGCGTGAGTTTGTAGCAGCTAATTGGCGGAGTGGTGGTCTTACAGACTATGCAGACTCCGTGA
    AGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCT
    GAAACCTGAGGACACGGCCGTTTATTACTGTGCCGCCGGGGGCGGTAGTCGCTGGTACGGGCGA
    ACAACCGCAAGTTGGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCT
    ACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    163 3yscf15 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCT
    GTGCAGTCTCTGGACGCACCTTCAGTAGATATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAG
    GAGCGTGAGTTTGTAGCAGCTATTAGCTGGAGTGGTAGTAGCACATATTATGCAGACTCCGTGAA
    GGGCCGATTCACCATCTCCAGAGACCACGCCAAGAACGTGATGTATCTGCAAATGAACGGCCTG
    AAACCTGAGGACACGGGTGTTTATGTCTGTGCAAGACCAGCGTACGGACTCCGCCCCCCGTATA
    ATTACCGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGA
    CTACGGTTCCGGCCGAGCATAG
    164 3yscf24 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAAACTCTCCT
    GCACAGCCTCTCAACGCACCTTCAGTCGCTATAGCTTGGGCTGGTTCCGCCAGGCTCCAGGTGAG
    GAGCGTGTTTTTGTAGCCGCTACTACATGGAGTGGTATAAGCAGTGACTATGCAGACTCCGTGAA
    GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGGGTATCTGCAAATGAACAATTTA
    AAACCTGAGGACACGGGCGTTTATTACTGTGCAGCAGGACGTAGTAGCTGGTTCGCCCCCTGGTT
    GACCCCCTATGAGTATGATTATTGGGGCCGGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACC
    CGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    165 3yscf142 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGACGCACCTTCAGTAGCCATGCCATGGCCTGGTTCCGCCAGGGTCCAGGAGA
    GGAGCGTCAGTTTCTAGCAGCTATTAGATGGAATGGTGATAACATACACTATTCAGACTCCGCGA
    AGGGCCGATTCACCATCTCCAGAGACCTCGCCAAGAACACGCTCTATCTGCAAATGAACAGCCT
    GAAACCTGAGGACACGGCCGTGTATTACTGTGCAAGGGGGGTGTATGACTACTGGGGCCAGGGG
    ACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGC
    ATAG
    166 3yscf75 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCGGATACTCTCCT
    GTACAGCCTCTGGACGCACCTTTGGACGCCCCTTCAGATATACCATGGGCTGGTTCCGCCGGGCT
    CCAGGGAAGGAGCGTGAGTTTGTAGGAGGTATTACAAGAAGTGGTAATAATATATACTATTCAG
    ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAAT
    GAACAGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTAACGCAGATTGGGGGTGGAGGAAC
    TACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACT
    ACGGTTCCGGCCGAGCATAG
    167 3yscf140 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCGCCT
    GTGCAGCCTCTGGAGAGACTGTCGATGATCTTGCCATCGGCTGGTTCCGCCAGGCCCCAGGGAA
    GGAGCGTGAGGAGATTTCATGTATTAGTGGTAGTGATGGTAGCACATACTATGCAGACTCCCTGT
    CGGGCCGATTCACCATCTCCAGGGACAACGTCAAGAACACGGTGTATCTGCAAATGAACAGCCT
    GAAACTTGAGGACACGGCCGTCTATTACTGTTATGCAGAGATTTACGATAGACGCTGGTATCGGA
    ACGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCC
    GGACTACGGTTCCGGCCGAGCATAG
  • TABLE 5
    Y. pestis F1 SAb Protein Sequences
    SEQ ID
    NO Name Sequence
    168 3F55 QVQLQESGGGLVQAGGSLRLSCAVSGMMYIREAIRWYRQAPGKQREWVAFVSSTGNPRYTDSVKG
    RFTISRDNAKNTVYLQMNSLTPEDTAVYYCNTYLGSRDYWGQGTQVTVSS
    169 3F85 QVQLQESGGGLVQPGGSLRLSCAVSGMMYIRYTMRWYRQAPGKQREWVAVVSSTGNPHYADSVK
    GRFTISRDNAKNTVYLQMNSLTPEDTAVYYCNTYLGSRDYWGQGTQVTVSS
    170 3F44 QVQLQESGGGLVRPGGSLRLSCAVSGRAVNRYHMHWYRQAPGKQREWVTFISVGGTTNYAGSVKG
    RFTVSRDNAKNTLYLQMNSLKPEDTAVYYCNSAEYWGQGTQVTVSS
    171 4H4 QVQLQESGGGSVQPGGSLSLSCSASGIIFSDYALTWYRQAPGKQREWVAQITRSQNINYTGSVKGRFT
    VSRDNAKNTVHLQMNSLKPEDTAVYYCHAYDGRRPPYWGQGTQVTVSS
    172 4F6 QVQLQESGGGSVQPGGSLSLSCSASGIIFSDYALTWYRQAPGKQREWVAQITRSQNINYTGSVKGRFT
    VSRDNAKNTVHLQMNSLKPEDTAVYYCHAYDGRRRTYWGQGTQVTVSS
    173 4F1 QVQLQESGGGLVQPGGSLSLSCSASGIIFSDYALTWYRQAPGKQREWVAQITRSQNINYTGSVKGRFT
    VSRDNAKNTVHLQMNSLKPEDTAVYYCHAYDGRRPPYWGQGTQVTVSS
    174 3H1 QVQLQESGGGLVQPGGSLSLScSASGIIFSDYALTWYRQAPGKQREWVAQITRRQNINYTGSVKGRF
    TVSRDNAKNTVHLQMNSLKPEDTAVYYCHAYDGRRSPYWGQGTQVTVSS
    175 3F61 QVQLQESGGGLVQPGGSLRLSCSASGIIFSDYALTWYRQAPGKQREWVAQITRSQNINYTGSVKGRF
    TVSRDNAKNTVHLQMNSLKPEDAAVYYCHAYDGRRPPYWGQGTQVTVSS
    176 4F27 QVQLQESGGGLVQPGGSLRLSCAASARIFSIYAMVWYRQAPGKQREWVAAITTGGTTNYADSVKGR
    FTISRDNAKNTVYLQMNSLKPEDTAVYYCNAPGYWGQGTQVTVSS
    177 3F26 QVQLQESGGGLVQPGGSLRLSCAASGVIASISVLRWYRQTPGKTRDWVAIITSGGNTRYADSVKGRF
    TTSRDNARNTVYLQMNSLKPEDTAVYYCNTLVGAKDYWGQGTQVTVSS
    178 4F59 QVQLQESGGGLVRPGGSLRLSCEASGTTFRSLVMKWYRQAPGKEREWVAFISSPGDRTRYTEAVKG
    RFTISRDNAKNALYLQMNGLKPEDTAVYYCNANGIYWGKGTQVTVSS
    179 3F5 QVQLQESGGGLVQSGDSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSTINSGGGSTSYAYSVKG
    RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKTASHIPLSQGTQVTVSS
    180 4F57 QVQLQESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSTINIGGGSTSYADSVKG
    RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKTASHIPLSQGTQVTVSS
    181 4F75 QVQLQESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSTINGGGGITSYADSVKG
    RFTISRDNAKNTMYLQMNSLKPEDTAVYYCAQTARDSRDSRGQGTQVTVSS
    182 3F59 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRLAPGKGLEWVSTINIAGGITSYADSVKGR
    FTISRDNAKNTLYLQMNSLKPEDTAVYYCAKTAANWSAQRGQGTQVTVSS
    183 4F78 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINMGGGTTSYADSVKG
    RFTISRHNAKNTLYLQMNSLKPEDTAVYYCAKTAGNWSAQRGQGTQVTVSS
    184 3F1 QVQLQESGGGLVQPGGSLRLSCAASGFTFSTSAMSWIRQPPGKAREVVATITSAGGSISYVNSVKGRF
    TISRDNAKNTLYLQMNMLKPEDTAVYYCARLVNLAQTGQGTQVTVSS
    185 3F65 QVQLQESGGGLVQPGGSLRLSCAASGFTFSTNAMSWIRQPPGKAREVVATITSAGGSISYVNSVKGRF
    TISRDNAKNTLYLQMNMLKPEDTAVYYCARLVNLAQTGQGTQVTVSS
  • TABLE 6
    Y. pestis F1 SAb DNA Sequences
    SEQ ID
    NO Name Sequence
    186 3F55 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCT
    GTGCAGTTTCTGGAATGATGTACATTAGGGAGGCTATACGCTGGTACCGCCAGGCTCCAGGGAA
    GCAGCGCGAGTGGGTCGCCTTTGTAAGTAGTACTGGTAATCCACGCTATACAGACTCCGTGAAG
    GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGA
    CACCTGAGGACACGGCCGTCTATTACTGTAATACATACTTGGGCTCGAGGGACTACTGGGGCCA
    GGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCC
    GAGCATAG
    187 3F85 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCT
    GTGCAGTTTCTGGAATGATGTACATTAGGTACACTATGCGCTGGTACCGCCAGGCTCCAGGGAAG
    CAGCGCGAGTGGGTCGCCGTTGTAAGTAGTACTGGTAATCCACACTATGCAGACTCCGTGAAGG
    GCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAC
    ACCTGAGGACACGGCCGTCTATTACTGTAATACATACTTGGGCTCGAGGGACTACTGGGGCCAG
    GGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCG
    AGCATAG
    188 3F44 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCGGCCTGGGGGGTCTCTGAGACTCTCCT
    GTGCAGTCTCTGGAAGAGCCGTCAATAGGTATCACATGCACTGGTACCGCCAGGCTCCAGGGAA
    GCAGCGCGAGTGGGTCACATTTATTAGTGTTGGTGGTACCACAAACTATGCAGGCTCCGTGAAG
    GGCCGATTCACCGTCTCCCGAGACAACGCCAAAAACACGCTGTATCTGCAAATGAACAGCCTGA
    AACCTGAGGACACGGCCGTCTATTACTGTAATTCAGCTGAATACTGGGGCCAGGGGACCCAGGT
    CACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    189 4F34 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGCCTGGGGGGTCTCTGAGCCTCTCCT
    GTTCAGCCTCTGGAATCATCTTCAGTGACTATGCCCTGACCTGGTACCGCCAGGCTCCAGGGAAG
    CAGCGCGAGTGGGTTGCACAGATTACGCGAAGTCAAAATATAAATTATACAGGATCCGTGAAGG
    GCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACAGTGCATCTGCAAATGAACAGCCTGAA
    ACCTGAGGACACGGCCGTCTACTATTGTCATGCATATGACGGTCGACGCCCACCCTACTGGGGCC
    AGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGG
    CCGAGCATAG
    190 4F6 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGCCTGGGGGGTCTCTGAGCCTCTCCT
    GTTCAGCCTCTGGAATCATCTTCAGTGACTATGCCCTGACCTGGTACCGCCAGGCTCCAGGGAAG
    CAGCGCGAGTGGGTTGCACAGATTACGCGAAGCCAAAATATAAATTATACAGGATCCGTGAAGG
    GCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACAGTGCATCTGCAAATGAACAGCCTGAA
    ACCTGAGGACACGGCCGTCTACTATTGTCATGCATATGACGGTCGACGCCGAACCTACTGGGGCC
    AGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGG
    CCGAGCATAG
    191 4F1 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGCCTCTCCT
    GTTCAGCCTCTGGAATCATCTTCAGTGACTATGCCCTGACCTGGTACCGCCAGGCTCCAGGGAAG
    CAGCGCGAGTGGGTTGCACAGATTACGCGAAGCCAAAATATAAATTATACAGGATCCGTGAAGG
    GCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACAGTGCATCTGCAAATGAACAGCCTGAA
    ACCTGAGGACACGGCCGTCTACTATTGTCATGCATATGACGGTCGACGCCCACCCTACTGGGGCC
    AGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGG
    CCGAGCATAG
    192 3F31 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGCCTCTCCT
    GTTCAGCCTCTGGAATCATCTTCAGTGACTATGCCCTGACCTGGTACCGCCAGGCTCCAGGGAAG
    CAGCGCGAGTGGGTTGCACAGATTACGCGAAGGCAAAATATAAATTATACAGGATCCGTGAAGG
    GCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACAGTGCATCTGCAAATGAACAGCCTGAA
    ACCTGAGGACACGGCCGTCTACTATTGTCATGCATATGACGGTCGACGATCACCCTACTGGGGCC
    AGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGG
    CCGAGCATAG
    193 3F61 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCT
    GTTCAGCCTCTGGAATCATCTTCAGTGACTATGCCCTGACCTGGTACCGCCAGGCTCCAGGGAAG
    CAGCGCGAGTGGGTTGCACAGATTACGCGAAGTCAAAATATAAATTATACAGGATCCGTGAAGG
    GCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACAGTGCATCTGCAAATGAACAGCCTGAA
    ACCTGAGGACGCGGCCGTCTACTATTGTCATGCATATGACGGTCGACGCCCACCCTACTGGGGCC
    AGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGG
    CCGAGCATAG
    194 4F27 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCT
    GTGCAGCCTCTGCCCGCATCTTCAGTATCTATGCCATGGTATGGTACCGCCAGGCTCCAGGGAAG
    CAGCGCGAGTGGGTCGCAGCTATTACTACTGGTGGTACCACAAACTATGCAGACTCCGTGAAGG
    GCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAA
    ACCTGAGGACACGGCCGTCTATTACTGTAATGCTCCGGGCTACTGGGGCCAGGGGACCCAGGTC
    ACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    195 3F26 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGAGTCATCGCCAGTATCTCCGTCCTGCGCTGGTACCGCCAAACACCAGGAAAG
    ACGCGCGACTGGGTCGCAATTATTACTAGTGGTGGCAACACACGCTATGCAGACTCCGTGAAGG
    GCCGATTCACCACCTCCAGAGATAACGCCAGGAACACGGTGTATCTGCAAATGAACAGCCTGAA
    ACCTGAGGACACGGCCGTCTATTACTGTAATACACTTGTAGGAGCCAAGGACTACTGGGGCCAG
    GGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCG
    AGCATAG
    196 4F59 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCGGCCTGGGGGATCTCTAAGACTCTCCT
    GTGAAGCCTCTGGAACCACCTTCAGAAGCCTCGTAATGAAATGGTACCGCCAGGCTCCAGGGAA
    GGAGCGCGAGTGGGTCGCATTTATTTCTAGTCCTGGTGATCGCACTCGCTACACAGAAGCCGTGA
    AGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACGCGCTGTATCTGCAAATGAACGGCCT
    GAAACCTGAGGACACGGCCGTGTATTATTGTAACGCGAACGGAATATACTGGGGCAAAGGGACC
    CAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATA
    G
    197 3F5 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAATCTGGGGATTCTCTGAGACTCTCCTG
    TGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGG
    GGCTCGAGTGGGTCTCAACTATTAATAGTGGTGGTGGTAGCACAAGCTATGCGTACTCCGTGAAG
    GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
    AACCTGAGGACACGGCCGTGTATTACTGTGCAAAGACGGCCTCTCACATACCCTTGAGCCAGGG
    GACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAG
    CATAG
    198 4F57 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGAAAG
    GGGCTCGAGTGGGTCTCAACTATTAATATTGGTGGTGGTAGCACAAGCTATGCAGACTCCGTGAA
    GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
    AAACCTGAGGACACGGCCGTGTATTACTGTGCAAAGACGGCCTCTCACATACCCTTGAGCCAGG
    GGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGA
    GCATAG
    199 4F75 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAGGAACTATGCAATGAGCTGGGTCCGTCAGGCTCCAGGAAA
    GGGGCTCGAGTGGGTCTCAACTATTAATGGTGGTGGTGGTATCACAAGCTATGCAGACTCCGTGA
    AGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACAATGTATCTGCAAATGAACAGCCT
    GAAACCTGAGGACACGGCCGTCTATTACTGTGCCCAAACCGCCCGCGATTCCCGCGATTCCCGGG
    GCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCC
    GGCCGAGCATAG
    200 3F59 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGAGCTGGGTCCGCCTGGCTCCAGGAAAG
    GGGCTCGAGTGGGTCTCAACTATTAATATCGCTGGTGGTATCACAAGCTATGCAGACTCCGTGAA
    GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
    AAACCTGAGGACACGGCCGTGTATTACTGTGCAAAAACGGCGGCCAACTGGAGCGCCCAGAGAG
    GCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCC
    GGCCGAGCATAG
    201 4F78 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGAAAG
    GGGCTCGAGTGGGTCTCAACTATTAATATGGGTGGTGGTACCACAAGCTATGCAGACTCCGTGA
    AGGGCCGATTCACCATCTCCAGACACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCT
    GAAACCTGAGGACACGGCCGTGTATTACTGTGCAAAAACGGCGGGCAACTGGAGCGCCCAGAG
    AGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGT
    TCCGGCCGAGCATAG
    202 3F1 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTGTCCT
    GTGCAGCCTCTGGATTCACCTTCAGTACAAGTGCCATGAGTTGGATCCGCCAGCCTCCAGGGAAG
    GCGCGCGAGGTGGTCGCAACTATTACTAGTGCTGGTGGTAGTATAAGTTATGTAAACTCCGTGAA
    GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACATGCTG
    AAACCTGAGGACACGGCCGTGTATTACTGTGCCCGACTGGTCAACCTTGCCCAGACCGGCCAGG
    GAACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGA
    GCATAG
    203 3F65 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCCTGGTGCAACCTGGGGGTTCTCTGAGACTGTCCT
    GTGCAGCCTCTGGATTCACCTTCAGTACAAATGCCATGAGTTGGATCCGCCAGCCTCCAGGGAAG
    GCGCGCGAGGTGGTCGCAACTATTACTAGTGCTGGTGGTAGTATAAGTTATGTAAACTCCGTGAA
    GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACATGCTG
    AAACCTGAGGACACGGCCGTGTATTACTGTGCCCGACTGGTCAACCTTGCCCAGACCGGCCAGG
    GGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGA
    GCATAG
  • TABLE 7
    Y. pestis LcrV SAb Protein Sequences
    SEQ ID
    NO Name Sequence
    204 1LCRV32 QVQLQESGGGMVEPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLERVSAINSDGDKTSYADSVKG
    RFTISRDNARNTLYLQMSNLKPEDTAVYYCADRDLYCSGSMCKDVLGGARYDFRGQGTQVTVSS
    205 2LCRV4 QVQLQESGGGLVEPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLERVSAINSDGDKTSYADSVKG
    RFTISRDNARNTLYLQMSNLKPEDTAVYYCADRDLYCSGSMCKDVLGGARYDFRGQGTQVTVSS
    206 2LCRV3 QVQLQESGGGMVEPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLERVSAINSDGDKTSYADSVKG
    RFTISRDNARNTLYLQMNNLKPEDTAVYYCADRDLYCSGSMCKDVLGGARYDFRGQGTQVTVSS
    207 1LCRV52 QVQLQESGGGLVQSGESLRLSCAASGLRFSSYAMSWVRQAPGKGLERVSAINSDGDKTSYADSVKG
    RFTISRDNARNTLYLQMSNLKPEDTAVYYCADRDLYCSGSMCKDVLGGARYDFRGQGTQVTVSS
    208 1LCRV4 QVQLQESGGGLVQPGGSLKLSCAASGFTFNWYTMAWYRQVPGEERKMVATITGASGDTKYADSVK
    GRFTISRDNAKNTVTLQMNSLKPGDAAVYYCHAYLTYDSGSVKGVNYWGQGTQVTVSS
    209 1LCRV13 QVQLQESGGGLVRPGGSLKLSCAASGFTFNWYTMAWYRQVPGEERKMVATITGASGDTKYADSVK
    GRFTISRDNAKNTVTLQMNSLKPGDTAVYYCHAYLTYDSGSVKGVNYWGQGTQVTVSS
    210 2LCRV1 QVQLQESGGGSVQPGGSLKLSCAASGFTFNWYTMAWYRQVPGEERKMVATITGASGDTKYADSVK
    GRFTISRDNAKNTVTLQMNSLKPGDTAVYYCHAYLTYDSGSVKGVNYWGQGTQVTVSS
    211 1LCRV81 QVQLQESGGGSVQPGGSLKLSCAASGFTFNWYTMAWYRQVPGEERKMVATITGASGDTKYADSVK
    GRSTISRDNAKNTVTLQMNSLKPGDTAVYYCHACLTYDSGSVKGVNYWGQGTQVTVSS
    212 1LCRV27 QVQLQESGGGFVQPGGSLKLSCAASGFTFNWYTMAWYRQVPGEERKMVATITGASGDTKYADSVK
    GRFTISRDNAKNTVTLQMNSLKPGDTAVYYCHAYLTYDSGSVKGVNYWGQGTQVTVSS
    213 1LCRV34 QVQLQESGGGLVQPGGSLKLSCAASGFTFNWYTMAWYRQVPGEERKMVATITGASGDTKYADSVK
    GRFTISRDNAKNTVTLQMNSLKPGDTAVYYCHAYLTYDSGSAKGVNYWGQGTQVTVSS
    214 1LCRV31 QVQLQESGGGLVQPGGSLGLSCAASGSLLNIYAMGWYRQAPGRQRELVATVTSSGTAEYADSVKGR
    FTISRDNAKNTVYLQMNSLRPEDTGVYYCNAHLRYGDYVRGPPEYNYWGQGTQVTVSS
    215 1LCRV28 QVQLQESGGGLVQPGGSLRLSCAASGGTLGYYAIGWFRQAPGKEREAVSCITSSDTSAYYADSAKGR
    FTISRDNAKNTMYLQMNNLKPEDTAVYYCAAGYYFRDYSDSYYYTGTGMKVWGKGTQVTVSS
    216 2LCRV11 QVQLQESGGGLVQPGGSTRLSCAASGFTLDIYAIGWFRQAPGKEHEGVSWIVGNDGRTYYIDSVKGR
    FTISRDNAKNTVYLEMNSLKPEDTAVYYCAAKFWPRYYSGRPPVGRDGYDYWGQGTQVTVSS
    217 1LCRV47 QVQLQESGGGLVQPGGSLILSCTISGASLRDRRVTWSRQGPGKSLEIIAVMAPDYGVHYFGSLEGRVA
    VRGDVVKNTVYLQVNALKPEDTAIYWCSMGNIRGLGTQVTVSS
  • TABLE 8
    Y. pestis LcrV SAb DNA Sequences
    SEQ ID
    NO Name Sequence
    218 1LCRV32 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCATGGTAGAACCTGGGGGTTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGATTCCGCTTCAGTAGTTATGCTATGAGTTGGGTCCGCCAGGCTCCAGGAAAG
    GGGCTCGAGCGGGTCTCGGCTATTAATAGTGATGGTGATAAAACAAGCTATGCAGACTCCGTGA
    AGGGCCGATTTACCATCTCCAGAGACAACGCCAGGAACACGCTGTATCTGCAAATGAGCAACCT
    GAAACCTGAAGACACGGCCGTGTATTACTGTGCAGACCGAGATTTGTACTGTTCAGGCTCTATGT
    GTAAGGACGTCTTGGGGGGAGCACGCTATGACTTTCGGGGCCAGGGGACCCAGGTCACCGTCTC
    CAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    219 2LCRV4 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTAGAACCTGGGGGTTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGATTCCGCTTCAGTAGTTATGCTATGAGTTGGGTCCGCCAGGCTCCAGGAAAG
    GGGCTCGAGCGGGTCTCAGCTATTAATAGTGATGGTGATAAAACAAGCTATGCAGACTCCGTGA
    AGGGCCGATTTACCATCTCCAGAGACAACGCCAGGAACACGCTGTATCTGCAAATGAGCAACCT
    GAAACCTGAAGACACGGCCGTGTATTACTGTGCAGACCGAGATTTGTACTGTTCAGGCTCTATGT
    GTAAGGACGTCTTGGGGGGAGCACGCTATGACTTTCGGGGCCAGGGGACCCAGGTCACCGTCTC
    CAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    220 2LCRV3 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCATGGTAGAACCTGGGGGTTCTCTGAGACTCTCTTGT
    GCAGCCTCTGGATTCCGCTTCAGTAGTTATGCTATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGG
    CTCGAGCGGGTCTCGGCTATTAATAGTGATGGTGATAAAACAAGCTATGCAGACTCCGTGAAGGGC
    CGATTCACCATCTCCAGAGACAACGCCAGGAACACGCTGTATCTGCAAATGAACAACCTGAAACCT
    GAAGACACGGCCGTGTATTACTGTGCAGACCGAGATTTGTACTGTTCGGGCTCTATGTGTAAGGAC
    GTCTTGGGGGGAGCACGCTATGACTTTCGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGC
    TACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    221 1LCRV52 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGTCTGGCGAGTCTCTCAGACTCTCCTG
    TGCAGCCTCTGGACTCCGCTTCAGTAGTTATGCTATGAGTTGGGTCCGCCAGGCTCCAGGAAAGG
    GGCTCGAGCGGGTCTCGGCTATTAATAGTGATGGTGATAAAACAAGCTATGCAGACTCCGTGAA
    GGGCCGATTTACCATCTCCAGAGACAACGCCAGGAACACGCTGTATCTGCAAATGAGCAACCTG
    AAACCTGAAGACACGGCCGTGTATTACTGTGCAGACCGAGATTTGTACTGTTCAGGCTCTATGTG
    TAAGGACGTCTTGGGGGGAGCACGCTATGACTTTCGGGGCCAGGGGACCCAGGTCACCGTCTCC
    AGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    222 1LCRV4 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAAACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAATTGGTATACCATGGCCTGGTATCGCCAGGTTCCAGGGGAG
    GAGCGCAAAATGGTCGCCACAATTACAGGTGCTAGTGGTGACACAAAATATGCAGACTCCGTGA
    AGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACGGTGACACTGCAAATGAACAGCCT
    TAAACCTGGAGACGCGGCCGTCTATTACTGTCATGCCTACCTAACCTACGACTCGGGGTCCGTCA
    AAGGAGTTAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGA
    CGTTCCGGACTACGGTTCCGGCCGAGCATAG
    223 1LCRV13 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCGGCCTGGGGGGTCTCTGAAACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAATTGGTATACCATGGCCTGGTATCGCCAGGTTCCAGGGGAG
    GAGCGCAAAATGGTCGCCACAATTACAGGTGCTAGTGGTGACACAAAATATGCAGACTCCGTGA
    AGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACGGTGACACTGCAAATGAACAGCCT
    TAAACCTGGAGACACGGCCGTCTATTACTGTCATGCCTACCTAACCTACGACTCGGGGTCCGTCA
    AAGGAGTTAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGA
    CGTTCCGGACTACGGTTCCGGCCGAGCATAG
    224 2LCRV1 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGCCTGGGGGGTCTCTGAAACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAATTGGTATACCATGGCCTGGTATCGCCAGGTTCCAGGGGAG
    GAGCGCAAAATGGTTGCCACAATTACAGGTGCTAGTGGTGACACAAAATATGCAGACTCCGTGA
    AGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACGGTGACACTGCAAATGAACAGCCT
    TAAACCTGGAGACACGGCCGTCTATTACTGTCATGCCTACCTAACCTACGACTCGGGGTCCGTCA
    AAGGAGTTAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGA
    CGTTCCGGACTACGGTTCCGGCCGAGCATAG
    225 1LCRV81 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGCCTGGGGGGTCTCTGAAACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAATTGGTATACCATGGCCTGGTATCGCCAGGTTCCAGGGGAG
    GAGCGCAAAATGGTCGCCACAATTACAGGTGCTAGTGGTGACACAAAATATGCAGACTCCGTGA
    AGGGCCGGTCCACCATCTCCAGAGACAATGCCAAGAACACGGTGACACTGCAAATGAACAGCCT
    TAAACCTGGAGACACGGCCGTCTATTACTGTCATGCCTGCCTAACCTACGACTCGGGGTCCGTCA
    AAGGAGTTAACTACTGGGGTCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGA
    CGTTCCGGACTACGGTTCCGGCCGAGCATAG
    226 1LCRV27 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTCGTGCAGCCTGGGGGGTCTCTGAAACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAATTGGTATACCATGGCCTGGTATCGCCAGGTTCCAGGGGAG
    GAGCGCAAAATGGTCGCCACAATTACAGGTGCTAGTGGTGACACAAAATATGCAGACTCCGTGA
    AGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACGGTGACACTGCAAATGAACAGCCT
    TAAACCTGGAGACACGGCCGTCTATTACTGTCATGCCTACCTAACCTACGACTCGGGGTCCGTCA
    AAGGAGTTAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGA
    CGTTCCGGACTACGGTTCCGGCCGAGCATAG
    227 1LCRV34 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAAACTCTCCT
    GTGCAGCCTCTGGATTCACCTTCAATTGGTATACCATGGCCTGGTATCGCCAGGTTCCAGGGGAG
    GAGCGCAAAATGGTCGCCACAATTACAGGTGCTAGTGGTGACACAAAATATGCAGACTCCGTGA
    AGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACGGTGACACTGCAAATGAACAGCCT
    TAAACCTGGAGACACGGCCGTCTATTACTGTCATGCCTACCTAACCTACGACTCGGGGTCCGCCA
    AAGGAGTTAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTACGA
    CGTTCCGGACTACGGTTCCGGCCGAGCATAG
    228 1LCRV31 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTAGGACTCTCCT
    GTGCAGCCTCTGGAAGCCTCTTAAATATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAGA
    CAGCGCGAGTTGGTCGCAACTGTAACGAGTAGTGGAACCGCAGAATATGCAGACTCCGTGAAGG
    GCCGATTCACCATCTCTAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAG
    ACCTGAGGACACGGGCGTCTATTACTGTAATGCACATCTCAGATATGGCGACTATGTCCGTGGCC
    CTCCGGAGTATAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGCGGCCGCTACCCGTA
    CGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    229 1LCRV28 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCT
    GTGCAGCCTCTGGAGGCACTTTGGGTTACTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAG
    GAGCGCGAGGCGGTCTCCTGTATTACTAGTAGTGACACTAGCGCATACTATGCAGACTCCGCGA
    AGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATGAACAACCT
    GAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCCGGTTACTATTTTAGAGACTATAGTGACA
    GTTACTACTACACGGGGACGGGTATGAAAGTCTGGGGCAAAGGGACCCAGGTCACCGTCTCCAG
    CGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    230 2LCRV11 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTACGAGACTCTCCT
    GTGCAGCCTCTGGATTCACTTTGGATATTTATGCTATAGGCTGGTTCCGCCAGGCCCCAGGGAAG
    GAGCATGAGGGGGTCTCGTGGATTGTTGGTAATGATGGTAGGACATACTACATAGACTCCGTGA
    AGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTTGAAATGAACAGCCT
    GAAACCTGAGGATACAGCCGTTTATTACTGCGCAGCTAAGTTCTGGCCCCGATATTATAGTGGTA
    GGCCTCCAGTAGGGAGGGATGGCTATGACTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCAG
    CGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
    231 1LCRV47 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGCGGGTCTCTGATACTCTCCTG
    TACAATCTCGGGAGCCTCGCTCCGAGACCGACGCGTCACCTGGAGTCGCCAAGGTCCAGGGAAA
    TCGCTTGAGATCATCGCAGTTATGGCGCCGGATTACGGGGTCCATTACTTTGGCTCCCTGGAGGG
    GCGAGTTGCCGTCCGAGGAGACGTCGTCAAGAATACAGTATATCTCCAAGTAAACGCCCTGAAA
    CCCGAAGACACAGCCATCTATTGGTGCAGTATGGGGAATATCCGGGGCCTGGGGACCCAGGTCA
    CCGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCATAG
  • TABLE 9
    F1 SAb Groups
    Group Name SEQ ID NO
    1 3F55, 3F85 168, 169
    2 3F44 170
    3 3F31, 3F61, 4F1, 4F6, 4F34 171-175
    4 4F27 176
    5 3F26 177
    6 4F59 178
    7 3F5, 4F57 179-180
    8 4F75 181
    9 3F59, 4F78 182-183
    10 3F1, 3F65 184-185
  • TABLE 10
    LcrV SAb Groups
    Group Name SEQ ID NO
    1 1LCRV32, 2LCRV4, 204-207
    2LCRV3, 1LCRV52
    2 1CLRV4, 1LCRV13, 208-213
    2LCRV1, 1LCRV81,
    1LCRV27, 1LCRV34
    3 1LCRV31 214
    4 1LCRV28 215
    5 2LCRV11 216
    6 1LCRV47 217
  • TABLE 11
    Binding Kinetics of LcrV and F1 Sabs
    SEQ BIACORE Microcal
    Name ID NO KD (nM) KD (nM)
    1LCRV13 209 0.00063 3.2
    1LCRV28 215 0.19 0.20
    1LCRV31 214 0.0019 0.76
    1LCRV32 204 22 26
    1LCRV47 217 >1000 no heat
    1LCRV81
    211 3.5 Error
    2LCRV11
    216 8.2 Error
    3F1
    184 97 110
    3F5 179 47 83
    3F26 177 — —
    3F44 170 — —
    3F55 168 2.2 190
    3F59 182 5.9 no heat
    3F61
    175 68 290
    3F85 169 15 110
    4F1 173 520 error
    4F6
    172 34 80
    4F27 176 — —
    4F34 171 390 error
    4F59
    178 27 83
    4F75 181 6/9  error
    4F78
    183 6/28 error
  • TABLE 12
    Binding Constants of LcrV SAbs
    SEQ
    ID ka KD
    Name NO (M−1 s−1) kd (s−1) (nM)
    1LCRV13 209 2.5 × 105 1.6 × 10−6 0.00063
    1LCRV28 215 4.3 × 105 8.1 × 10−5 0.19
    1LCRV31 214 1.7 × 105 3.1 × 10−7 0.0019
    1LCRV32 204 3.4 × 105 7.3 × 10−3 22
    1LCRV47 217 n.b. n.b. —
    1LCRV81 211 1.8 × 105 6.3 × 10−4 3.5
    2LCRV11 216 8.8 × 105 7.2 × 10−3 8.2
  • Although specific embodiments have been described in detail in the foregoing description and illustrated in the drawings, various other embodiments, changes, and modifications to the disclosed embodiment(s) will become apparent to those skilled in the art. All such other embodiments, changes, and modifications are intended to come within the spirit and scope of the appended claims.

Claims (10)

What is claimed is:
1. A single-domain antibody against Yersinia pestis (Y. pestis) LcrV protein comprising:
a first framing region (“FR”) sequence comprising SEQ ID No:96 or SEQ ID No:99;
a first complementarity determining region (“CDR”) sequence comprising SEQ ID No:22;
a second FR sequence comprising SEQ ID No:116, the first CDR sequence being positioned between the first FR sequence and the second FR sequence;
a second CDR sequence comprising SEQ ID No:49;
a third FR sequence comprising SEQ ID No: 141, the second CDR sequence being positioned between the second FR sequence and the third FR sequence;
a third CDR sequence comprising SEQ ID No.73 or SEQ ID No.75; and
a fourth FR sequence comprising SEQ ID No:148, the third CDR sequence being positioned between the third FR sequence and the fourth FR sequence.
2. The single-domain antibody of claim 1, wherein the at least one single-domain antibody further comprises:
at least one of a protein tag, a protein domain tag, or a chemical tag.
3. The single-domain antibody of claim 1, further comprising:
a plurality of single-domain antibodies, wherein the single-domain antibodies of the plurality are against the Y. pestis LcrV protein and a Y. pestis YscF protein or a Y. pestis F1 protein.
4. A polypeptide comprising:
a plurality of the single-domain antibodies of claim 1 organized into a chain.
5. The polypeptide of claim 4, wherein at least a portion of the plurality of single-domain antibodies comprising the polypeptide is against a different epitope on the LcrV protein.
6. The polypeptide of claim 4, wherein the plurality of single-domain antibodies comprising the polypeptide is against the Y. pestis LcrV protein and a Y. pestis YscF protein or a Y. pestis F1 protein.
7. The polypeptide of claim 4, further comprising:
a fusion protein.
8. The polypeptide of claim 4, further comprising:
a multivalent protein complex such that the single-domain antibodies of the plurality are joined together with at least one linker molecule.
9. The polypeptide of claim 4, wherein at least one of the plurality of single-domain antibodies comprising the polypeptide further comprises:
at least one of a protein tag, a protein domain tag, or a chemical tag.
10. At least one isolated nucleotide sequence encoding the at least one single-domain antibody of claim 1, wherein the isolated nucleotide sequence is selected from the group consisting of SEQ ID No:212 or SEQ ID No:213.
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