ANTIBODIES AGAINST DISEASE CAUSING AGENTS OF POULTRY AND USES THEREOF
20220242941 · 2022-08-04
Inventors
- Hamlet ABNOUSI (North Vancouver, CA)
- Slade LOUTET (Shuniah, CA)
- Filip VAN PETEGEM (Vancouver, CA)
- Tsz Ying Sylvia CHEUNG (Richmond, CA)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
A61P33/02
HUMAN NECESSITIES
International classification
Abstract
Described herein are methods and antibodies useful for reducing, eliminating, or preventing infection with a parasite population in an animal. Also described herein are antigens useful for targeting by heavy chain antibodies and V.sub.HH fragments for reducing a parasite population in an animal.
Claims
1. A polypeptide comprising at least one variable region fragment of a heavy chain antibody (V.sub.HH), wherein the at least one V.sub.HH specifically binds a disease-causing agent, wherein the disease-causing agent is a parasite from the Apicomplexa phylum.
2. The polypeptide of claim 1, wherein the polypeptide comprises a plurality of V.sub.HHs.
3.-5. (canceled)
6. The polypeptide of claim 1, wherein the variable region fragment of the heavy chain antibody comprises an amino acid sequence at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID Nos: 1 to 25 or 111 to 167.
7. The polypeptide of claim 1, wherein the variable region fragment of the heavy chain antibody comprises a complementarity determining region 1 (CDR1) as set forth in any one of SEQ ID Nos: 26 to 50 or 168 to 224, a complementarity determining region 2 (CDR2) as set forth in any one of SEQ ID Nos: 51 to 75 or 225 to 281, and a complementarity determining region 3 (CDR3) as set forth in any one of SEQ ID Nos: 76 to 100 or 282 to 338.
8.-10. (canceled)
11. The polypeptide of claim 1, wherein the Apicomplexan parasite is from the Aconoidasida class, the Gregarinasina subclass, or the Coccidia subclass.
12.-13. (canceled)
14. The polypeptide of claim 1, wherein the disease-causing agent comprises a species of Eimeria.
15. The polypeptide of claim 14, wherein the species of Eimeria is Eimeria tenella or Eimeria maxima.
16. The polypeptide of claim 14, wherein the V.sub.HH specifically binds an Eimeria virulence factor.
17. The polypeptide of claim 15, wherein the V.sub.HH specifically binds an antigen or polypeptide at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ IDs Nos: 101 to 110 or combinations thereof.
18. The polypeptide of claim 16, wherein the Eimeria virulence factor is EmMIC1 polypeptide, EmMIC1-like polypeptide, EtMIC1 polypeptide, EtMIC1-like polypeptide, EmMIC2 polypeptide, EmMIC2-like polypeptide, EtMIC2 polypeptide, EtMIC2-like polypeptide, EmAMA1 polypeptide, EmAMA1-like polypeptide, EtAMA1 polypeptide, EtAMA1-like polypeptide, EmRON2 polypeptide, EmAMA1-binding EmRON2 peptide, EmAMA1-binding EmRON2-like peptide, EmRON2-like polypeptide, EtRON2 polypeptide, EtRON2-like polypeptide, EtAMA1-binding EtRON2 peptide, or EmAMA1-binding EmRON2-like peptide.
19. A nucleic acid encoding the polypeptide of claim 1.
20. (canceled)
21. An expression vector comprising the nucleic acid of claim 19.
22. A cell comprising the nucleic acid of claim 19. of nucleic acids of claim 20.
23.-30. (canceled)
31. The polypeptide of claim 1 further comprising a vitamin, an antibiotic, a hormone, an antimicrobial peptide, a steroid, a probiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1,3-glucan, vegetable extracts, peptone, shrimp meal, krill, algae, B-cyclodextran, alginate, gum, tragacanth, pectin, gelatin, an additive spray, a toxin binder, a short chain fatty acid, a medium chain fatty acid, yeast, a yeast extract, sugar, a digestive enzyme, a digestive compound, an essential mineral, an essential salt, or fibre.
32. A method of producing the polypeptide of claim 1, comprising (a) incubating a cell in a medium suitable for secretion of the polypeptide from the cell; and (b) purifying the polypeptide from the medium.
33. A method of reducing or preventing a poultry-associated parasite infection in a non-human animal comprising administering the polypeptide of claim 1 to the non-human animal.
34. A method for reducing transmission or preventing transmission of a poultry-associated parasite from a poultry species to a non-human animal comprising administering the polypeptide of claim 1 to the poultry species.
35. The method of claim 34, wherein the non-human animal is a species of a chicken, turkey, duck, quail, pigeon, squab, ostrich, pheasant, or goose.
36. The method of claim 34, wherein the non-human animal is a cow, sheep, goat, fish, rabbit, camel, llama, guanaco, alpaca, or vicuna.
37. The method of claim 34, wherein the polypeptide is formulated for introduction to the alimentary canal orally or rectally, provided to the exterior surface, provided to the medium in which the animal dwells, provided by injection, provided intravenously, provided via the respiratory system, provided via diffusion, provided via absorption by the endothelium or epithelium, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects to a host.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0006]
[0007]
[0008]
[0009]
DEFINITIONS
[0010] In describing the present invention, the following terminology is used in accordance with the definitions below.
[0011] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.
[0012] Host
[0013] As referred to herein, “host”, “host organism”, “recipient animal”, “host animal” and variations thereof refer to the intended recipient of the product. In certain embodiments, the host is from the superorder Galloanserae. In certain embodiments, the host is a poultry animal. In certain embodiments, the poultry animal is a chicken, turkey, duck, quail, pigeon, squab, pheasant or goose. In certain embodiments, the poultry animal is a chicken. In certain embodiments, the host is a mammal. In certain embodiments, the mammal is a cow, sheep, pig, goat, horse, primate, marsupial, dog, donkey, reindeer, caribou, or deer. In certain embodiments, the mammal is a human. In certain embodiments, the host is an invertebrate.
[0014] Pathogens
[0015] As referred to herein, “pathogen”, “pathogenic”, and variations thereof refer to virulent microorganisms, that can be associated with host organisms, that give rise to a symptom or set of symptoms in that organism that are not present in uninfected host organisms, including the reduction in ability to survive, thrive, reproduce. Without limitation, pathogens encompass parasites, bacteria, viruses, prions, protists, fungi and algae. In certain embodiments, the pathogen is a parasite belonging to the Apicomplexa phylum (
[0016] “Virulence”, “virulent” and variations thereof refer to a pathogen's ability to cause symptoms in a host organism. “Virulence factor” refers to nucleic acids, plasmids, genomic islands, genes, peptides, proteins, toxins, lipids, macromolecular machineries or complexes thereof that have a demonstrated or putative role in infection.
[0017] “Disease-causing agent” refers to a microorganism, pathogen or virulence factor with a demonstrated or putative role in infection.
[0018] Parasite
[0019] As referred to herein, “parasite”, “parasitic” and variations thereof refer, without limitation, to Eimeria species, or any other parasitic species associated with host organisms. In certain embodiments, bacteria may not be virulent in all host organisms it is associated with.
[0020] Antibodies
[0021] A schematic of camelid heavy chain only antibodies and their relationship to V.sub.HH domains and complementarity determining regions (CDRs) is shown in
[0022] As referred to herein “V.sub.HH” refers to an antibody or antibody fragment comprising a single heavy chain variable region which may be derived from natural or synthetic sources. NBXs referred to herein are an example of a V.sub.HH. In a certain aspect a V.sub.HH may lack a portion of a heavy chain constant region (CH2 or CH3), or an entire heavy chain constant region.
[0023] As referred to herein “heavy chain antibody” refers to an antibody that comprises two heavy chains and lacks the two light chains normally found in a conventional antibody. The heavy chain antibody may originate from a species of the Camelidae family or Chondrichthyes class. Heavy chain antibodies retain specific binding to an antigen in the absence of any light chain
[0024] As referred to herein “specific binding”, “specifically binds” or variations thereof refer to binding that occurs between an antibody and its target molecule that is mediated by at least one complementarity determining region (CDR) of the antibody's variable region. Binding that is between the constant region and another molecule, such as Protein A or G, for example, does not constitute specific binding.
[0025] As referred to herein “antibody fragment” refers to any portion of a conventional or heavy chain antibody that retains a capacity to specifically bind a target antigen and may include a single chain antibody, a variable region fragment of a heavy chain antibody, a nanobody, a polypeptide or an immunoglobulin new antigen receptor (IgNAR).
[0026] As referred to herein an “antibody originates from a species” when any of the CDR regions of the antibody were raised in an animal of said species. Antibodies that are raised in a certain species and then optimized by an in vitro method (e.g., phage display) are considered to have originated from that species.
[0027] As referred to herein “conventional antibody” refers to any full-sized immunoglobulin that comprises two heavy chain molecules and two light chain molecules joined together by a disulfide bond. In certain embodiments, the antibodies, compositions, feeds, products, and methods described herein do not utilize conventional antibodies.
[0028] Production System
[0029] As referred to herein, “production system” and variations thereof refer to any system that can be used to produce any physical embodiment of the invention or modified forms of the invention. Without limitation, this includes but is not limited to biological production by any of the following: bacteria, yeast, algae, arthropods, arthropod cells, plants, mammalian cells. Without limitation, biological production can give rise to antibodies that can be intracellular, periplasmic, membrane-associated, secreted, or phage-associated. Without limitation, “production system” and variations thereof also include, without limitation, any synthetic production system. This includes, without limitation, de novo protein synthesis, protein synthesis in the presence of cell extracts, protein synthesis in the presence of purified enzymes, and any other alternative protein synthesis system.
[0030] Product
[0031] As referred to herein, “product” refers to any physical embodiment of the invention or modified forms of the invention, wherein the binding of the V.sub.HH to any molecule, including itself, defines its use. Without limitation, this includes a feed, a feed additive, a nutritional supplement, a premix, a medicine, a therapeutic, a drug, a diagnostic tool, a component or entirety of an in vitro assay, a component or the entirety of a diagnostic assay (including companion diagnostic assays).
[0032] Feed Product
[0033] As referred to herein, “feed product” refers to any physical embodiment of the invention or modified forms of the invention, wherein the binding of the V.sub.HH to any molecule, including itself, defines its intended use as a product that is taken up by a host organism. Without limitation, this includes a feed, a pellet, a feed additive, a nutritional supplement, a premix, a medicine, a therapeutic or a drug.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Descriptions of the invention provided are to be interpreted in conjunction with the definitions and caveats provided herein.
[0035] For many years, the agriculture industry has utilized antibiotics and anticoccidials to control pathogenic bacteria and parasites, respectively. Some of these molecules also acted as growth promoters. This approach has contributed greatly to the spread of antibiotic and anticoccidial resistance amongst pathogenic organisms. The use of antibiotics as growth promoters in animal feed has already been banned in Europe (effective from 2006) in an effort to phase out antibiotics for non-medicinal purposes and limit antimicrobial resistance. Widespread protection of farmed animals through vaccination has failed due to the short lifespan of many agriculturally important animals, logistical challenges with vaccination of industrial-sized flocks, and high costs. The withdrawal of prophylactic antibiotics and anticoccidials in animal feed and the failure of vaccination to offer widespread protection underpins the need for the development of non-antibiotic products to administer to agricultural animals to prevent infection and promote growth.
[0036] Significant pathogens affecting poultry animals include parasites, such as members of the Eimeria genus, as well as bacteria, such as members of the Clostridium and Salmonella genera.
[0037] Eimeria parasites, particularly Eimeria tenella, are the causative agent of coccidiosis in chickens. This disease is estimated to cause €10 billion in poultry losses globally(1). Coccidiosis is characterized by reduced weight gain and feed conversion, malabsorption, cell lysis of cells lining the epithelium, and diarrhea(3). Motility, cell adhesion, and tight junction formation are all thought to be important for Eimeria pathogenesis(4).
[0038] Intestinal damage caused by Eimeria parasites, particularly Eimeria maxima, is one of the most important predisposing factors for a second disease, chicken necrotic enteritis(5). Losses due to necrotic enteritis are estimated at $6 billion(2) USD per annum. Necrotic enteritis can lead to significant mortality in chicken flocks(3). At subclinical levels, damage to the intestinal mucosa caused by C. perfringens leads to decreased digestion and absorption, reduced weight gain and increased feed conversion ratio (6).
[0039] Prior arts relating to the field of this invention rely on the host organism to generate protection against disease-causing agents. This approach is often limited by the short lifespan of the host organisms affected by the pathogens listed above, which do allow the host organism's immune system sufficient time to generate long-lasting immunity. Furthermore, the effectiveness of prior arts is limited by technical challenges associated with widespread vaccination of large flocks of host organisms. These problems are circumvented by introducing exogenous peptides that neutralise the virulence and spread of the disease-causing agent into the host via feed without eliciting the host immune response. Moreover, the methods described herein provide scope for the adaptation and refinement of neutralising peptides, which provides synthetic functionality beyond what the host is naturally able to produce.
[0040] Antibody heavy chain variable region fragments (V.sub.HHs) are small (12-15 kDa) proteins that comprise specific binding regions to antigens. When introduced into an animal, V.sub.HHs bind and neutralise the effect of disease-causing agents in situ. Owing to their smaller mass, they are less susceptible than conventional antibodies, such as previously documented IgYs, to cleavage by enzymes found in host organisms, more resilient to temperature and pH changes, more soluble, have low systemic absorption and are easier to recombinantly produce on a large scale, making them more suitable for use in animal therapeutics than conventional antibodies.
[0041] Antibodies for Preventing or Reducing Virulence (Summary)
[0042] In one aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents to reduce the severity and transmission of disease between and across species. In certain embodiments, the V.sub.HH is supplied to host animals. In certain embodiments, the V.sub.HH is an ingredient of a product.
[0043] Binding to Reduce Virulence
[0044] In another aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents, and in doing so, reduce the ability of the disease-causing agent to exert a pathological function or contribute to a disease phenotype. In certain embodiments, binding of the V.sub.HH(s) to the disease-causing agent reduces the rate of replication of the disease-causing agent. In certain embodiments, binding of the V.sub.HH(s) to the disease-causing agent reduces the ability of the disease-causing agent to bind to its cognate receptor. In certain embodiments, binding of the V.sub.HH(s) to the disease-causing agent reduces the ability of the disease-causing agent to interact with another molecule or molecules. In certain embodiments, binding of the V.sub.HH(s) to the disease-causing agent reduces the mobility or motility of the disease-causing agent. In certain embodiments, binding of the V.sub.HH(s) to the disease-causing agent reduces the ability of the disease-causing agent to reach the site of infection. In certain embodiments, binding of the V.sub.HH(s) to the disease-causing agent reduces the ability of the disease-causing agent to cause cell death.
[0045] Antibodies Derived from Llamas
[0046] In a further aspect, the present invention provides a method for the inoculation of Camelid or other species with recombinant virulence factors, the retrieval of mRNA encoding V.sub.HH domains from lymphocytes of the inoculated organism, the reverse transcription of mRNA encoding V.sub.HH domains to produce cDNA, the cloning of cDNA into a suitable vector and the recombinant expression of the V.sub.HH from the vector. In certain embodiments, the camelid can be a dromedary, camel, llama, alpaca, vicuna or guacano, without limitation. In certain embodiments, the inoculated species can be, without limitation, any organism that can produce single domain antibodies, including cartilaginous fish, such as a member of the Chondrichthyes class of organisms, which includes for example sharks, rays, skates and sawfish. In certain embodiments, the heavy chain antibody comprises a sequence set forth in Table 1. In certain embodiments, the heavy chain antibody comprises an amino acid sequence with at least 80%, 90%, 95%, 97%, or 99% identity to any sequence disclosed in Table 1. In certain embodiments, the heavy chain antibody possess a CDR1 set forth in Table 2. In certain embodiments, the heavy chain antibody possess a CDR2 set forth in Table 2. In certain embodiments, the heavy chain antibody possess a CDR3 set forth in Table 2.
TABLE-US-00001 TABLE 1 Unique SEQ IDs for VHH antibodies of this disclosure SEQ ID NBX Amino acid sequence Antigen 1 NBX0707 QVQLQESGGGLVQAGGSLRLSCAASGSIFSRDTMA EmMIC2 WYRQVPGEQRELVAFITNFGGTNHADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAGLVSLRT HTWEYWGQGTQVTVSS 2 NBX0708 QVQLQESGGGLVQAGDSLSLSCVDSKRTADRYVM EmMIC2 HWFRQVPGKDREFVAAISGNGLVRNYADSVKGRF TISRDNAKNMVYLQMKSLKPEDTAVYYCAADFVL DSRWRYVPYWGQGTQVTVSS 3 NBX0709 QVQLQESGGGLVQAGGSLRLSCAASGSIFSRDTMA EmMIC2 WYRQVPGEQRELVAYITNFGGTNHADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAGLVSLHT HTWEYWGQGTQVTVSS 4 NBX0710 QVQLQESGGGLVQAGGSLRLSCAASGSIFSINAMG EmMIC2 WYRQAPGKQRELVAGITSGGSTNYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAADYVGLG DDAFYVPYWGQGTQVTVSA 5 NBX0711 QVQLQQSGGGLVQPGGSLRLSCAASRSIRSADAMA EmMIC2 WYRQAPGKQRVWVATITSGGSTYYTDSVKGRFTIS RDNAKSTVYLQMSSLKPEDTAVYYCNAVGDRTSY WGQGTQVTVAS 6 NBX0712 QVQLQESGGGLVQTGGSLRLSCAASGRTFTRNAM EmMIC2 GWFRQAPGKAREFVAVISWSGNRAYSDSVKGRFTI SRDNGKNLVSLQMNSLKPEDTAVYYCAAAREAITS TYYTPHVLTDYDAWGQGAQVTVSS 7 NBX0713 QVQLQESGGGLVQPGGSLRLSCAASGRTFRRNAM EmMIC2 GWFRQAPGKAREFVATISWSGNRAYSDSVKGRFTI SRDNGKNLVSLQMNSLKPEDTAVYYCAAARERITS TYYTPHVLTDYDAWGQGAQVTVSS 8 NBX0714 QVQLQESGGGLVQAGGSLRLSCAASGSIFSRDTMA EmMIC2 WYRQVPGEQRELVAYITNFGGTNHAGSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAELVSLRT HTWEYWGQGTQVTVSS 9 NBX0715 QVQLQQSGGGLVQAGGSLRLSCAASGRTFSRNAM EmMIC2 GWFRQAPGKAREFVATISWSGNRAYSDSVKGRFTI SRDNEKNLVSLHMNSLKPEDTAVYYCAAARERITS TYYTPHVLTDYDAWGQGAQVTVSS 10 NBX0716 QVQLQESGGGLVQAGGSLSLSCTASGRTFSTFPVA EmMIC2 WFRQAPGKEREFVARINWFTTDTYYADSVKGRFTI SRDSAKNTVYLQMNALKPEDTAIYYCAAARSNTG TSRFDYWGQGTQVTVSS 11 NBX0717 QVQLQESGGGLVQAGGSLRLSCAASTNIATFTTMG EmMIC2 WYRQAPGKERELVATTTPWGATTSYADSVKGRFTI SNDNAKNTVNLQMNSLKPEDTAVYYCNAQQDIPT TQTYWGQGTQVTVSS 12 NBX0718 QVQLQESGGGLVQAGGSLRLSCAANGRTFSSYAM EmMIC2 AWFRQAPGKEREFVAAVGWSGGRTYYTDSVKGRF TISRDNAKDTVYLQMNSLKPEDTAVYYCAATRGW AQATLLYDYDYWGQGTQVTVSS 13 NBX0726 QVQLQQSGGGLVQAGGSVRLSCTANGLTFGNYAM EtMIC2 AWFRRTPGKERAFVGGMSASGAGTYYLDSVKGRF TISRDTAKNTVYLEMNSLKAEDTAVYYCAANSIYP GRRWASYDYWGQGTQVTVSS 14 NBX0727 QVQLQQSGGGLVQAGGSVRLSCAASGLTLGNYAL EtMIC2 AWFRRTPGKEREFVAGMSGSGAGTYYLDSVNGRF TISRDNAKNTLYLQMNSLKAEDTAVYYCAANSIYP GRRWASYDYWGQGTQVTVSS 15 NBX0728 QVQLQESGGGLVQAGGSLRLSCAASASIFSRDTMA EtMIC2 WYRQVPGEQRELVAYVTNFGGTNHADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCAADLISLRT HTWEYWGQGTQVTVSS 16 NBX0731 QVQLQESGGGLVQPGGSLRVSCAASGFTFSRDTMA EmMIC2 WYRQVPGEQRELVAYITNFGGTNHADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAGLVSLRT HTWEYWGQGTQVTVSS 17 NBX0732 QVQLQESGGGLVQTGGSLRLSCAASGRTFRRNAM EmMIC2 GWFRQAPGKAREFVATITWSGNRAYSDSVKGRFTI SRDNEKNLVSLQMNSLKPEDTAVYYCAAARERITS TYYTPHVLTEYDAWGQGAQVTVSS 18 NBX0733 QVQLQQSGGGLVQPGGSLRLSCAASGSIFSVDAMA EmMIC2 WYRQAPGKQREWVATFTKGGSTYYAGSVKGRFTI SRDNAKNTVYLQMSSLKPEDTAVYYCNAVGDRTG AWGQGTQVTVAS 19 NBX0734 QVQLQESGGGLVQAGGSLRLSCAASGSIFSRDTMA EmMIC2 WYRQVPGEQRELVAYITNFGGTNHADSVKGRFTIS RDNAKNTVYLQMNSLKPEETAVYYCAAGLVSLRT HTWEYWGQGTQVTVSS 20 NBX0740 QVQLQESGGGLVQAGGSLRLSCAASGRTFVNYNM EmMIC2 GWFRQAPGKEREFVATINVVSGGSTYYAGSVKGRF TISRDSAKNTVYLQMNKLKPEDTAVYYCAAEVGY GYQGPPLTTPSMYDYWGQGTQVTVST 21 NBX0748 QVQLQESGGGLVQAGGSLRLSCVASGSIFSRDTMA EtMIC2 WYRQVPGEQRELVAYITNFGGTNHADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAGLLSLRS HTWELWGQGTQVTVSS 22 NBX0749 QVQLQESGGGLVQAGGSLRLSCATSGSIFSRDTMA EtMIC2 WYRQVPGEQRELVAYITNFGGTNHADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAGLLSLRT HTWEYWGQGTQVTVSS 23 NBX0750 QVQLQESGGGLVQPGDSLRLSCAPSGRTFSGYVTG EmMIC2 WFRQAPGMEREFVAAITWSGDSTYYADSVKGRFQI SRDSAKNTVYLQMNSLKPEDAGVYYCAVKSQTYS TDYVQPRRYAYWGQGTQVTVSS 24 NBX0751 QVQLQESGGGLVQPGGSLRLSCAVSGTIFSITPMG EmMIC2 WYRQAPGKQRELVASISGGGSTNYTDPVKGRFTIS RDKARNTVYLQMNNLKPEDTAVYYCNAASLAIVR GINNYWGQGTQVTVSS 25 NBX0752 QVQLQESGGGLVQAGGSLRLSCAASGSIFSRDNMA EmMIC2 WYRQVPGEQRELVALITNFGGTNHADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAGLVSLLT HTWEYWGQGTQVTVSS 111 NBX16011 QVQLQESGGGLVQAGGSLRLSCKISGNTFSSYTMG EmAMA1 WYRRPPGKQRELVAQLTKGGSTNYADSVKDRFTIS TDNAKNTVYLQMDSLKPEDTAVYYCNMKTAWTI GTRGYDYWGQGSQVTVSS 112 NBX16012 QVQLQESGGGLVQAGGSLRLSCAISRSTFSSYTMG EmAMA1 WYRRPPGKERELVAQITNGGSTNYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCNMKTAWTI GTRGYDYWGQGSQVTVSS 113 NBX16013 QVQLQESGGGLVQAGGSLRLSCVASGSSISSYAMA EmAMA1 WYRQAPGQQRELVAGISSGGATEYADSAKARFTIS RDNAKNTVYLQLYNLKPEDTAVYYCNMRQLPRW SQLLGSWGQGTQVSVSS 114 NBX16014 QVQLQQSGGGLVQAGGSLRLSCSASGSTISSYAMA EmAMA1 WYRQAPGQQRELVAGISSGGGTQYADSAKGRFTIS RDNAKNTVYLQLHTLKPEDTAVYYCNMRQLPRWS LLLGAWGQGTQVSVSS 115 NBX16015 QVQLQQSGGGLVQAGGSLRLSCAASGSTLTRYSVS EmAMA1 WYRQAPGNEREVVSRILKGGSTHYADSVKGRFTIS RDNAKNTVSLQMNSLKPEDTAVYYCHLDWTDFW GQGTQVTVSS 116 NBX16016 QVQLQQSGGGLVQAGGSLRLSCAASGSSFSSYAM EmAMA1 GWYRQAPGKQREWVAGIGSLGSPNYADSVKGRFV MSRDNAKNTVYLQMNSLKPEDTAVYYCNMRLLT TWYNLLGSWGQGTQVTVSS 117 NBX16017 QVQLQESGGGLVEAGGSLRLSCVASRNIFGVAHMT EmAMA1 WYRQAPGKERELVATVSSSGTTNHVDSVKGRFTIS RDNSKTALYLQMNTLKPEDTAVYVCRIATNVPPYN YVVGQGIQVTVSS 118 NBX16018 QVQLQESGGGLVQPGGSLRLSCAASGSSISNYAMA EmAMA1 WYRQAPGKQREWVAGISQRMDTIYADSVKGRFTIS RDNAKNTVSLQMNSLKPEDTAVYYCNIRILPTWAT TVGSWGQGTQVTVSS 119 NBX16019 QVQLQESGPGLVKPSQTLSLTCTVSGGSITTNRYY EmMIC1 WSWIRQPPGKGLEWMGAIAYDGSTYYSPSLKSRTA ISRDTSKNQFSLQLSSVTPEDTAVYYCTRGGDYSSN DYYGMDYWGKGTLVTVSS 120 NBX16020 QVQLQESGGGLVQAGGSLRLSCAASGSSLSRYSVS EmAMA1 WYRQAPGDEREVVSRLTSRGDNFYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCHLDWTDFW GQGTQVTVSS 121 NBX16021 QVQLQESGGGLVQAGGSLRLSCVASGSDVSTYAL EmAMA1 GWYRQAPGKQREWVAGISRGGDTNYADSVKGRF TISRDYAKNTVYLQMNSLKPEDTAVYYCNIRILPN WATTVGRWGQGTQVTVSS 122 NBX16022 QVQLQESGGGLVQAGGSLRLSCVASGSSGSDYALG EmAMA1 WYRQAPGKQREWVAGISRGGDTNYADSVQGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCNMRILPNW NPIVGRWGQGTQVTVSS 123 NBX16023 QVQLQESGGGLVQAGGSLRLSCAAPVRTFSNYAM EmAMA1 GWYRQAPGKQRELVAGISSNGRTDYVDSVKGRFTI SRDNAKNTVSLQMNSLKPEDTAVYYCNMRLTTSW ERLLGAWGQGTQVTVSS 124 NBX16024 QVQLQESGGGLVQAGGSLRLSCSASGSTFSTFSRY EmAMA1 AMAWYRQAPGQQREWVAGISSGGSTTYADSAKG RFTISRDNAKNTVYLQLHSLKPEDTAVYYCNMRQL PSWSQLLGSWGQGTQVSVSS 125 NBX16025 QVQLQESGGGLVQTGGSLRLACAASGDSFILYTAG EmAMA1 WYRQAPGKERELVAQITSGGTTNYGDFAKGRFTIF RDDAKNMVILQMASLKPEDTAVYYCNAKKVRSW PITEEPRDYWGQGTQVTVSS 126 NBX16026 QVQLQESGGGLVQPGGSLRLSCAASGSTLTRYSVS EmAMA1 WYRQAPGNERDVVARILKGGSTHYSDSVKGRFTIS RDNAKNTVSLQMNSLGPEDTAVYYCHLDWTDFW GPGTQVTVSS 127 NBX16027 QVQLQESGGGLVQAGGSLRLSCAASGSSISSYAMG EmAMA1 WYRQAPGKQREWVAVIGSGGTTWYADSVKGRSTI SRDNAKNTVYLQMNSLKPEDTAVYYCNMRLSTLS LHRALGAWGQGTRVTVSS 128 NBX16028 QVQLQESGGGLVQAGGSLRLSCSASGSTFTSSGSTF EmAMA1 SRYAMAWYRQATGQQRELVAGISSGGSTQYADFA RGRFTISRDNAKDTVYLQMNNLQPEDTAVYYCNIR ILPRWDQTVGSWGQGTQVTVSS 129 NBX16029 QVQLQESGGGLVQAGGSLRLSCVASGSIFTAGSTF EmAMA1 NTYAMAWYRQAPGQSRELVAGISSSGSTEYADSV KGRFTISRDNAKNTVYLQLHSLKPEDTAVYYCNM RQLPQWSTLLGAWGQGTQVSVSS 130 NBX16030 QVQLQQSGGGLVQAGGSLTLSCAISGNTYNAHTM EmMIC1 AWYRQAPGKQRELVARMDFNGESNYDNSVKGRF TISRDNAENTLFLQMNSLRPDDTAVYYCKGSWYFL GDYWGQGTQVTVSS 131 NBX16033 QVQLQESGGGTVQAGGSLRLSCAVSGSTFSSYSMN EtRON2 WYRQGPGNQREWVGGIRSNDSTYYADSVKGRFTI SRDNAKNAWNLQMNSLKSEDTAVYYCNFWDGSF NQYWGQGTQVTVSA 132 NBX16034 QVQLQESGGGVVQAGGSLRLSCVASGSDTSVYAM EtRON2 RWWRQVPGKERLLLASITSGGTVRYAESVKGRFTI SRDDAKNTLYLQMNSLKTEDTAVYLCNAERVLAR QNYWGQGTQVTVSS 133 NBX16035 QVQLQESGGGLVQAGGSLRLSCAASGSIFSINVIGW EtRON2 YRQVPGKERELVANSGSTTKYADSVKGRFTISRDN AKNTVDLQMNSLKPEDTAVYYCNAVVTSRTSLGL RSYVYWGQGTQVTVSS 134 NBX16036 QVQLQESGGGLVQAGGSLRLSCVALGNIGWINDM EtRON2 GWYRQAPGKERELVARISSAEATNYADAVKGRFTI SRDDAKNTVYLQMNSLKPEDTAVYSCHTRRWYD DGYDYDTWGQGTQVTVSS 135 NBX16037 QVQLQQSGGGLVQAGGSLRLSCAASGSIFSINVIGW EtRON2 YRQVPGKERELVAKSDRTTKYADSVKGRFTISRDN AKNTVDLQMNSLKPEDTAVYYCNAVVTSRTSQGL RSYAYWGQGTQVTVSS 136 NBX16038 QVQLQQSGGGLVQSGGSLRLSCTATRSTISGYGGR EtRON2 WYRQAPGKQREFVALLSSGGMTRYADSVMGRFTI SRDDIKNTLYLQMNTLKPEDTAVYYCNTFDGAWG QGTQVTVSS 137 NBX16039 QVQLQQSGGGLVQAGGSLRLSCTTSGIIFSFSLLNV EtRON2 GWYRQAPGKERELVARMSTAGNTYYSTSVKDRFT ISRDNGKNTVDLQMNSLNADDTAVYYCNLRRSWA PSDSGFWGQGTQVFVSS 138 NBX16040 QVQLQESGGGMVQAGGSLRLSCAASGTTITINTMR EtRON2 WYRQPPGKERELVASIVPSGKTYYADSVKGRFTISR DNHKNTMYLQMNSLKPEDTAVYYCNSDGRFRGIG TYWGQGTQVTVSS 139 NBX16041 QVQLQESGGGLVQAGGSLRLSCAASGSIFSINVIGW EtRON2 YRQAPGKERELVANSGSTTKYADSVKGRFTISRDN AKNTVDLQMNSLKPEDTAVYYCNAVVTSRTSLGL RSYAYWGQGTQVTVSS 140 NBX16042 QVQLQESGGGLVQAGGSLRLSCVASGITFNFNYYV EtRON2 MGWYRQAPGKQRELVARISDGGNTNYADSVKGRF TISRDNTKNTGYLQMNSLKPEDTAIYYCNLRRSWA PADNGHWGQGTQVTVSS 141 NBX16043 QVQLQESGGGLVQAGGSLRLSCAASGIAFSMFDM EtRON2 VWYRQAPGKQRELVASVSDGGSTAYADSVKGRFT ISRDNAKKMVYLQMNSLKPEDTAVYYCKAARSW AYGSGYWGQGIQVTVSS 142 NBX17001 QVQLQQSGGGLVQAGGSLRLSCAASGITPSRHPMT EtAMA1 WYRQVPGKKRELVASLTDADSTYYTDSVKGRFTIL RDSAANTVYLQLNGLKPEDTAVYYCFAILGGRSY WGQGTQVTVSS 143 NBX17002 QVQLQESGGGLVQAGGSLRLSCAASQSITSTYGMA EtAMA1 WYRQAPGKQREWVATINHTGNTNYVASVKGRFAI SRDNSDYTVYLQMSDLKPEDTAVYYCSPAVWTGL RPWGQGTQVTVSS 144 NBX17003 QVQLQESGGGLVQAGGSLRLSCAASGSTPSRHPMT EtAMA1 WYRQAPGKSRELLASITNVDSTYYADSVKGRATIF RDNAASTVYLQLNGLKLEDTAVYSCFAVLDGRSY WGQGTQVTVSS 145 NBX17004 QVQLQESGGGLVNPGGSLRLSCAASVSTTSTYGMT EtAMA1 WYRQAPGKQREWVATINHTGGTNYADSVKGRVAI SIDNAKNTVYLQMSDLKPEDTAVYYCSPPVWTGL RPWGQGTQVTVSS 146 NBX17005 QVQLQESGGGTVQAGESLTLSCAASGTIFRFTVMG EtAMA1 WYRQVPGKEREFVASITYTDATDYADSVKGRFTIS RDNAKNTAYLQMNSLKPEDTAVYSCFAHYAGYY YGQGTQVTVSP 147 NBX17006 QVQLQQSGGGLVQAGGSLRLSCTASGITSSTYAMS EtAMA1 WYRQAPGKEREPVASMASSGSTFYADSVKGRFTIS RDNAKNMVYLQMNSLKPGDTAVYYCKVPRFGGS DYVVGQGTQVTVSS 148 NBX17007 QVQLQQSGGGLVQPGGSLRLSCTASGITSSTYAMS EtAMA1 WYRQAPGKQREPVASIASSGSTFYAESVKGRFTISR DNAKNMAFLQMNSLEPGDTAVYYCKVPRYGGSD YVVGQGTQVTVSS 149 NBX17008 QVQLQESGGGLVQAGGSLRLSCAASRSSLSTYAMG EtMIC1 WYRQAPGKQRELIATITTGGVTQYVDFVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCVRSPRTSWT SWGQGTQVTVSS 150 NBX17009 QVQLQQSGGGLVQAGGSLRLSCVASASALRMGW EtMIC1 YRQAPGEQRELVATLDNAGKTNYAASVKGRFTISR DSAKNTVYLQMNSLKPEDTAVYYCQAHRWTFDG WQDYWGQGTQVTVSS 151 NBX17010 QVQLQESGGGLVQAGGSLRLSCTASRSTLSTYAMG EtMIC1 WYRQAPGEQRELVATITTGHITQYADFVKGRFTISR DNTKNTVYLQMNSLKPEDTAVYYCVRSPRTSWTS WGQGTQVTVSS 152 NBX17011 QVQLQESGGGLVQAGGSLRLSCAASASALRMGWY EtMIC1 RQAPGESRELVATIDNAGKTNYADSVKGRFTIAKD SAKNTVYLQMNSLKPGDTAVYYCQAHRWTFDGW QDYWGQGTQVTVSS 153 NBX17012 QVQLQESGGGLVQAGGSLRLSCASSASALRMGWY EtMIC1 RQAPGEQRELVATMDNAGSTNYAGSVKGRFTISRD SAKNTVYLQMNSLKPGDTAVYYCQARRWTFDGW QDYWGQGTQVTVSS 154 NBX17013 QVQLQQSGGGLVQAGGSLRLSCAASGSTLSTYAM EtMIC1 GWYRQAPGKQRELVATITTGRVTQYADFVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCVRSPRTPW SSWGQGTQVTVSS 155 NBX17014 QVQLQQSGGGLVQAGGSLRLSCATRSSLSTYAMG EtMIC1 WYRQAPGKQRELVATITTGHITQYADFVKGRFTIS RDNAKNMVYLQMNSLKPEDTAVYYCARSPRTSW TVWGQGTQVTVSS 156 NBX17015 QVQLQESGGGLVQAGGSLRLSCAASGFSLASYHIT EtMIC1 WFRQAPGKEREGVSCIGYNSGWTDYGEFVKGRFTI SRDNDKNTVYLQMNNLQPEDSAVYYCAARWSFG GQCSYGTHNVQRYRGQGTQVTVSS 157 NBX17016 QVQLQQSGGGKVQPGGSLRLSCVASRPVFSMAWY EtMIC1 RQAPGKQRVMVASTTNGKEPNYEDSVQGRFTISRD NAKNAVYLQMNSLKPEDTAIYSCKARHWEFDGVK EYWGQGTQVTVSS 158 NBX17017 QVQLQESGGGLGQAGGSLRLSCEASGRAFSTYHM EtAMA1 GWYRQAPGKQRELVATITSSGNINYADSVKGRFTIS RDNAKNTVNLQMNNLKPDDTAVYYCNRGVLSPSD VYWPSTTWGQGTQVTVSS 159 NBX17018 QVQLQESGGGTVQAGESLTLSCAASGSIFRFTVMG EtAMA1 WYRQVPGKEREFVASITYPGGTEYVDSVKGRFTIS RDNAKNTAYLQVSSLKPEDTAVYYCFAHYGSYYY GQGTQVTVSP 160 NBX17019 QVQLQESGGGLVQAGGSLRLSCTGPGSTFSSYAVG EmRON2 WYRQAPGGNREWVASISSSGEITRYADSVTGRFTIS RDNAKNTVDLQMNSLRPEDTAVYYCAIGTMARGK GTLVTVSS 161 NBX17020 QVQLQQSGGGLVQAGGSLRLSCAGSGRPFGSYVM EmRON2 GWYRQAPGEQREMVARMTSAGSGGVADYGESVK GRFAISRDYAKNMVFLQMNSLKPEDTAVYYCWSA LGYWGQGTQVTVSS 162 NBX17021 QVQLQQSGGGLVQAGGSLRLSCAASRSALSMGWY EtMIC1 RQAPGEQRELVATKDNAGVTTYADSVKGRFTVSR DSAKNTVYLQMNSLKPEDTAVYYCQARRWTLDG WQDYWGQGTQVTVSS 163 NBX17022 QVQLQESGGGLVQAGGSLRLSCAASASALRMGWY EtMIC1 RQAPGEQRELVATIDSAGNTNYAGSVKGRFTISRDS AKNTVYLQMNSLKPGDTAVYYCQARRWTFDGWQ DYVVGQGTQVTVSS 164 NBX17023 QVQLQESGGGLVQAGGSLRLSCTASRSTLSTYAMG EtMIC1 WYRQAPGKQRELVATITTGRITQYADFVKGRFTISR DNDKNTVYLQMNSLKPEDTAVYYCARSPRTSWIL WGQGTQVTVSS 165 NBX17024 QVQLQESGPGLVKPSQTLSLTCTVSGGSITTSYSTW EmRON2 SWIRQPPGKGLEWMGVIADDGSAEYSPSLKSRTSIS LDTSKNQFSLQLSSVTPEDTAVYYCARRGGAWGS NVVWAYHMDDWGKGTLVTVSS 166 NBX17034 QVQLQESGPSLVRPSQTLTLTCTLSGGSITDDHYY EtMIC1 FTWIRQLPGKELEWLGTIAAAGNIFPSPSFESRTS ISRDTSSNQFTLRLNSATPEDTAVYYCARYLKLG LSGMDYVVGKGILVTVSS 167 NBX17035 QVQLQESGGGLVQPGGSLRVSCVASGFTFSAAYMS EtMIC1 WVRQAPGKGLEWVSTIYSDGTRTYYADSVKGRFTI SRDNTKNTVYLEMNSLKPEDTALYYCSRDNFGLG DYVVGQGTQVTVSS
TABLE-US-00002 TABLE 2 Unique SEQ IDs for V.sub.HH CDRs of this disclosure CDR2 CDR3 CDR1 CDR1 CDR2 SEQ CDR3 SEQ Amino Acid SEQ ID Amino Acid ID Amino Acid ID NBX Sequence NO: Sequence NO: Sequence NO: Antigen NBX0707 GSIFSRDT 26 ITNFGGT 51 AADFVLDS 76 EmMIC2 M RWRYVPY NBX0708 KRTADRY 27 ISGNGLVR 52 AADFVLDS 77 EmMIC2 VM RWRYVPY NBX0709 GSIFSRDT 28 ITNFGGT 53 AAGLVSLH 78 EmMIC2 M THTWEY NBX0710 GSIFSINA 29 ITSGGST 54 AADYVGLG 79 EmMIC2 M DDAFYVPY NBX0711 RSIRSADA 30 ITSGGST 55 NAVGDRTS 80 EmMIC2 M Y NBX0712 GRTFTRN 31 ISWSGNR 56 AAAREAIT 81 EmMIC2 AM STYYTPHV LTDYDA NBX0713 GRTFRRN 32 ISWSGNR 57 AAARERIT 82 EmMIC2 AM STYYTPHV LTDYDA NBX0714 GSIFSRDT 33 ITNFGGT 58 AAELVSLR 83 EmMIC2 M THTWEY NBX0715 GRTFSRN 34 ISWSGNR 59 AAARERIT 84 EmMIC2 AM STYYTPHV LTDYDA NBX0716 GRTFSTFP 35 INWFTTDT 60 AAARSNTG 85 EmMIC2 V TSRFDY NBX0717 TNIATFTT 36 TTPWGAT 61 NAQQDIPT 86 EmMIC2 M T TQTY NBX0718 GRTFSSYA 37 VGWSGGR 62 AATRGWA 87 EmMIC2 M T QATLLYDY DY NBX0726 GLTFGNY 38 MSGSGAG 63 AANSIYPG 88 EtMIC2 AM T RRWASYDY NBX0727 GLTLGNY 39 MSGSGAG 64 AANSIYPG 89 EtMIC2 AL T RRWASYDY NBX0728 ASIFSRDT 40 VTNFGGT 65 AADLISLRT 90 EtMIC2 M HTWEY NBX0731 GFTFSRDT 41 ITNFGGT 66 AAGLVSLR 91 EmMIC2 M THTWEY NBX0732 GRTFRRN 42 ITWSGNR 67 AAARERIT 92 EmMIC2 AM STYYTPHV LTEYDA NBX0733 GSIFSVDA 43 FTKGGST 68 NAVGDRTGA 93 EmMIC2 M NBX0734 GSIFSRDT 44 ITNFGGT 69 AAGLVSLR 94 EmMIC2 M THTWEY NBX0740 GRTFVNY 45 INWSGGS 70 AAEVGYG 95 EmMIC2 NM T YQGPPLTT PSMYDY NBX0748 GSIFSRDT 46 ITNFGGT 71 AAGLLSLR 96 EtMIC2 M SHTWEL NBX0749 GSIFSRDT 47 ITNFGGT 72 AAGLLSLR 97 EtMIC2 M THTWEY NBX0750 GRTFSGY 48 ITWSGDST 73 AVKSQTYS 98 EmMIC2 VT TDYVQPRR YAY NBX0751 GTIFSITP 49 ISGGGST 74 NAASLAIV 99 EmMIC2 M RGINNY NBX0752 GSIFSRDN 50 ITNFGGT 75 AAGLVSLL 100 EmMIC2 M THTWEY NBX16011 GNTFSSYT 168 LTKGGST 225 NMKTAWTI 282 EmAMA1 M GTRGYDY NBX16012 RSTFSSYT 169 ITNGGST 226 NMKTAWTI 283 EmAMA1 M GTRGYDY NBX16013 GSSISSYA 170 ISSGGAT 227 NMRQLPR 284 EmAMA1 M WSQLLGS NBX16014 GSTISSYA 171 ISSGGGT 228 NMRQLPR 285 EmAMA1 M WSLLLGA NBX16015 GSTLTRYS 172 ILKGGST 229 HLDWTDF 286 EmAMA1 V NBX16016 GSSFSSYA 173 IGSLGSP 230 NMRLLTT 287 EmAMA1 M WYNLLGS NBX16017 RNIFGVA 174 VSSSGTT 231 RIATNVPP 288 EmAMA1 HM YNY NBX16018 GSSISNYA 175 ISQRMDT 232 NIRILPTWA 289 EmAMA1 M TTVGS NBX16019 GGSITTNR 176 IAYDGST 233 TRGGDYSS 290 EmMIC1 YYW NDYYGMDY NBX16020 GSSLSRYS 177 LTSRGDN 234 HLDWTDF 291 EmAMA1 V NBX16021 GSDVSTY 178 ISRGGDT 235 NIRILPNW 292 EmAMA1 AL ATTVGR NBX16022 GSSGSDY 179 ISRGGDT 236 NMRILPNW 293 EmAMA1 AL NPIVGR NBX16023 VRTFSNY 180 ISSNGRT 237 NMRLTTS 294 EmAMA1 AM WERLLGA NBX16024 GSTFSTFS 181 ISSGGST 238 NMRQLPS 295 EmAMA1 RYAM WSQLLGS NBX16025 GDSFILYT 182 ITSGGTT 239 NAKKVRS 296 EmAMA1 A WPITEEPR DY NBX16026 GSTLTRYS 183 ILKGGST 240 HLDWTDF 297 EmAMA1 V NBX16027 GSSISSYA 184 IGSGGTT 241 NMRLSTLS 298 EmAMA1 M LHRALGA NBX16028 GSTFTSSG 185 ISSGGST 242 NIRILPRWD 299 EmAMA1 STFSRYA QTVGS M NBX16029 GSIFTAGS 186 ISSSGST 243 NMRQLPQ 300 EmAMA1 TFNTYAM WSTLLGA NBX16030 GNTYNAH 187 MDFNGES 244 KGSWYFLG 301 EmMIC1 TM DY NBX16033 GSTFSSYS 188 IRSNDST 245 NFWDGSFN 302 EtRON2 M QY NBX16034 GSDTSVY 189 ITSGGTV 246 NAERVLAR 303 EtRON2 AM QNY NBX16035 GSIFSINVI 190 SGSTT 247 NAVVTSRT 304 EtRON2 SLGLRSYV Y NBX16036 GNIGWIN 191 ISSAEAT 248 HTRRWYD 305 EtRON2 DM DGYDYDT NBX16037 GSIFSINVI 192 SDRTT 249 NAVVTSRT 306 EtRON2 SQGLRSYAY NBX16038 RSTISGYG 193 LSSGGMT 250 NTFDGA 307 EtRON2 G NBX16039 GIIFSFSLL 194 MSTAGNT 251 NLRRSWAP 308 EtRON2 NV SDSGF NBX16040 GTTITINT 195 IVPSGKT 252 NSDGRFRG 309 EtRON2 M IGTY NBX16041 GSIFSINVI 196 SGSTT 253 NAVVTSRT 310 EtRON2 SLGLRSYAY NBX16042 GITFNFNY 197 ISDGGNT 254 NLRRSWAP 311 EtRON2 YVM ADNGH NBX16043 GIAFSMFD 198 VSDGGST 255 KAARSWA 312 EtRON2 M YGSGY NBX17001 GITPSRHP 199 LTDADST 256 FAILGGRS 313 EtAMA1 M Y NBX17002 QSITSTYG 200 INHTGNT 257 SPAVWTGL 314 EtAMA1 M RP NBX17003 GSTPSRHP 201 ITNVDST 258 FAVLDGRS 315 EtAMA1 M Y NBX17004 VSTTSTYG 202 INHTGGT 259 SPPVWTGL 316 EtAMA1 M RP NBX17005 GTIFRFTV 203 ITYTDAT 260 FAHYAGY 317 EtAMA1 M Y NBX17006 GITSSTYA 204 MASSGST 261 KVPRFGGS 318 EtAMA1 M DY NBX17007 GITSSTYA 205 IASSGST 262 KVPRYGGS 319 EtAMA1 M DY NBX17008 RSSLSTYA 206 ITTGGVT 263 VRSPRTSW 320 EtMIC1 M TS NBX17009 ASALRM 207 LDNAGKT 264 QAHRWTF 321 EtMIC1 DGWQDY NBX17010 RSTLSTYA 208 ITTGHIT 265 VRSPRTSW 322 EtMIC1 M TS NBX17011 ASALRM 209 IDNAGKT 266 QAHRWTF 323 EtMIC1 DGWQDY NBX17012 ASALRM 210 MDNAGST 267 QARRWTF 324 EtMIC1 DGWQDY NBX17013 GSTLSTYA 211 ITTGRVT 268 VRSPRTPW 325 EtMIC1 M SS NBX17014 SSLSTYA 212 ITTGHIT 269 ARSPRTSW 326 EtMIC1 M TV NBX17015 GFSLASY 213 IGYNSGW 270 AARWSFG 327 EtMIC1 HI T GQCSYGTH NVQRY NBX17016 RPVFSM 214 TTNGKEP 271 KARHWEF 328 EtMIC1 DGVKEY NBX17017 GRAFSTY 215 ITSSGNI 272 NRGVLSPS 329 EtAMA1 HM DVYWPSTT NBX17018 GSIFRFTV 216 ITYPGGT 273 AHYGSYY 330 EtAMA1 M NBX17019 GSTFSSYA 217 ISSSGEIT 274 AIGTMA 331 EmRON2 V NBX17020 GRPFGSY 218 MTSAGSG 275 WSALGY 332 EmRON2 VM GVA NBX17021 RSALSM 219 KDNAGVT 276 QARRWTL 333 EtMIC1 DGWQDY NBX17022 ASALRM 220 IDSAGNT 277 QARRWTF 334 EtMIC1 DGWQDY NBX17023 RSTLSTYA 221 ITTGRIT 278 ARSPRTSW 335 EtMIC1 M IL NBX17024 GGSITTSY 222 IADDGSA 279 ARRGGAW 336 EmRON2 STW GSNWWAY HMDD NBX17034 GSITDDHY 223 IAAAGNI 280 ARYLKLGL 337 EtMIC1 YF SGMDY NBX17035 GFTFSAA 224 IYSDGTRT 281 SRDNFGLG 338 EtMIC1 YM DY
[0047] Antibodies Recombinantly Expressed
[0048] In another aspect, the present invention provides a method for producing V.sub.HH in a suitable producing organism. Suitable producing organisms include, without limitation, bacteria, yeast and algae. In certain embodiments, the producing bacterium is Escherichia coli. In certain embodiments, the producing bacterium is a member of the Bacillus genus. In certain embodiments, the producing bacterium is a probiotic. In certain embodiments, the yeast is Pichia pastoris. In certain embodiments, the yeast is Saccharomyces cerevisiae. In certain embodiments, the alga is a member of the Chlamydomonas or Phaeodactylum genera.
[0049] Antibodies Added to Feed
[0050] In yet another aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents and are administered to host animals via any suitable route as part of a feed product. In certain embodiments, the animal is selected from the list of host animals described, with that list being representative but not limiting. In certain embodiments, the route of administration to a recipient animal can be, but is not limited to: introduction to the alimentary canal orally or rectally, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the animal dwells (including air based media), provided by injection, provided intravenously, provided via the respiratory system, provided via diffusion, provided via absorption by the endothelium or epithelium, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects. In certain embodiments, the host is from the superorder Galloanserae. In certain embodiments, the host is a poultry animal. In certain embodiments, the poultry animal is a chicken, turkey, duck, quail, pigeon, squab, pheasant or goose. In certain embodiments, the poultry animal is a chicken.
[0051] Feed Product
[0052] In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents and are administered to host animals in the form of a product. The form of the product is not limited. In certain embodiments, the product is feed, pellet, nutritional supplement, premix, therapeutic, medicine, or feed additive, but is not limited to these forms.
[0053] Feeding Dosage
[0054] In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents and are administered to host animals as part of a product at any suitable dosage regime. In practice, the suitable dosage is the dosage at which the product offers any degree of protection against a disease-causing agent, and depends on the delivery method, delivery schedule, the environment of the recipient animal, the size of the recipient animal, the age of the recipient animal and the health condition of the recipient animal among other factors. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration in excess of 1 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration in excess of 5 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration in excess of 10 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration in excess of 50 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration in excess of 100 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration less than 1 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration less than 500 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration less than 100 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animal at a concentration less than 50 mg/kg of body weight. In certain embodiments, V.sub.HHs are administered to recipient animals at a concentration less than 10 mg/kg of body weight.
[0055] Feeding Frequency
[0056] In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents and are administered to host animals as part of a product at any suitable dosage frequency. In practice, the suitable dosage frequency is that at which the product offers any protection against a disease-causing agent, and depends on the delivery method, delivery schedule, the environment of the recipient animal, the size of the recipient animal, the age of the recipient animal and the health condition of the recipient animal, among other factors. In certain embodiments, the dosage frequency can be but is not limited to: constantly, at consistent specified frequencies under an hour, hourly, at specified frequencies throughout a 24-hour cycle, daily, at specified frequencies throughout a week, weekly, at specified frequencies throughout a month, monthly, at specified frequencies throughout a year, annually, and at any other specified frequency greater than 1 year.
[0057] Feed Additives
[0058] In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents and are administered to host animals as part of a product that also comprises other additives or coatings. In practice, the most suitable coating or additive depends on the method of delivery, the recipient animal, the environment of the recipient, the dietary requirements of the recipient animal, the frequency of delivery, the age of the recipient animal, the size of the recipient animal, the health condition of the recipient animal In certain embodiments, these additives and coatings can include but are not limited to the following list and mixtures thereof: a vitamin, an antibiotic, a hormone, an antimicrobial peptide, a steroid, a probiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1,3-glucan, vegetable extracts, peptone, shrimp meal, krill, algae, B-cyclodextrin, alginate, gum, tragacanth, pectin, gelatin, an additive spray, a toxin binder, a short chain fatty acid, a medium chain fatty acid, yeast, a yeast extract, sugar, a digestive enzyme, a digestive compound, an essential mineral, an essential salt, or fibre.
[0059] Non-Feed Uses
[0060] In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents, and can be used in a non-feed use, such as but not limited to: a diagnostic kit, an ELISA-based assay, a western blot assay, an immunofluorescence assay, or a FRET assay, in its current form and/or as a polypeptide conjugated to another molecule. In certain embodiments, the conjugated molecule is can be but is not limited to: a fluorophore, a chemiluminescent substrate, an antimicrobial peptide, a nucleic acid or a lipid.
[0061] Antigens
[0062] In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a V.sub.HH or V.sub.HHs that bind disease-causing agents, produced by a species of Eimeria. In certain embodiments, the species does not belong to the Eimeria genus but is capable of harbouring disease-causing agents shared by Eimeria species. In certain embodiments, the Eimeria species refers to both current and reclassified organisms. In certain embodiments, the Eimeria species is Eimeria tenella. In certain embodiments, the Eimeria species is Eimeria maxima.
[0063] In certain embodiments, the V.sub.HH or plurality thereof is capable of binding to one or more disease-causing agents, originating from the same or different species. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria maxima MIC1 (EmMIC1, SEQ ID 101). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria tenella MIC1 (EtMIC1, SEQ ID 102). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria maxima MIC2 (EmMIC2, SEQ ID 103). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria tenella MIC2 (EtMIC2, SEQ ID 104). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria maxima AMA1 (EmAMA1, SEQ ID 105). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria tenella AMA1 (EtAMA1, SEQ ID 106). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria maxima RON2 (EmRON2, SEQ ID 107). In certain embodiments, the disease-causing agent is a peptide with 80% or greater amino acid sequence identity to a peptide from EmRON2 that binds EmAMA1 (SEQ ID 108). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Eimeria tenella RON2 (EtRON2, SEQ ID 109). In certain embodiments, the disease-causing agent is a peptide with 80% or greater amino acid sequence identity to a peptide from EtRON2 that binds EtAMA1 (SEQ ID 110). In certain embodiments, the disease-causing agent is an exposed peptide, protein, protein complex, nucleic acid, lipid, or combination thereof, that is associated to the surface of the Eimeria parasite. In certain embodiments, the disease-causing agent is an exposed peptide, protein, protein complex, nucleic acid, lipid, or combination thereof, that is deposited on the surface of host cells by the Eimeria parasite. In certain embodiments, the disease-causing agent is the Eimeria parasite.
TABLE-US-00003 Antigen Sequences EmMIC1 >AAA29076.1 em100 gene is homologous the Eimeria tenella gene et100 (accession number M73495) encoding the microneme protein Etp100 [Eimeria maxima] (SEQ ID 101) MALLPTQRLAPGWALSLLVFLAAGLTFHSSHAAASSEADQVCTRLLDVMLVVD ESGSIGTSNYGKVRSFISNFAGTMPLSPDDVRVGLVTFGTSAVTRWDLSDSRAQNADLL AAAAKKLPYAAGSTYTHLGLAKAEEILFSFQKGGRDNAPKMILVMTDGASSRRSQTLS AAEKLRNRGVIIVVLGVGTGVNSAECRSIAGCDTSDTVECPRYLQSNWGGVSSQINGIIK AACKDLAKDAVCSEWSEYGPCEGECGTEGTRTSTRVEIAPPRPGTPPCPTCEAPQGRSC AQQPPGLMRTEQCTMPACKIDAHCGDFGPWSEWSTTCGSATRQRVRQGYEDPPASGG GLSCIDQNPPKYAKEVEVVQKSPCPVQQQPGPWSDWSDCSATCGGGTRYREREGYPQE GELFGGQTLQAQGLDVRETDTCNENPCPVDATCGEWTEFSDCSRVCGGGTKERRREP WLDNAQFGGRSCSQQHPEGPTESVECNEHPCPVDEVVGEWEDWGPCSEQCGRGRQFR YRGPSLQQAMFGGKTIEEQNAGVPEEQKILKVEERPCNDVPCGPCTLPFTEWTACESCS GTRTRDSMVAFDYDDRQCQNPTHEEESCDAVCEESASGGGVGGGAGGAGGGGGGSA GGEGSNGAGPGEDKEEESKGFPTAAVAGGVAGGVLAIAAGAGAFYGLSGGAASAAGG AAAEVMVESGTANPPEVEKESLITAGEQSEMWAS EtMIC1 >AIR96017.1 microneme protein Mic-1 [Eimeria tenella] (SEQ ID 102) MAPLPRRRLAPCRALSLLVGLLAASFAFSSLQPGATTSSGQDQVCTSLLDVMLV VDESGSIGTSNFRKVRQFIEDFVNSMPIPPEDVRVGLITFATRSKVRWNLSDPKATNPSL AISAARSLSYSTGVTYTHYGLQDAKKLLYDTNAGARNNVPKLVLVMTDGASNLPSQTR SSAAALRDAGAIVVVLGVGSGVNSSECRSIAGCSTSNCPRYLQSNWSNVTQQVNGIIKA ACKDLAKDAVCSEWSEYGPCVGECGKEGVQTSTRVEISPQKPGSPPCPTCEAPRGRSCA EQPPGLTRTQPCTMPVCKTDAHCGEFGAWSEWSTTCGTATRKRQREGYNSPPAAGGG LSCMEQNPPKHEFEVETVQKSPCPVQQQPGPWSEWTECSATCGGGTKHREREGLPQEG ELYGGQTLEQQGIAVRETASCSENPCPIGATCGEWTEYSACSRTCGGGTQERKREPWLD NAQHGGRTCMEQYPDGPISVRECNTQPCPVDEVVGDWEDWGQCSEQCGGGKRTRNR GPSKQEAMFGGKTVAQQNVELPEGEKIEVVQEGGCNEVPCGPCTLPFSEWTECESCSG HRTRESAVAFDYTDRMCSGDTHEVHSCEEYCSQNAGGGAGGDGGAGGGTGGSGEEEG KEESSGFPTAAVAGGVAGGVLAIAAGAGAFYGLSGGSAAAATEAGAEVMTEAGTSNA AEVEKESLISAGEQSEMWAS EmMIC2 >CBX60033.1 Microneme protein 2 [Eimeria maxima] (SEQ ID 103) MARALSLIALGLLFSLPSTSAVRTKVPGDEPSGPDSALTAGSPTRGNHGVGGFAE AHCNRLTVRGGLQEKEAVKVTANGWGKGDADFFVELVTDNTRGMLQIRESQTEAGPG LAGGGGQEGGSTEGSKTDEGPVKEQPSIPIVGVRIPGSANENGESRKPAVLVYGEGESAP KEFPLDSPAGPTSPFMVVLQQKSPTEMTVRLFTWIPNGSGGDGSWHETFVDVGVGINHR DVMVVVSDCVPHSLRIYGSSSADLVTADEKTCQATEPQLVNLTSPPENRTHPGAETSTT TSVSS EtMIC2 >AAD05559.1 microneme protein Etmic-2 [Eimeria tenella] (SEQ ID 104) MARALSLVALGLLFSLPPSSAVRTRVPGEDSFSPESGVLSGTDAPERRPIVPGLVE GNCGRLTVRNGPSVDETIKVTSAGWTKSERDFIVSLVADETRKVVQLRESEGASGASGP GPAPAEKPPSGQGSAEEAPKGEGGQEKPSVPLIAVRIHGSGGDKGESAPQSAVLLYGND ESEPTEVPLETAAGPTTPLMVLITQQNPKEVEVRVLAWISTDATTGKGSWKENSVVVGS SLSGRDLTVNLSDCGPSSLRVYGSASADLVTVKEGMCEADDPELIALTRPHTSAASPLP AEEGDVAQDAQQSAGAQQEAEAQEVGEPQQEAAAAEQGSSAAESDTQQSS EmAMA1 >SNT95431.1 Apical membrane antigen 1 [Eimeria maxima] (SEQ ID 105) MCGLRAAFTEAVCLGLLSLGSTVVQGIKDKVHQGHTEAAATAAAGNLSAELHA ALHQPNDNPFLVPPLSDFMDRFNIPKVHGSGIYVDLGGDKEVDGRTYREPSGLCPVFGK TIVLYQPQNNPNYKNDFLDDMPTKQQSDAVGHPLPGGFNNSFKMPDKSPYSPMSAQKL NSYKQLKANTPMGKCAEMSFMTTAGKNSSYRYPWVYDTKRDLCYFLYLPVQRLMGE RYCSVDGKPDGMTWYCFEPHKALDSRPELVYGSAYVGRDPDYWETHCPNKAVKDAV FGVWVSGRCTEHKHLDGAKKEKVNSKAECWSLAFENPEVASDHPVTEDENFGTYGYF FPSTEPNQPKSGGEGVNFASFYPGSMECWLSGEIPTCLVPLEGAAAFTALGSLEEETAPC TDSFPQTKTPCDRNTCTQIVATCVSGTLVSEEVPCSPEDGTRCEGGFPKGVMIGLAAAG GILLLLLGGGGFLLYRSRMRPAAKGDEATRSDYVQEEAAANRRKQRQSDLVQQAEPSF WEEAEADEAETGESTHVLVDQDY EtAMA1 >XP_013229486.1 apical membrane antigen-1 [Eimeria tenella] (SEQ ID 106) MRRLSPALGLLAAALSCAGPAAGVQHKLQHRQQQQQQHSHASTSHAAAVLAA SSDASTDSNPFMQPPYAEFMARFNIPKVHGSGVYVDLGNDKEVKGKMYREPGGRCPVF GKNIEFYQPLDSDLYKNDFLENVPTEEAAAAAKPLPGGFNNNFLMKDKKPFSPMSVAQ LNSYPQLKARTGLGKCAEMSYLTTAAGSSYRYPFVFDSKKDLCYLLLVPLQRLMGERY CSTRGSPPGLSHFCFKPLKSVSLRPHLVYGSAYVGERPDDWETKCPNKAVKDAVFGVW EGGRCEEQRLRLGAQTAAAAAKEDCWALAFNNPFAASDQPTSQDEAATSPGYYFPSIT PSQPKSGGVGVNFASYYPSGECVLSGEVPTCLLPRQGAAAFTSVGSLEEEELPHCDPTFP ASLGSCDPSSCKAILTECRGGRLVEQQTDCVPEDGSKCESKGGGVFIGLAVAGGLLLLL LTGGAFFIYKQRQKALPKESSPQRTDFVQDEAATGRGKKRQSDLVQQAEPSFWEEAEA DEPHADENTQVLLDQEY EmRON2 >XP_013337434.1 hypothetical protein, conserved [Eimeria maxima] (SEQ ID 107) MFRLLYLPGLVTILSVSQRTPEVKITMLGAFSIFALSTLLSLPPYSWRMAAMADS LSVTPEYEVEGPTNFLTPSLVTLDSALEGLDSGGTPGFSGQYADLLDRICPADSPALDLP QQPTREQNIGELELMLSDNDLGEATNKLWLAFYGHEVPKAASSVEELSAQFLELVGQV RVAFQDVHHHMVKQEGHPEFHNSQLPRSIVPIVGARNPLMLGLFWNLLIAYTGFDSYF GEDSITLPFFSWPSLLASLGGQSSSHIDAMCSVQRSRSLTEKFFKWRSPRGIQQNRRHKR VSSLRESSHRTFCELIDRLISSLGEFVAGHVTTLAAAGVPVELGISPLQNMKRLHAETCLP GEDGHILAPCIFEESRLALDSQQLVKMEDDTDQQIRHSAQAELLKQAHTTISGLGALDC KLSSRKWRALPYTPMPRLFKLPELAQLRKDWILERLNLAVATMLTTSSLDNASNYWLA FDRRNFKRAAQDFKKHTIDVLRSVGMEAIGRRLLQSSGSEISDDEVQAAASRLKDLRAV SDGMRVALALYAILNAGRHAQSAHVGSQGHAIHAQCYGTCCSTAEPYAYVGVRVPEL SGRDAQEHERNQDPESQKGAWSKIDLNGLPLPDLTDWWSRLNYIVVQALESYLVLEKH LEFLEDTVFTLVSDSLRKLKADAGQTLFFTRIAHQKRQGPLQRLWEGAKKSLMGFLYRS PGRQHGIWFGVTVDFEQLHDLLGQLKLVIEAEPRLTIKINMQEALLREMETRIRVEGSDI SRVPPLVERNMGMLGVRRAYGTLSEGLRDTEFQRSMCIDHCRGLWQMALSTMLPSML SPDIFRKYEKAFGTPWALEHLSDPALVNSRRMVLKSDAALNFFDHSTPKEVREELKGLE SGQASMFAYYMLFSSRAHQVLGNHYLGLYLRQQAPFMGNMVVDWISTRRKHAVAAI VSSFVLTFMGIYAVMSFVDILQNLIVSGAPPPFDCLWNPVFGEMACSPVPGGASLGTAW ATAIEQVFLIGLFSGTAGGFSLFLTINSAIAVVVNQSKTLMRLQMCLGSAISRLLRRGKRS FSRIREYFIKRSSVKRVMLQRAVAGMKAGSSTATMSNSETLEAADQLLDRLTRTGRANP LKASGGKPAFK EmAMA1-binding EmRON2 peptide SEQ ID 108) DILQNLIVSGAPPPFDCLWNPVFGEMACSPVPGGASL EtRON2 >XP_013231132.1 rhoptry neck protein 2, putative, partial [Eimeria tenella] (SEQ ID 109) MGVFSRLLSAALLCAAASPALLSPQAAAAAAAPAEGSTSFTLPSGGSLHVQVLR GPQQQQQQQQQGPWGSTMSFAGPTGATGGYSVTDSATLRSSAGPPAAAAAAAAAEGV YELPTPPTPENPISKTQVLFGWVPEAAAAASLQAVNAEELNNALRSQAFVRKLRTRALV EKSCMIPLASDRRSYLEAFWSAAAAERKFRAEAESQRAAIEEELKQQQMAAPDMSYAR SVATRANIESGASPAAAAAAAAGAAAAAAAGEEAAAAALRYQETVDRVLANRIEVLM TCKLAALLGQPGIFLNDKIAAAEVLDLVATALGIGNTKEEAAAAAASPIVPMFGGLEPFL LASNPLIVGHVLTLLIAHIDREAFFGEAARKAFYSFTSLAAAAGGEGAVGMLDEMCDRD RGPRLAAPLLGKYRPKGRGGKPRKRGSSSPWFVRGASRVHRNKLQPELLRAYCDATE MILNALMLKQEDVQQEMLKFSLPVEPLVDPATNASRIQTKTCRGGAPVCEFENSILSPIA NPLDPQVLEQNTNTRAAFNLYAGLASAQLGNLLEETGSKYYDVRADQWLEIISKPTAH SDILEKVFFYDNREFLASKRSKLIKSFETNAKKIAALAGKPAVSISEAAAAYEEIQRSVGS QTGKKGAAAAKKNLFPTAATLLLKSTLACDMTSSFAAKLESFTQFMFKAKAASGRRQA PLQRTLMAFIRTGKGSALQSMCSYDPLTFNYLFLYRFVLLGSDPAKTHDKQHAAVSKTP LTTRLLGSTWTPSILKRVLRGVKRKSSILKAKQLLLESLDAGVFPLLLTSFEWIVHTQAA LQVNQNSEMFHELEAQQPRALPAEPQKAAEAKLHEGGLVKRTDEQLAEWAEFGVPAA LREQLRRGEKLPKAVALERIPTPDLTQWEQQLNRKWLAALEAYLRHPYGAAAAAARD PVALLVQRSRDRLEAELDSTIFLGRIVGAPRGPRRGRRALRAVGGLFRALLRAPRQSEFA VWMGVKVHADRVLRVLHAVHAAAEVAKTAGLFEHVREAFLELVRDAVLGSLQNQLR VPGFDTFAAVEAGLRGGEVSAAAAAAAAAAAAARRSAGFLSVHPELAALGPAAREAE FQNSMCMDHCEALWTLVTSLVFSALQNPRKWRDYEKEVGGAAAAAALADPRRVNSF RLGLSVQTDFFENVLDKKSKRNIQKMKFGGGSWFAYALLLAARLHRGLGHSDLATFFS FQAPYLGHFVLQWQQQRREARRKALLSMLSLGFFFAYTFISVSDITQHLNDSGLGPAVE CLENLVVGPVCPAAVVAPAVRSAAAAAAADVFKVGLFGLLTPYLVWPMAAAAAWQL LRSEFKVLLQFEMSLKSLFSRFSSWVRRPFARWWQQRRRLKQLLLQSAAAKFRTEKKR LHGRDPPPRDF EtAMA1-binding EtRON2 peptide (SEQ ID 110) DITQHLNDSGLGPAVECLENLVVGPVCPAAVVAPAVR
Examples
[0064] The following illustrative examples are representative of the embodiments of the applications, systems and methods described herein and are not meant to be limiting in any way.
[0065] While preferred embodiments of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
[0066] Production of Antigens
[0067] Recombinant antigens can be purified from an E. coli expression system. For example, an antigen can be expressed at 18° C. in E. coli BL21 (DE3) cells grown overnight in autoinducing media (Formedium). Cells are then lysed by sonication in buffer A (250 mM NaCl, 50 mM CaCl.sub.2, 20 mM imidazole and 10 mM HEPES, pH 7.4) with 12.5 □g/ml DNase I, and 1× Protease inhibitor cocktail (Bioshop). The lysate is cleared by centrifugation at 22000×g for 30 minutes at 4° C., and is then applied to a 5 ml HisTrap HP column (GE Healthcare) pre-equilibrated with buffer A, washed with ten column volumes of buffer A and eluted with a gradient of 0% to 60% (vol/vol) buffer B (250 mM NaCl, 50 mM CaCl.sub.2, 500 mM imidazole and 10 mM HEPES, pH 7.4). The protein is then dialyzed overnight in the presence of TEV against buffer C (250 mM NaCl, 10 mM HEPES, pH 7.4 and 5 mM □-mercaptoethanol) at 4° C. The dialyzed protein is applied to a HisTrap HP column (GE Biosciences) pre-equilibrated with buffer C. 6×His-tagged TEV and 6×His-tag are bound to the column and the antigen is collected in the flowthrough. The sample is dialyzed overnight against buffer D (5 mM NaCl and 10 mM Tris pH 8.8) and then applied to a 5 ml HiTrap Q HP column (GE Healthcare). The protein is eluted with a gradient of 0% to 50% (vol/vol) buffer E (1.0 M NaCl and 10 mM Tris pH 8.8). Lastly, the elution is loaded onto a Superdex 75 Increase 10/300 GL gel filtration column (GE Healthcare) using buffer F (400 mM NaCl and 20 mM HEPES pH 7.4). The protein sample is then concentrated to 1 mg/mL using Amicon concentrators with appropriate molecular weight cutoff (MWCO; Millipore). The purified protein is stored at −80° C.
[0068] Alternatively, the EmAMA1-binding peptide of EmRON2 (SEQ ID 108) and the EtAMA1-binding peptide of EtRON2 (SEQ ID 110) were expressed in E. coli as fusions at the C-terminus of glutathione S-transferase (GST). They were purified as described as above without TEV cleavage.
[0069] Production of NBXs and Panning
[0070] Llama Immunisation
[0071] A single llama is immunized with purified disease-causing agents, such as the antigens listed, which may be accompanied by adjuvants. For SEQ ID 108 and 110, the antigenic peptides were provided to the llama as fusions to GST. The llama immunization is performed using 100 μg of each antigen that are pooled and injected for a total of four injections. At the time of injection, the antigens are thawed and the volume increased to 1 ml with PBS. The 1 ml antigen-PBS mixture is then mixed with 1 ml of Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (IFA) for a total of 2 ml. A total of 2 ml is immunized per injection. Whole llama blood and sera are then collected from the immunized animal on days 0, 28, 49, 70. Sera from days 28, 49 and 70 are then fractionated to separate V.sub.HH from conventional antibodies. ELISA can be used to measure reactivity against target antigens in polyclonal and V.sub.HH-enriched fractions. Lymphocytes are collected from sera taken at days 28, 49, and 70.
[0072] Panning
[0073] RNA isolated from purified llama lymphocytes is used to generate cDNA for cloning into phagemids. The resulting phagemids are used to transform E. coli TG-1 cells to generate a library of expressed V.sub.HH genes. The phagemid library size can be ˜2.5×10.sup.7 total transformants and the estimated number of phagemid containing V.sub.HH inserts can be estimated to be ˜100%. High affinity antibodies are then selected by panning against the antigens used for llama immunization. Two rounds of panning are performed and antigen-binding clones arising from round 2 are identified using phage ELISA. Antigen-binding clones are sequenced, grouped according to their CDR regions, and prioritized for soluble expression in E. coli and antibody purification.
[0074]
[0075]
[0076] Purification of V.sub.HHs from E. coli
[0077] TEV protease-cleavable, 6×His-thioredoxin-NBX fusion proteins are expressed in the cytoplasm of E. coli grown in autoinducing media (Formedium) for 24 hours at 30° C. Bacteria are collected by centrifugation, resuspended in buffer A (10 mM HEPES, pH 7.5, 250 mM NaCl, 20 mM imidazole) and lysed using sonication. Insoluble material is removed by centrifugation and the remaining soluble fraction is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A. The protein is eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 500 mM NaCl, 500 mM Imidazole). The eluted protein is dialyzed overnight in the presence of TEV protease to buffer C (10 mM HEPES, pH 7.5, 250 mM NaCl). The dialyzed protein is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer C. 6×His-tagged TEV and 6×His-tagged thioredoxin are bound to the column and purified NBX is collected in the flowthrough. The NBX-containing flowthrough is dialyzed to buffer D (10 mM HEPES, pH 7.0) and applied to a HiTrapSP ion exchange column (GE Biosciences. Highly purified NBX protein is eluted from the column using a linear gradient from buffer D to buffer E (10 mM HEPES, pH 7.0, 500 mM NaCl) NBX proteins are dialyzed overnight to buffer F (20 mM HEPES, pH 7.4, 150 mM NaCl) and concentrated to ˜10 mg/ml.
[0078] Purification of V.sub.HHs from P. pastoris
[0079] Pichia pastoris strain GS115 with constructs for the expression and secretion of 6×His-tagged V.sub.HH are grown for 5 days at 30° C. with daily induction of 0.5% (vol/vol) methanol. Yeast cells are removed by centrifugation and the NBX-containing supernatant is spiked with 10 mM imidazole. The supernatant is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A (10 mM HEPES, pH 7.5, 500 mM NaCl). The protein is eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 500 mM NaCl, 500 mM imidazole). NBX proteins are dialyzed overnight to PBS and concentrated to ˜10 mg/ml.
[0080] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document is specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
[0081] The following references are incorporated by reference in their entirety. [0082] (1) Van Meirhaeghe, H. & De Gussem, M. (2014). Coccidiosis a major threat to the chicken gut. Retrieved on May 25, 2018 from: https://www.poultryworld.net/Home/General/2014/9/Coccidiosis-a-major-threat-to-the-chicken-gut-1568808W/?dossier=35765&widgetid=1. [0083] (2) Wade, B. & Keyburn, A. (2015). The true cost of necrotic enteritis. Retrieved on May 25, 2018 from: https://www.poultryworld.net/Meat/Articles/2015/10/The-true-cost-of-necrotic-enteritis-2699819W/. [0084] (3) Chapman, H. D. (2014). Milestones in avian coccidiosis research: a review. Poultry Science, 93(3), pp. 501-511. [0085] (4) Boucher, L. E. & Bosch, J. (2015). The apicomplexan glideosome and adhesins-structures and function. Journal of Structural Biology, 190(2), pp. 93-114. [0086] (5) Moore, R. J. (2016). Necrotic enteritis predisposing factors in broiler chickens. Avian Pathology, 45(3), pp. 275-281. [0087] (6) Abid, S. A. et al. (2016). Emerging threat of necrotic enteritis in poultry and its control without use of antibiotics: a review. The Journal of Animal and Plant Sciences, 26(6), pp. 1556-1567.