Title:
HIV Antigens and Antibodies
Kind Code:
A1


Abstract:
The present invention relates to a method for reducing the occurance and/or severity of viral infections. The method embodies procedures for expanding HIV from the blood of HIV antibody negative donors and deriving a non-infectious virus particle product that is antigenic. The procedures for deriving the antigenic, non-infectious virus particle product are optimally designed to maintain the integrity of the envelope proteins while maximizing the depletion of capsid proteins and RNA. The resulting virus particle product, when introduced into humans or non-human animals, enables the production of antibodies that target the natural envelope macromolecular structure that is required for infectivity. The present invention can be applied to producing virus stocks from the blood of HIV-seronegative donors, for deriving non-infectious virus particles that retain intact envelope proteins, for producing anti-viral antibodies, and for administering anti-virus antibodies to patients.



Inventors:
Killian, Scott M. (San Francisco, CA, US)
Szakal, Evelin (San Francisco, CA, US)
Vyas, Girish N. (San Francisco, CA, US)
Application Number:
14/771337
Publication Date:
01/07/2016
Filing Date:
02/28/2014
Assignee:
THERABIOLI, INC.
Primary Class:
Other Classes:
424/136.1, 424/139.1, 424/188.1, 435/7.1, 435/69.6, 435/235.1, 435/238, 530/387.3, 530/387.9, 424/133.1
International Classes:
A61K39/21; C07K16/10; C07K14/005; C07K16/00; C12N7/00; G01N33/569
View Patent Images:



Other References:
Pancera et al., Retrovirology, 2012, 9(Suppl 2):P74.
Walker et al., Science, 2009, 326:285-289.
Primary Examiner:
KINSEY WHITE, NICOLE ERIN
Attorney, Agent or Firm:
BOZICEVIC, FIELD & FRANCIS LLP (REDWOOD CITY, CA, US)
Claims:
What is claimed is:

1. A composition of matter comprising isolated antigen binding proteins (ABPs), wherein said isolated ABPs selectively bind to an epitope on an HIV-1 trimeric envelope glycoprotein subunit (TEGS).

2. The composition of matter of claim 1, wherein said TEGS is prepared by a process comprising: obtaining infectious HIV-1 virus particles from human CD4+ cell culture grown in serum-free media; contacting said infectious HIV-1 virus particles with agents that selectively remove from said particle viral RNA and viral capsid protein while retaining viral envelope protein in a non-denatured conformation, wherein said agents do not chemically fix or cross-link said envelope protein; and isolating protein from said contacted infectious HIV-1 virus particles wherein said isolated protein comprises non-infectious complexes comprising a trimeric envelope glycoprotein subunit, said subunit comprising HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA.

3. The composition of matter of claim 2, wherein said infectious HIV-1 virus particles are Fiebig I/II isolates or founder virus.

4. The composition of matter of any of claims 2-3, wherein said agents comprise cyclodextrin.

5. The composition of matter of any of claims 1-4, wherein said ABPs neutralize infectious HIV-1 particles.

6. The composition of matter of any of claims 1-5, wherein said ABPs neutralize infectious HIV-1 particles from at least one HIV-1 R5 strain and at least one HIV-1 X4 strain.

7. The composition of matter of any of claims 1-6, wherein said ABPs compete in binding to TEGS with a reference antibody or antibody fragment comprising a heavy chain CDR selected from the group consisting of SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 73, and a light chain CDR selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO:74.

8. The composition of matter of any of claims 1-6, wherein said ABPs compete in binding to TEGS with a reference antibody or antibody fragment having a nucleotide sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112.

9. The composition of matter of any of claims 1-6, wherein said ABPs compete in binding to TEGS with a reference antibody or antibody fragment having a nucleotide sequence comprising at least a portion of one of the SEQ ID NOs in Table 2.

10. The composition of matter of claim 7, wherein said reference antibody or antibody fragment is selected from the group consisting of: recombinant antibody, chimeric antibody, humanized antibody, single-chain antibody, synthetic antibody, CF antibody and fragments thereof.

11. The composition of matter according to any of claims 1-10, wherein said ABPs are selected from the group consisting of: monoclonal antibody, antibody fragment, recombinant antibody, chimeric antibody, humanized antibody, single-chain antibody, synthetic antibody, CF antibody and fragments thereof.

12. The composition of matter according to any of claims 1-10, wherein said ABPs are polyclonal antibodies.

13. The composition of matter of claim 12, wherein said ABPs are human polyclonal antibodies.

14. The composition of matter of claim 1, wherein said ABPs are bispecific antibodies.

15. The composition of matter according to any of claims 1-14, wherein said ABPs are derived from a phage display.

16. An immunotherapeutic method comprising administering to a subject a therapeutic amount of said composition of matter of any of claims 1-12.

17. A method of producing said composition of matter of claim 1, said method comprising: obtaining a sequence of said ABPs; and synthesizing said ABPs based on said obtained sequence, said synthesis occurring in a cell-free protein expression system.

18. A method for preparing a non-infectious antigenic composition capable of producing neutralizing HIV-1 antibodies comprising: obtaining infectious HIV-1 virus particles from human CD4+ cell-culture grown in serum-free media; contacting said infectious HIV-1 virus particles with agents that selectively remove from said particle viral RNA and viral capsid protein while retaining viral envelope protein in a non-denatured conformation, wherein said agents do not chemically fix or cross-link said envelope protein; and isolating protein from said contacted infectious HIV-1 virus particles wherein said isolated protein comprises non-infectious complexes comprising a trimeric envelope glycoprotein subunit, said subunit comprising HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA.

19. The method of claim 18, wherein said infectious HIV-1 virus particles are Fiebig I/II isolates or founder virus.

20. The method of any of claims 18-19, wherein said agents comprise cyclodextrin.

21. An antigenic composition produced according to said method of any one of claims 18-20.

22. An antigenic composition capable of producing neutralizing HIV-1 antibodies comprising a non-infectious HIV-1 trimeric envelope glycoprotein subunit, said subunit comprising HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA, wherein said composition is substantially free of serum proteins.

23. The antigenic composition of claim 22 that is capable of eliciting antibodies that neutralizes infectious HIV-1 particles.

24. The antigenic composition of claim 22 that is capable of eliciting antibodies that neutralizes infectious HIV-1 particles from at least one HIV-1 R5 strain and at least one HIV-1 X4 strain.

25. The antigenic composition of claim 22, wherein said antigenic composition is capable of detecting in an immunoassay anti-TEGS antibodies in serum from an animal immunized with said composition.

26. A method comprising administering an amount of said antigenic composition of any of claims 22-24 to a subject wherein a neutralizing HIV-1 antibody is produced.

27. The method of claim 26, wherein said subject is non-human.

28. The method of claim 26, wherein said subject is human.

29. The method of claim 28, wherein said human subject has a homozygous deletion of 32 base pairs in a gene encoding CCR5 coreceptor for HIV-1.

30. The method of any of claims 26-29, further comprising recovering polyclonal antibodies from said subject wherein said polyclonal antibodies neutralize HIV-1.

31. The method of any of claims 26-29, further comprising preparing a cell line comprising B cells isolated from said subject, and said cell line producing neutralizing HIV-1 antibody.

32. The method of claim 31, wherein said cell line is a hybridoma, and further comprising growing said hybridoma in cell culture, and recovering a neutralizing HIV-1 antibody from said cell culture.

33. The method of claim 31, wherein said cell line is a hybridoma, and further comprising isolating therefrom a nucleic acid encoding at least one CDR from a gene encoding a neutralizing HIV-1 antibody.

34. The method of claim 31, wherein said cell line comprises immortalized cells or transformed cells.

35. A method of producing ABPs that neutralize HIV-1, comprising culturing a cell comprising a gene encoding at least part of said ABPs under conditions wherein said gene is expressed and said ABPs are recovered.

36. The method of claim 35, wherein said gene comprises at least one CDR having a sequence obtained from a nucleic acid isolated according to claim 33.

37. The method of claim 35, wherein said ABP is selected from the group consisting of: chimeric antibody, humanized antibody, single-chain antibody, and fragments thereof.

38. In an immunoassay method for the detection of anti-HIV-1 antibodies, the improvement comprising using at least one antigen therein which is an HIV-1 trimeric envelope glycoprotein subunit isolated from an immunogenic composition according to any of claims 21-24.

39. An immunoassay method according to claim 38, wherein said antigen is bound to an immunoassay support.

40. An immunoassay method according to claim 38, wherein said antigen is in solution.

41. An immunoassay method according to claim 40, wherein said antigen comprises a detectable label.

42. A method for preparing an immunogenic, inactivated virus composition, comprising: obtaining an infectious virus particle, said particle comprising RNA, an envelope protein, a capsid protein, a reverse transcriptase, a gp120 protein, and a gp41 protein; and contacting said infectious virus particle with agents that selectively remove from said particle said RNA and said capsid protein and said reverse transcriptase, while retaining said envelope protein in a non-denatured conformation, wherein said agents do not chemically fix or cross-link said envelope protein, thereby producing an immunogenic, inactivated virus composition.

43. The method of claim 42, wherein said immunogenic, inactivated virus composition comprises a trimeric envelope glycoprotein subunit (TEGS), said subunit comprising said envelope, said gp120, and said gp41 proteins.

44. The method of claim 42, wherein said infectious virus particle is a Fiebig I/II isolate or transmitted founder virus.

45. The method of claim 42, wherein said infectious virus particle is obtained from a mammalian subject that lacks antibodies against said infectious virus particle.

46. The method of claim 45, wherein said infectious virus particle is selected from the group consisting of an HIV particle, an FIV particle, and an EIAV particle.

47. The method of claim 46, wherein said infectious virus particle is an HIV particle.

48. The method of claim 44, wherein said infectious virus particle comprises an HIV-1 or an HIV-2 particle.

49. The method of claim 42, wherein said agents comprise cyclodextrin

50. The method of claim 42, wherein said agents comprise Benzonase.

51. The method of any one of claims 42-49, wherein said infectious virus particle comprises a plurality of distinct HIV isolates.

52. The method of claim 51, wherein said distinct HIV isolates are selected from the group consisting of distinct HIV types, distinct HIV groups, and distinct HIV clades.

53. The method of claim 43, wherein said preparation of said TEGS further comprises: obtaining a DNA sequence of said TEGS; cloning said DNA sequence into an expression vector; and expressing said cloned DNA sequence to obtain TEGS proteins.

54. A product produced according to said method of any one of claims 42-52.

55. The product of claim 54, wherein said product is capable of eliciting antibodies against said trimeric envelope glycoproteins in a mammalian subject inoculated with said product.

56. The product of claim 55, wherein said antibodies are virus neutralizing antibodies

57. A method of quantifying antibodies present in a sample from a subject, comprising: contacting said sample with said product of claim 54, and determining said specific binding of antibodies in said sample to said product.

58. A method of generating an immune response in a subject, comprising: administering an immunogenic amount of said product of claim 54 to said subject.

59. The method of claim 58, wherein said subject is genetically resistant to viral infection.

60. The method of claim 58, wherein said subject is receiving antiretroviral therapy.

61. The method of claim 58, wherein said immune response comprises a neutralizing response against said infectious virus particle.

62. A method of generating an antibody, comprising: administering to a subject an immunogenic amount of said product of claim 54, and isolating said antibody, wherein said antibody specifically binds to said product of claim 54.

63. A method of generating a monoclonal antibody, comprising: administering to a subject an immunogenic amount of said product of claim 54; producing a hybridoma using B cells isolated from said immunized subject, wherein said hybridoma produces a monoclonal antibody that specifically binds to said product of claim 54; and isolating said monoclonal antibody.

64. A monoclonal antibody that specifically binds to said product of claim 54.

65. The monoclonal antibody of claim 64, wherein said antibody is capable of neutralizing in vivo said infectious virus particle.

66. A polyclonal antibody preparation that specifically binds to said product of claim 54.

67. The polyclonal antibody preparation of claim 66, wherein said antibody preparation is capable of neutralizing in vivo said infectious virus particle.

68. An immunotherapeutic method, comprising administering to a subject a therapeutic amount of said monoclonal antibody of any of claims 64-67.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/770,974, filed Feb. 28, 2013. The entire teachings of these applications are incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel HIV-1 antigenic compositions that can be used to produce antibodies that neutralize multiple strains of HIV-1, the use of such antigenic compositions to induce an immune response in a subject, and the use of such antibodies in prophylactic and therapeutic methods.

2. Description of the Related Art

Acquired immune deficiency syndrome (AIDS) is a life-threatening clinical condition caused by infection with the Human Immunodeficiency Virus 1 (HIV-1) (Killian M S, Levy J A, European Journal of Immunology 2011, 41: 3401-3411). AIDS was first described in a set of unusual clinical cases the early 1980s and its causative agent was identified shortly thereafter. Since then, major efforts have been made to develop therapies and vaccines for HIV/AIDS. To date, clinically beneficial antiretroviral drugs have been developed that delay the onset of AIDS, but an effective vaccine remains elusive, as do immune-based therapies and a cure for the infection.

Importantly, the current standard treatment of care for HIV-infected individuals has significant limitations (Reust C E, American Family Physician 2011, 83: 1443-1451). The clinically approved antiretroviral drugs require daily life-long application; non-adherence is common. They do not cure HIV-1 infection. Drug-resistant HIV-1 strains can emerge in the presence of antiretroviral drugs. Numerous toxicities caused by the long-term usage of antiretroviral drugs have been reported. HIV-1-infected individuals can progress to AIDS while receiving antiretroviral medication. Antiretroviral drugs are expensive and their monitored administration poses a difficult challenge in many parts of the world.

HIV-1 infection creates significant challenges for the development of a vaccine and immune-based therapies (Q Yu et al., Cellular and Molecular Immunology 2010, 7: 334-340). Unlike many other viruses that infect humans, HIV permanently integrates its viral nucleic acid genome into host cell chromosomes. Following the initial (acute) infection, HIV-1 spreads throughout the host while its genetic composition mutates at a high rate. Thus, vaccines and other immune-based clinical interventions must be effective against a variety of HIV-1 strains or genetic variants. Therefore, a necessary feature of an effective vaccine for HIV/AIDS is the ability to elicit broadly neutralizing antibodies (BNAb), or antibodies that target genetically distinct HIV-1 strains.

Among the proteins expressed by HIV-1, the envelope (Env) proteins are displayed on the surface of the virus, enable the virus to attach to host cells, and are indispensible for infectivity (Q Yu et al., Cellular and Molecular Immunology 2010, 7: 334-340). The HIV-1 genome encodes a single envelope nucleic acid sequence that is transcribed and translated as two glycoproteins: a transmembrane gp41 (˜41 kDa) and an external gp120 (˜120 kDa). Both proteins assemble as trimeric subunits, with the gp120 subunits being noncovalently attached to the membrane-bound gp41 subunits. Together, these gp41 and gp120 subunits constitute an envelope ‘spike’.

Several features of the envelope spike make it particularly challenging for vaccine and antibody development (Q Yu et al., Cellular and Molecular Immunology 2010, 7: 334-340). First, the amino acid sequence of the envelope spike is highly variable, due to the high mutation rate of HIV-1. Second, HIV-1 displays a relatively low number of envelope spikes (˜10) per virion. Third, its noncovalent assembly renders the envelope spike physically unstable. Fourth, its dual-trimeric assembly creates a complex protein structure. And fifth, the envelope proteins are heavily glycosylated, masking potential antibody binding sites.

A variety of methods are currently being used to generate antibodies specific for HIV. Because it is difficult to produce large amounts of natural HIV-1 in primary cell culture and because the envelope proteins on the virus are “low-abundance” proteins, the field is largely using recombinant proteins, recombinant viruses and/or long-term cell lines to elicit and to screen for anti-HIV-1 proteins. Recombinant proteins are unproven to possess the native conformational determinant(s) that can be targeted by highly effective antibodies or used for effective vaccination. Another approach used in the field is to isolate antibodies directly from the blood of persistently HIV-1-infected individuals. The limitation of such antibodies is implied in the nature of their source. Screening for anti-HIV-1 envelope antibodies is frequently performed using recombinant viruses, virus lysates, and/or recombinant proteins and peptides. These viruses and virus products are unproven to contain the native conformational determinant(s) that can be targeted by highly effective antibodies.

SUMMARY OF THE INVENTION

Embodiments of the present disclosure include methods (and corresponding products) of reducing HIV-1 transmission and disease progression by providing a system for producing a retroviral particle depleted of capsid proteins and RNA, using said retroviral particles to elicit antibodies in a subject, and administering said antibodies and/or said retroviral particles to a subject.

Accordingly one aspect of the invention is a composition of matter including isolated antigen binding proteins (ABPs). The isolated ABPs selectively bind to an epitope on an HIV-1 trimeric envelope glycoprotein subunit (TEGS). In an embodiment, the TEGS is prepared by obtaining infectious HIV-1 virus particles from human CD4+ cell culture grown in serum-free media. In an embodiment, the infectious particles are Fiebig I/II isolates or founder virus. The TEGS is further prepared by contacting the infectious HIV-1 virus particles with agents that selectively remove viral RNA and viral capsid protein while retaining viral envelope protein in a non-denatured conformation, such that the agents do not chemically fix or cross-link the envelope protein. In one embodiment, the agents include cyclodextrin. The TEGS is further prepared by isolating protein from the HIV-1 virus particles such that the isolated protein includes non-infectious complexes comprising TEGS, which includes HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA.

1. In some embodiments, the ABPs neutralize infectious HIV-1 particles, such as particles from an HIV R5 strain and an HIV X4 strain. In another embodiment, the ABPs compete in binding to TEGS with a reference antibody or antibody fragment comprising a heavy chain CDR selected from the group consisting of SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 73, and a light chain CDR selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO:74.

In another embodiment, the ABPs compete in binding to TEGS with a reference antibody or antibody fragment having a nucleotide sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112.

The reference antibody or ABPs may be recombinant antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, synthetic antibodies, CF antibodies, polyclonal antibodies, human polyclonal antibodies, bispecific antibodies, or fragments thereof. In a further embodiment, the ABPs are administered to a subject at a therapeutic amount. In yet another embodiment, the ABPs are derived from a phage display.

Another aspect of the invention is an antigenic composition capable of producing neutralizing HIV-1 antibodies including non-infectious complexes. The composition includes a TEGS that comprises HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA, such that the composition is non-infectious and substantially free of serum proteins. In a further embodiment, the composition is capable of eliciting antibodies that neutralize infectious HIV-1 particles, such as particles from an HIV-1 R5 strain and an HIV-1 X4 strain. In another embodiment, the composition is administered to a subject such that a neutralizing HIV-1 antibody is produced. The subject can be human or non-human. In a further embodiment, a human subject has a homozygous deletion of 32 base pairs in the gene encoding CCR5 coreceptor for HIV-1. In some embodiments, polyclonal antibodies that neutralize HIV-1 are recovered from the subject.

In an embodiment, a cell line that produces neutralizing HIV-1 antibody is prepared, and the cell line includes splenocytes or B cells isolated from the subject. In some embodiments, the cell line includes immortalized cells or transformed cells. In a further embodiment, the cell line is a hybridoma that is grown in cell culture, and neutralizing HIV-1 antibody is recovered from the cell culture. In a further embodiment, a nucleic acid encoding at least one CDR from a gene encoding a neutralizing HIV-1 antibody is isolated from the hybridoma. Another aspect of the invention is a method of producing ABPs that neutralize HIV-1, by culturing a cell comprising a gene encoding at least part of the ABPs under conditions such that the gene is expressed and the ABPs are recovered.

Another aspect of the invention is an improvement of an immunoassay method for the detection of anti-HIV-1 antibodies. The improvement includes using at least one antigen that is an HIV-1 TEGS isolated from an immunogenic composition as described above. In an embodiment, the antigen is bound to an immunoassay support. In another embodiment, the antigen is in solution. In a further embodiment, the antigen includes a detectable label.

Another aspect of the invention is a method for preparing an immunogenic, inactivated virus composition, by obtaining an infectious virus particle that includes RNA, an envelope protein, a capsid protein, a reverse transcriptase, a gp120 protein, and a gp41 protein. In some embodiments, the virus composition includes TEGS, which includes the envelope, the gp120 protein, and the gp41 protein. In other embodiments, the virus particle is a Fiebig I/II isolate or transmitted founder virus, or the virus particle is obtained from a mammalian subject that lacks antibodies against the virus particle, or the virus particle is an HIV particle, an FIV particle, or an EIAV particle. In some embodiments, the virus particle comprises an HIV-1 or an HIV-2 particle. The method for preparing the immunogenic, inactivated virus composition further involves contacting the infectious virus particle with agents that selectively remove the RNA, the capsid protein and the reverse transcriptase, while retaining the envelope protein in a non-denatured conformation, such that the agents do not chemically fix or cross-link the envelope protein, thus producing an immunogenic, inactivated virus composition. In an embodiment, the agents include cyclodextrin and/or Benzonase.

In one embodiment, the infectious virus particle includes multiple distinct HIV isolates. In a further embodiment, the distinct HIV isolates are distinct HIV types, distinct HIV groups, or distinct HIV clades. In another embodiment, preparation of the TEGS comprises obtaining a DNA sequence of the TEGS, cloning the DNA sequence into an expression vector, and expressing the cloned DNA sequence to obtain TEGS proteins. In another embodiment, a product is produced by one of the above methods, such that the product is capable of eliciting antibodies against the trimeric envelope glycoproteins in a mammalian subject inoculated with the product. In a separate embodiment, the antibodies are virus neutralizing antibodies. In one embodiment, the antibodies present in a sample from a subject are quantified by contacting the sample with the product, and determining the specific binding of antibodies in the sample to the product. In another embodiment, an immune eresponse is generated in a subject by administering an immunogenic amount of the product to the subject. In a further embodiment, the subject is genetically resistant to viral infection. In a further embodiment, the subject is receiving antiretroviral therapy. In a further embodiment, the immune response includes a neutralizing response against the infectious virus particle.

In another embodiment, an antibody is generated by administering to a subject an immunogenic amount of the above product, and isolating the antibody, such that the antibody specifically binds to the product. In a separate embodiment, an antibody is generated by administering to a subject an immunogenic amount of the above product, producing a hybridoma using splenocytes or B cells isolated from the immunized subject, such that the hybridoma produces a monoclonal antibody that specifically binds to the product, and isolating the monoclonal antibody. In another embodiment, a monoclonal antibody specifically binds to the above product. In a further embodiment, the antibody is capable of neutralizing in vivo the infectious virus particle. In another embodiment, a polyclonal antibody preparation specifically binds to the above product. In a further embodiment, the antibody preparation is capable of neutralizing in vivo the infectious virus particle.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

FIG. 1 illustrates a flowchart that summarizes a method for producing TEGS for generating neutralizing antibodies, in accordance with an embodiment of the invention.

FIG. 1A illustrates a method for producing virus stocks for use in preparing a virus particle product, in accordance with an embodiment of the invention.

FIG. 2 illustrates results of an RT-PCR assay for validation and optimization for measuring virus, in accordance with an embodiment of the invention.

FIG. 3 illustrates a diagram of a particle that features multiple virus particle products, in accordance with an embodiment of the invention.

FIG. 4 illustrate (left) RT-PCR data and (right) a bar graph that demonstrate that a virus particle product can be sued to capture, detect, and measure anti-virus particle product ABPs, in accordance with an embodiment of the invention.

FIG. 5 shows SDS-PAGE gels demonstrating that rabbits immunized with TEGS produce TEGS-specific antibodies, in accordance with an embodiment of the invention.

FIG. 6 is a graph demonstrating that the antibody response elicited by immunizing rabbits with TEGS is dose dependent, in accordance with an embodiment of the invention.

FIG. 7 illustrates (left) a representative amplification profile, and (right) neutralization results, that illustrate that TEGS anti-serum neutralizes HIV-1, in accordance with an embodiment of the invention.

FIG. 8 is a chart demonstrating that select recombinant HIV-1 envelope proteins do not block neutralizing activity of TEGS antiserum from a rabbit, in accordance with an embodiment of the invention.

FIG. 9 shows (left) a gel, and (right) Western blot results, which illustrate refinement of a virus particle product, in accordance with an embodiment of the invention.

FIG. 10 shows a plasmid in which an envelope gene from a virus particle product was cloned and sequenced, in accordance with an embodiment of the invention.

FIG. 11 illustrates results of (left) an ELISA and (right) a Western blot, showing that supernatants from mouse hybridoma cells produce virus particle produce-specific antibodies, in accordance with an embodiment of the invention.

FIG. 12 illustrates an antibody-phage display approach used with splenocytes and bone marrow derived from rabbits immunized with TEGS, in accordance with an embodiment of the invention.

FIG. 13 illustrates (left) a TEGS-specific ABP and (right) Western blot results, showing that the TEGS-specific ABP was produced and validated, in accordance with an embodiment of the invention.

FIG. 14 shows a compilation of a similarity matrix between heavy chain variable region amino acid sequences, and a similarity matrix between light chain variable region amino acid sequences, in accordance with an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

The term “vector” means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.

The term “expression vector” or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.

The term “host cell” means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

The term “transformation” refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid. A cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.

The terms “polypeptide” or “protein” means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally-occurring and non-recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein.

The term “isolated protein” means that a subject protein (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Typically, an “isolated protein” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. In some embodiments, an isolated protein has undergone post-translational modifications and is glycosylated.

A “variant” of a polypeptide (e.g., an antigen binding protein, or an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

The term “naturally occurring” as used throughout the specification in connection with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials which are found in nature or a form of the materials that is found in nature.

The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An “antibody” is a species of an antigen binding protein. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies as described further below. The antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length “light” (in certain embodiments, about 25 kDa) and one full-length “heavy” chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 10 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, typically, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair typically form the antigen binding site.

The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989), or International Immunogenetics Information System (IMGT; Lefranc et al., Nucleic Acids Research 2009 37: D1006-D1012; doi:10.1093/nar/gkn838).

In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the IMGT definition, and the contact definition.

The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

IMGT®, the international ImMunoGeneTics information System® (http://www.imgt.org), is a global reference in immunogenetics and immunoinformatics, created in 1989 by Marie-Paule Lefranc (Université Montpellier 2 and CNRS). IMGT® is a high-quality integrated knowledge resource specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH) of human and other vertebrate species, and in the immunoglobulin superfamily (IgSF), MH superfamily (MhSF) and related proteins of the immune system (RPI) of vertebrates and invertebrates. IMGT® provides a common access to sequence, genome and structure Immunogenetics data, based on the concepts of IMGT-ONTOLOGY and on the IMGT Scientific chart rules. IMGT® works in close collaboration with EBI (Europe), DDBJ (Japan) and NCBI (USA). IMGT® consists of sequence databases, genome database, structure database, and monoclonal antibodies database, Web resources and interactive tools.

By convention, the CDR regions in the heavy chain are typically referred to as H1, H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as L1, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxyl-terminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

Some species of mammals also produce antibodies having only a single heavy chain.

Each individual immunoglobulin chain is typically composed of several “immunoglobulin domains,” each consisting of roughly 90 to 110 amino acids and having a characteristic folding pattern. These domains are the basic units of which antibody polypeptides are composed. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically comprises one or more domains that can be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. IgG heavy chains, for example, contain three C region domains known as CH1, CH2 and CH3. The antibodies that are provided can have any of these isotypes and subtypes.

The term “variable region” or “variable domain” refers to a portion of the light and/or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen binding protein (including, e.g., an antibody or immunological functional fragment thereof). In some embodiments, the antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen.

The term “epitope” includes any determinant capable being bound by an antigen binding protein, such as an antibody or to a T-cell receptor. An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

An “antigen binding protein” (“ABP”) as used herein means any protein that binds a specified target antigen. “Antigen binding protein” includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments. Peptibodies are another example of antigen binding proteins. The term “immunologically functional fragment” (or simply “fragment”) of an antibody or immunoglobulin chain (heavy or light chain) antigen binding protein, as used herein, is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to a given epitope. In some embodiments, the fragments are neutralizing fragments. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), Fab′, F(ab′)2, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life. In some embodiments, antibody fragments are produced by treatment of an immunoglobulin with a protease such as pepsin or papain. The immunoglobulin may be digested before and after a disulfide bond between two H chains in a hinge region. As will be appreciated by one of skill in the art, an antigen binding protein can include nonprotein components.

Certain antigen binding proteins are antibodies or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen binding proteins is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. These various antigen binding proteins are further described herein.

An “Fc” region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

A “Fab fragment” comprises one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” comprises one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule.

A “F(ab′)2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab′)2 fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains.

The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region.

A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

A “bivalent antigen binding protein” or “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. Bivalent antigen binding proteins and bivalent antibodies can be bispecific, see, infra. A bivalent antibody other than a “multispecific” or “multifunctional” antibody, in certain embodiments, typically is understood to have each of its binding sites identical.

A “multispecific antigen binding protein” or “multispecific antibody” is one that targets more than one antigen or epitope.

A “bispecific,” “dual-specific” or “bifunctional” antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites. Bispecific antigen binding proteins and antibodies are a species of multispecific antigen binding protein antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes, which can reside on the same or different protein targets.

An “immobilized antibody” is an antibody supported on an insoluble carrier via physical adsorption, chemical bond, or some other method. An immobilized antibody can be used to detect, quantify, separate, or purify an antigen contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, or a centrifuged supernatant.). In one embodiment, an insoluble carrier used to immobilize the antibody includes a plastic consisting of a polystyrene resin, a polycarbonate resin, a silicon resin, or a nylon resin; a plate consisting of a substance insoluble in water, such as glass; a product having an inner volume, such as a test tube; and either beads, a ball, a filter, or a membrane. In another embodiment, an insoluble carrier used to immobilize the antibody includes insoluble carriers used in affinity chromatography, such as a cellulose carrier, an agarose carrier, a polyacrylamide carrier, a dextran carrier, a polystyrene carrier, a polyvinyl alcohol carrier, a polyamino acid carrier, or a porous silica carrier. An immobilized antibody can be sensitized to an insoluble carrier such as a solid phase carrier, or as a labeled antibody that is labeled with some labeling substance.

The term “labeled antibody” is an antibody that is labeled with a labeling substance. A labeled antibody can be used to detect or quantify an antigen contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, or a centrifuged supernatant). The labeling substance is not particularly limited, as long as it is able to bind to an antibody via a physical bond, such as a chemical bond. The labeling substance can be an enzyme, a fluorescent substance, a chemoluminescent substance, biotin, avidin, or a radioisotope, for example. More specifically, the labeling substance can be an enzyme such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholine esterase; a fluorescent substance such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate; a radioisotope such as 3H, 14C, 125I or 131I; or biotin, avidin or chemoluminescent substances. Methods for binding a labeling substance to an antibody include a glutaraldehyde method, a maleimide method, a pyridyl disulfide method or a periodic acid method.

A labeling substance such as a radioisotope or a fluorescent substance generates detectable signals on its own. Other labeling substances such as enzymes, chemoluminescent substances, biotin, and avidin are not able to generate detectable signals on their own, but generate detectable signals by reacting with one or more types of other substances via a colorimetric method, a fluorescence method, a bioluminescence method, or a chemoluminescence method, for example. In an embodiment, biotin is used as a labeling substance, and reacts with at least avidin or enzyme-modified avidin.

An antigen binding protein is “selective” when it binds to one target more tightly than it binds to a second target. Methods of determining ABP selectivity or binding specificity are well-known in the art.

The term “recombinant antibody” refers to an antibody engineered by recombinant DNA technology in a cell line, without involving the use of animals. The term “complement-fixing (CF) antibody” refers to an antibody that is combined with an antigen, leading to opsonization or cell lysis.

The term “neutralizing antigen binding protein” or “neutralizing antibody” refers to an antigen binding protein or antibody, respectively, that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking a binding site on the ligand or by binding to the ligand and altering the ligand's ability to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments, the term can also denote an antigen binding protein that prevents the protein to which it is bound from performing a biological function. In assessing the binding and/or specificity of an antigen binding protein, e.g., an antibody or immunologically functional fragment thereof, an antibody or fragment can substantially inhibit binding of a ligand to its binding partner when an excess of antibody reduces the quantity of binding partner bound to the ligand by at least about 1-20, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured in an in vitro competitive binding assay). In some embodiments, the neutralizing ability is characterized and/or described via a competition assay. In some embodiments, the neutralizing ability is described in terms of an IC50 or EC50 value. The term “broadly neutralizing HIV-1 antibody” refers to a neutralizing antibody that neutralizes more than one HIV-1 strain.

Preferably, the neutralizing antigen binding proteins or neutralizing antibodies of the present invention neutralize infectivity; i.e., the antigen binding protein or antibody reduces or eliminates viral infectivity. Infectivity neutralization can be tested in a standard assay, for example, by incubating the antibody or ABP of interest with viral isolates and susceptible human cells, and then measuring viral levels in a RT-PCR assay. Such an assay is described in Example 8 (below).

In an embodiment, neutralizing antibodies to an HIV-1 virus particle product are generated by immunizing a subject with a concentrated HIV-1 virus particle product, such as TEGS. In a preferred embodiment, the concentrated HIV-1 virus particle product was produced by propagating the virus in serum-free medium, concentrating the virus, and treating the virus with agents to inactivate the virus. In some embodiments, the concentration step involves using molecular weight cutoff (MWCO) filters. In other embodiments, the agents for inactivating the virus can include a cyclodextrin or Benzonase.

The term “target” refers to a molecule or a portion of a molecule capable of being bound by an antigen binding protein. In certain embodiments, a target is an antigen. The use of “antigen” in the phrase “antigen binding protein” simply denotes that the moiety that comprises the antigen can be bound by an antibody.

The term “compete” when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen. Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (ETA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

As used herein, “substantially pure” means that the described species of molecule is the predominant species present, that is, on a molar basis it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition wherein the object species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the object species is purified to essential homogeneity wherein contaminating species cannot be detected in the composition by conventional detection methods and thus the composition consists of a single detectable macromolecular species.

The term “virus” refers to a small infectious agent that only replicates inside the cell of another organism. A virus particle consists of genetic material made from either DNA or RNA, a protein coat, and optionally a lipid envelope. The term “retrovirus” refers to a virus that uses RNA as its genetic component.

The term “expansion” refers to propagation of the virus and or cells in vitro.

The term “trimeric envelope glycoprotein subunit” or “TEGS” refers to protein complexes of trimeric gp120 bound to the membrane-anchored trimeric gp41. Monomeric gp120 and gp 41 are HIV envelope proteins having masses of 120 and 41 kilodaltons (kDa) respectively. Thus, TEGS are predicted to have a mass of several hundred kDa. TEGS function in the attachment and entry of HIV into the cell. Proposed is that TEGS contain a highly conserved protein shape or ‘conformational determinant’ that is required for HIV attachment and entry. Antibodies that bind and obscure the ‘conformational determinant’ can be broadly neutralizing antibodies. Non-neutralizing antibodies that bind to TEGS can also help to reduce infectious virus particles and therefore can also be clinically beneficial.

The term “non-infectious” refers to the infectivity state of all components of the fluids harvested from cultures containing virus-infected cells. Non-infectious material, when used in standard infectivity assays with blood cells, permissive cell lines, and/or animal models, produces no detectable virus replication.

The term “fluid” refers to cell culture conditioned medium. In addition to the input cell culture medium, a “fluid” can contain modifications to the constituents of the input cell culture medium, cells, cellular debris, products secreted by cells, products of cellular breakdown, intact viruses, partial viruses, virus proteins, and contaminants.

The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

The term “biological sample”, as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such substances include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.

The term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.

The term “enhancing antibody” refers to an antibody that can promote the replication of a virus, virus subset, or molecular virus construct. This phenomenon facilitates antibody-dependent enhancement (ADE) of infection and can involve complexes of microorganisms and antibodies, interactions of antibodies with Fc receptors (FcR), complement proteins, and complement receptors (CR).

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Methods of the Invention

Viruses and virus derivatives, natural or synthetic, when used for immunization are appreciated for their abilities to elicit protective immune responses. The resulting protection can be attributed to the production of antibodies that are specific for components of the immunogen. Thus, the use of viruses or virus products can enable the production of virus-specific antibodies. The viruses and virus derivatives can be useful in the form of an immunogen or vaccine. The resulting antibodies can also be used for research and clinical benefit. The present virus particle product can be used to elicit virus-particle-specific antibodies. The present invention relates to: producing trimeric envelope glycoproteins derived from HIV, using these glycoproteins for use in clinical and research assays for immunization purposes, and producing antigen-binding proteins (ABPs) (for example, antibodies) that have a neutralizing activity on a wide range of HIV strains. The present invention is not limited to any known variant of HIV and it can include future strains that can arise from recombination of present circulating forms.

Practices of the method of the present invention include extracting cholesterol from a virus membrane and depleting capsid proteins and RNA, and creating a virus particle product. In some examples, the virus is an infectious retroviral particle that comprises RNA, an envelope protein, a capsid protein, a gp120 protein, and a gp41 protein. In one embodiment, the virus particle product is prepared from HIV-1 cultured in primary human cells. The HIV stocks can be prepared from HIV isolated from individuals undergoing acute HIV infection (such that the individuals' blood is HIV RNA positive and HIV antibody negative). In a further embodiment, the primary human cells are CD4+ blood cells cultured in a serum-free medium.

In an embodiment, the virus particle product is processed to render the HIV-1 non-infectious. In one embodiment, the virus particle product is not exposed to chemical compounds, such as formaldehyde, ethyleimine, or beta-propiolactone that can modify or disrupt the natural envelope protein structure. The virus particle product retains TEGS (trimeric envelope glycoprotein subunit) in its natural state as evidenced by its ability to bind both anti-gp120 and anti-gp41 antibodies (Vyas G N et al., Biologicals, 2012 40: 15-20). One method of preparing the virus particle product includes contacting an infectious retroviral particle with an agent (for example, a cyclodextrin) that selectively removes RNA, reverse transcriptase and capsid protein while retaining the envelope protein in a non-denatured conformation, wherein said agent does not chemically fix or cross-link said envelope protein, thereby producing an immunogenic, inactivated retroviral composition. Any residual DNA/RNA is degraded by hydrolysis with protease-free Benzonase®. Thus, the immunogenic, inactivated retroviral composition comprises a trimeric envelope glycoprotein subunit, said subunit comprising said envelope, said gp120, and said gp41 proteins. The native HIV-1 envelope structure is retained, and said gp120 subunits are noncovalently attached to said gp41 proteins. In another embodiment, supernatant from the serum-free cell culture of HIV-infected CD4+ cells is concentrated and treated with beta-cyclodextrin and with or without Benzonase to render the HIV non-infectious.

The virus particle product can be derived from HIV obtained from HIV-infected by seronegative subjects, or further derived from a plurality of HIV types, including HIV-1, HIV-2, groups, and clades. The virus particle product can also be obtained from a mammalian subject that lacks antibodies against said infectious retroviral particle, or from animal lentiviruses such as Feline Immunodeficiency Virus (FIV) or Equine Infectious Anemia (EIAV). The virus particle product can also be a Fiebig I/II isolate or founder virus.

The virus particle product is capable of eliciting neutralizing antibodies or enhancing antibodies in a mammalian subject inoculated with said product. For example, an immunogenic amount of the virus particle product can be administered to a subject to generate an anti-virus particle product antibody (for example, a TEGS-specific antibody), after which antibody can then be isolated. In some cases, said subject is a camelid subject. In a preferred embodiment, the anti-virus particle product antibody has neutralizing activity against at least one HIV strain, and more preferably has neutralizing activity against a broad range of HIV strains, including the R5 strain and the X4 strain.

In an embodiment, virus-particle-specific antibodies are elicited in animals. For example, to elicit antibodies, animals (e.g., mice, rabbits, camelids) can be immunized with TEGS or iTEGS. During the antibody production protocol (e.g., 8+ weeks), animals receive multiple (e.g., 3-5) immunizations. The resulting animal serum can contain virus-particle-specific antibodies. Compounds such as Freund's Adjuvant, aluminum salts, CpG, and other adjuvants can be administered along with the immunization to enhances the production of antibodies.

In the case of FIG. 6, a diagram (ptbi-env11) is shown that demonstrates the ability to genetically engineer components of natural viruses for the production of virus particle products. In this case, the envelope gene was PCR amplified from primary cells infected with a founder/transmitted HIV-1 isolate (HIV-1TBI-11, or TBI01280) and then cloned into a plasmid. The insert was confirmed by sequence analysis to be a full-length HIV-1 envelope gene.

In some cases, the anti-virus particle product antibodies are quantified by contacting a sample from a subject with the virus particle product, and determining the specific binding of antibodies in said sample to the virus particle product. One example includes a method of generating an immune response in a subject by administering an immunogenic amount of the virus particle product to said subject. The subject may be genetically resistant to viral infection, or the subject may be receiving anti-retroviral therapy. In some cases, the immune response includes a neutralizing response against the virus particle product.

Monoclonal antibodies can be generated from lymphocytes (for example, B lymphocytes) that have been isolated from the blood, blone marrow, or spleen of an immunized animal or human and immortalized using hybridoma and/or transformation procedures. By culturing the obtained cell derivatives, monoclonal antibodies can be obtained from the cells. In one embodiment, the antibodies are purified by methods such as DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, or ethanol fractionation. Some of the monoclonal antibodies of the present invention comprise VH and VL amino acid sequences as listed in Table 1. An oligopeptide or polypeptide is within the scope of the invention if it has an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a portion of at least one of the amino acid sequences of Table 1, preferably to achieve maximal levels of sequence identity. Some of the monoclonal antibodies of the present invention comprise VH, VL and linker nucleic acid sequences as listed in Table 2. An oligonucleotide is within the scope of the invention if it has a nucleic acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a portion of at least one of the nucleic acid sequences of Table 2, preferably to achieve maximal levels of sequence identity.

In an embodiment, monoclonal antibodies are generated when an immunogenic amount of the virus particle product is administered to a subject, and a hybridoma is produced using a splenocyte isolated from said immunized subject. Thehybridoma produces a monoclonal antibody that specifically binds to the virus particle product, and the monoclonal antibody is isolated. In an embodiment, said antibody is capable of neutralizing in vivo the virus particle product. Furthermore, a polyclonal antibody can be prepared that specifically binds to the virus particle product, where the antibody is capable of neutralizing in vivo the virus particle product Immunotherapeutic methods are possible which involve administering a therapeutic amount of the aforementioned monoclonal antibody or polyclonal antibody to a subject.

Enhancing antibodies are reported for a variety of viruses and infections, including HIV, hepatitis C virus, and Ebola (Fust G. Parasitology 1997, 115: S127-140) (Meyer K et al., PloS one 2011, 6:e23699). These enhancing antibodies can be elicited in subjects and/or measured using the embodied procedures. A preferred application of the elicited or otherwise obtained enhancing antibodies is for clinical use in exposing reservoirs of HIV, where virus replication levels are too low to be detected and/or targeted by natural immune responses or clinical interventions. Another preferred application is the use of enhancing antibodies in clinical and laboratory settings where elevated levels of virus infectivity and/or replication are desired, such as for gene therapy, gene delivery using viral vectors, or maximizing the production of virus particle products.

To generate an enhancing antibody, an immunogenic amount of the virus particle product is administered to a subject. In an embodiment, said antibody is capable of activating replication in vivo of said infectious retroviral particle Immunotherapeutic methods are possible which involve administering a therapeutic amount of the enhancing antibody to a subject. Also, a protein or protein fragment that binds to the enhancing antibody can be produced, and immunotherapeutic methods are possible which involve administering a therapeutic amount of said protein or protein fragment to a subject.

In one embodiment, an anti-virus particle product antibody is a bispecific antibody having two different antigen binding sites. The bispecific antibody binds two different epitopes. In a further embodiment, the bispecific antibody that selectively recognizes TEGS, has functional properties that differ from either of the corresponding monospecific antibodies. Further disclosure for producing bispecific antibodies is found in Rouet R and Christ D, Nature Biotechnology 2014, 32: 136-137; and Kontermann R, Landes Biosciences 2012, 4: 182-197.

A genetic sequence encoding an anti-virus particle product antibody can be obtained by producing a cDNA library using mRNA derived from immunized animals that produce TEGS-specific monoclonal antibodies as described above, and isolating a plasmid containing cDNA encoding a monoclonal antibody. In an embodiment, the mRNA from a specimen containing B cells is prepared by dissolving the cells in a guanidinium isothiocyanate solution, followed by mRNA extraction. The cDNA is produced using the extracted mRNA as a template, and the cDNA, or PCR amplified cDNA, is incorporated into vectors to produce a cDNA library. A gene encoding an anti-virus particle product monoclonal antibody is obtained using the cDNA library. Amino acid sequences of TEGS-specific antibody regions are in Table 1, and nucleic acid sequences of TEGS-specific antibody regions are in Table 2. The PCR primers used for this purpose are in Table 3.

TABLE 1
TEGS-specific antibody region amino acid sequences
Antibody
Regionand
Clone
SEQ ID NONumberAmino Acid Sequence
SEQ ID NO: 1VHQSLEESGGGLFKPTDTLTLTCTVSGFSLSNYGVVWVR
TBIfabT203QAPGNGLEWIGIIDHHGIPYYATWAKSRSTITRNTNLD
TVTLKMTSLTAADTATYFCAR
SEQ ID NO: 2VLELVMTQTESPVSAAVGSTVTINCQASQSVYSNNNLAW
TBIfabT203FQKKPGQPPKRLIHSASTLASGVPSRFKGSGSGTQFTLT
ISDLECDDAATYYCAGVFSGSISVFGGGTEVVVK
SEQ ID NO: 3VHVGEGVRGGLLKPTDTLTLTCTVSGFSLNSYAVFWVRQ
TBIfabT205APGNGLEWIGTVSSVGDTYFATWAKSRSTITRNTNLN
TVTLKMTSLTAADTATYFCA
SEQ ID NO: 4VLQPVLTQSPSVSAALGSSAKLTCTLSSAHKTYYIEWYQQ
TBIfabT205QQGEAPRYLMQLESDGSYTKGTGVPDRFSGSSSGADR
YLIISSVQAEDEADYYCGADYSGGFVFGGGTQLTVT
SEQ ID NO: 5VHQSVEESGGGLFKPTDTLTLTCTVSGFSLSGYGVSWVR
TBIfabT206QAAGNGLEWIGAISSGGSAYYARWAKSRSTITRNTNL
NTVTLKMTSLTAADTATYFCAR
SEQ ID NO: 6VLELVMTQTEPPVSAPVGGTVTINCQASQNIGSSYLSWYQ
TBIfabT206QKPGQPPKLLIYQASTLASGVPSRFKGGGSGTDYSLTIS
GVQCADAATYYCQSTFYSSGTGYAFGGGTELEIL
SEQ ID NO: 7VHQSVKESEGGLFKPTDTLTLTCTASEFTIGSYSSGWVRQ
TBIfabT208APGKELEWIGTLSSTGSAHYANWAKGRSTITRNTNEN
TVTLKMASLTAADTATYFCAR
SEQ ID NO: 8VLPVLTQSPSVSAALGASAKLTCTLSSGHKTYTIDWYQQ
TBIfabT208QQQGEAPRYLMQIGSDGSYTKGTGVPDRFSGSSSGTD
RYLIISSVQAEDEADYYCGADYSGGFVFGGGTQLTVT
SEQ ID NO: 9VHQSVEESGGGLFKPTDTLTLTCTVSGIDLSRNGVTWVRQ
TBIfabT209APGSGLEWIGVINSHGDSDYATWANSRSTITRNTNLNT
VTLKMTSLTAADTATYFCA
SEQ ID NO:VLELVMTQTPSSVSAAVGGTVTINCQASQTINNLLAWYQ
10TBIfabT209QKPGQPPKLLIYGASTLASGVPSRFSGSGSGTQFILTISG
MKAEDAATYYCQSAYYNAGATFGAGTNVEIK
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFSLSNSAMSWVRQ
11TBIfabT210APGNGLEWIGDIDSSGSAYYASWAKSRSTITRNTNLNT
VTLKMTSLTAADTATYFCAR
SEQ ID NO:VLPVLTQSPSVSAALGASAKFTCTLSSGHKTYTIDWYQQ
12TBIfabT210QQQGEAPRYLMQIGSDGSYTKGTGVPDRFSGSSSGTD
RYLIISSVQAEDEADYICGVTGSNVYAQDPADRH
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFDISGVYMSWVRQ
13TBIfabT216APGNGLEWIGAIDRGGGTYYASWAIGRSTITRNTNDN
TVTLEMTSLTAADTATYFCAK
SEQ ID NO:VLELVMTQTPSSVSAAVGGTVTINCQASESISNYLAWYQ
14TBIfabT216QKPGQPPKLLTYDASDLASGVPSRFSGSGYGTEFTLTIS
GVKAEDAATYYCQSGYVSAGTFGAGTNVEIK
SEQ ID NO:VHQSLEESGGGLIKPTDMLTLTCTVSGFSLSNYGVMWVR
15TBIfabT222QAPGNGLESIGYIGSGGDTSYASWAKSRSTIARNTNEN
TVSLLMNGLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSVSAALGASAKLTCTLSSAHKTYTIDWYQ
16TBIfabT222QQQGEAPRYLMHLKSDGTYTKGTGVPDRFSGSSSGAD
RYLIIPSVRTDDEADYYCGTDYSGGYVFGGGTQLTVT
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFSLSIYGVSWVRQ
17TBIfabT226APGNGLEWVGAIGSGGSAYYATWAKSRSTITRNTNLN
TVTLKMASLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSASAALGSSAKLTCTLSSAHKTYYIDWYQ
18TBIfabT226QQQGEAPRYLMQVKSDGSYTRGTGVPDRFSGSSSGAD
RYLIIPSVQADDEADYYCGSDYSGGYVFGGGTQLTVT
SEQ ID NO:VHQSVEESRGGLFKPTDTLTLTCTVSGFSLSTYNIQWVRQ
19TBIfabT229APGNGLEYIGTIGSSGSAYYASRAKSRSTITRNTALNTV
SLQVDSLTDADTATYFCAR
SEQ ID NO:VLELDLTQTPSSVSAAVGGTVTINCQASQSVSNLLAWYQ
20TBIfabT229QKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEFTLTIS
DVVCDDAATYYCAGHKSSSTDGTAFGGGTELEIL
SEQ ID NO:VHQSVKESEGGLFKPTDTLTLTCTVSGFTVSNNAISWVRQ
21TBIfabT232APGNGLEWIGAISYGGNTYYANWPKSRSTITRNTNLN
TVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSASAALGSSAKLTCTLSSAHKTYYIDWYQ
22TBIfabT232QQQGEAPRYLMQVKSDGSYTKGTGVPDRFSGSSSGA
DRYLIIPSVQADDEADYYCGSDYSGGYVFGGGAQLTVT
SEQ ID NO:VHQSVKESEGGLFKPTDTLTLTCTVSGFSLSNYGVSWVR
23TBIfabT234QAPGKEVEWIGYINSGGSTNYASWAKSRSTITRNTNL
NTVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELDMTQTPSSVSAAVGDTVTINCQASQSVTNLLAWYQ
24TBIfabT234QKPRQPPKLLIYDASNLESGVPSRFRGSGSGTEFTLTIS
GMKAEDAATYYCQSGYYSAGATFGAGTNVEIK
SEQ ID NO:VHQSVKESEGGLFKPMDSMTLTCTVSGFSLSSYGVSWVR
25TBIfabT236QAPGNGLEWIGAISSGGSAYYARWAKSRATITRNTNL
NTVTLKMASLTAADTATYFCAR
SEQ ID NO:VLELVLTQTPSPVSAAVGGTVTINCQSSQSVYSNNRLAW
26TBIfabT236YQQKPGQPPKQLIYYASTLASGVSSRFKGSGSGTQFTL
TISDVVCDDAATYYCAGYKNSGIDEHAFGGGTELEIL
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFSLNRYDMSWVR
27TBIfabT239QAPGNGLEWIGVINSGGFTYYASWAKSRSTITRNTNE
NTVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSVSAALGASAKLTCTLSSGHKTYTIDWYQ
28TBIfabT239QQQGEAPRYLMQLGSDGSYTKQTGVPDRFSGSSSGAD
RYLIISSVQADDEADYYCGADYSGGFVFGGGTQLTVT
SEQ ID NO:VHQSVKESEGGLFKPTDTLTLTCTVSGFSLSNNAINWVRQ
29TBIfabT240APGNGLEWIGAVGSGGRAYYAGWAKSRSTITRNTNL
NTVTLKMTNLTAADTATYFCAR
SEQ ID NO:VLELVLTQTPSSVSAAVGGTVSISCQSSQSVYSNYLAWYQ
30TBIfabT240QKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTIN
GVQCDDAATYYCQGTFDDGLYKAFGGGTELEIL
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFSLSNYGMGWVR
31TBIfabT241QAPGNGLEYIGFISSGGNTYYASWAKSRSTITRDTNLN
TVTLKMSSLTAADTATYFCAR
SEQ ID NO:VLELVMTQTPSSVSAAVGGTVTINCQASQSVYNLLAWYQ
32TBIfabT241QKPGQPPKLLTHGTSNLESGVPSRFRGSGSGTEFTLTIS
GMKAEDAATYYCQSGYYSTGATFGAGTNVEIK
SEQ ID NO:VHQSLEESGGGLFKPTDPLTLTCTVSGFSINDYNMQWVR
33TBIfabT247QAPGIGLEWIGAINAWGDTYYTSWAKSRSTITRDTNL
NTVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELDLTQTPSSVSAAVGGTVTINCQSSQSVDSNNYLSW
34TBIfabT247YQQKPGQPPKLLIYDASTLASGVPSRFSGSGSGTQFTL
TISEVQCDDAATYYCQGSYYSGDWYGAFGGGTELEIL
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTASGFTVTSNAISWVRQ
35TBIfabT249APGNGLEYIGFIGAAGNANYASWAKSRSTITRNTNLNT
VTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELVMTQTPASVSEPVGGTVTISCQASQGVYSDRLAWY
36TBIfabT249QQKPGQPPKLLMYYASDLSSGVPSRFKGSGSGTEFTLT
ISDLECADAATYYCQSNYGSLSSSYTFGGGTEVVVK
SEQ ID NO:VHQSVEESRGGLFKPTDTLTLTCTVSGFTIDTYGVTWVRQ
37TBIfabP202APGNGLEYIGFISSGGAAYYASWAKSRSTITRNTNLNT
VTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELVMTQTPPSLSASVGETVRIRCLASENVYSAVAWYQ
38TBIfabP202QKPGKPPTLLISGASNLESGVPPRFSGSGSGTDYTLTIG
GVQAEDAATYFCQGYSSYLTFGAGTNVEIK
SEQ ID NO:VHQSLEESGGGLIKPTDTLTLTCTVSGFSLSIYDISWVRQA
39TBIfabP210PGNGLEWIGAIGSGDTTYYASWAKSRSTITRNTYLNTV
TLKMTSLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSASAALRSSAKLTCTLSSAHKSYDIDWYQQ
40TBIfabP210QSGEAPRYLMRLRSDGKYTKGTGVPDRFSGSSSGADR
YLIIPSVQADDGADYYCGTDYSGGYVFGGGTQLTVT
SEQ ID NO:VHEQLVESEGGLFKPTDTLTLTCTVSGFSLNNYGVTWVR
41TBIfabP214QAPGRGLEWIGAVWSGATTDYASWAKSRSTITRNTNE
NTVTLKMSSLTAADTATYFCA
SEQ ID NO:VLELVMTQTESPVSAAVGGTVTINCQASQSISSWLAWYQ
42TBIfabP214QKPGKPPTLLISGASNLESGVPPRFSGSGSGTDYTLTIG
GVQAEDAATYYCLGGYSYSSIGTTFGAGTNVEIK
SEQ ID NO:VHQEQLEESGGGLVQPGGSLKLSCKASGFDFINYGVIWV
43TBIfabP217RQAPGKGLEWIGYIDPIFGNTIYASWVNDRFTISSHNA
QNTLYLQLNSLTAADTATYFCAR
SEQ ID NO:VLELVMTQTPSSVSAAVGGTVTINCQASQSVNNLLAWYQ
44TBIfabP217QKPGQPPKLLTYGTSNLESGVPSRFRGSGSGTEFTLTIS
GMKAEDAATYYCQSGYYSAGLTFGAGTNVEIK
SEQ ID NO:VHQSLEESGGGLIKPTDTLTLTCTVSGFSLSTNGVSWVRQ
45TBIfabP219APGSGLEWIGAIDLYGATYYATWAKSRSTITRNTNLN
TVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELVLTQTPASVSEPVGGTVTIKCQASQNIYSGISWYQQ
46TBIfabP219KPEKPPTLLISGASNLEPGVPPRFSGSGSGTDYTLTIGG
VQAGDAATYYCLGVYSFGSTDLTFGAGTNVEIK
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFSLSSYAISWVRQ
47TBIfabP220APGNGLEWIGYINYDGIAYYASWAKSRSTITRNTNLNT
VTLKMTGLTAADTATYFCAR
SEQ ID NO:VLELDLTQTPSSVSAAVGGTVSISCQSSQSVYNNYLAWY
48TBIfabP220QQKPGQPPKLLIYYASKLASGVPSRFKGSGSGTQFTLTI
SDVQCDDAATYYCQGTFDNGLYKAFGGGTELEIL
SEQ ID NO:VHQSLEESGGGLVQPGGSLKLSCKGSGFDLDSNAMCWV
49TBIfabP221RQAPGSGLEWIGTITSGGSAYYASWAKSRSTITRNTNL
NTVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELDMTQTPSSVSAAVGGTVTINCQASESISNLLAWYQ
50TBIfabP221QKPGQPPKLLIYSASTLASGVPSRFRGSGSGTEFTLTIS
GMKAEDAATYYCQSGYYSTGATFGAGTNVEIK
SEQ ID NO:VH TBIfabT7QEQLEESGGRLVKPDETLTLTCTVSGLSLNNFGVSWV
51RQAPGNGLEWIRAIDFGSGSAYYANWAKSRSTITSNTR
LNTVTLKMISLTAADTATYFCSR
SEQ ID NO:VL TBIfabT7QPVLTQSPSVSAALGASAKLTCTLSSAHKTYTIDWYQ
52QQSGEAPRYLMQLKSDGNYTKGTGVPDRFSGSSSGAD
RYLIIPSVQADDEADYYCGADYSGGYVFGGGTQLTVT
SEQ ID NO:VHQSVKESEGGLFKPTDTLTLTCTVSGFSLSNYAINWVRQ
53TBIfabT14APGEGLEWIGYIDPTFGSTYYASWVNDRFTISSHNAQN
TLYLQLNSLTPADTATYFCAR
SEQ ID NO:VLELDMTQTPSSVSAAVGGTVTISCQASQSVYNNNNLSW
54TBIfabT14YQQKPGQPPKLLIYDASKLASGVPSRFKGSGSGTQFTL
TISDLECDNAATYFCQQGYDGSDVDNVFGGGTEVVVK
SEQ ID NO:VH TBIfabP9QSLEESGGGLFKPGGSLTLTCTVSGFTITSYHMCWVRQ
55APGNGLGWIGAVSASGHTYYANWAKSRSTITRDTNLN
TMTLKMTSLTAADTATYFCA
SEQ ID NO:VL TBIfabP9ELVLTQTPPSLSASVGETVRIRCLASEFLFNAVSWYQQ
56KPEKPPTLPIYGASNLESGVPPRFSGSGSGTQFTLTISDL
ECDDAATYYCAGDYSDWIYAFGGGTEVVVK
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFSLSSYGITWVRQ
57TBIfabP12APGNGLEWIGAIGSDAKTYYASWAKGRSTITGDTNLN
TVTLRMTSLTAADTATYFCAR
SEQ ID NO:VLELVLTQTPSSVPAAVGGTVTIDCQSSESVYNNNNLAW
58TBIfabP12YQQKPGQPPKLLIYGASTLASGVSSRFKGSGSGTEFTL
TISDLECADAATYYCQQGYSIGNVDNAFGGGTELEIL
SEQ ID NO:VHQSVKESEGGLFKPTDTLTLTCTVSGFSLSSYGVSWVRQ
59TBIfabP18APGNGLEWIGAISSGGDAYYASWATSRSTITRNTNLNT
VTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELVMTQTPASVEVAVGGTVTIKCQASQSISSYLAWYQ
60TBIfabP18QKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTIS
DVVCDDAATYYCAGYKGGSSDGSAFGGGTELEIL
SEQ ID NO:VHQSVEESGGGLFKPADTLTLTCTVSGFSLSYPGVSWVRQ
61TBIfabP19APGNGLEYIGFINADGDSYYPTWAKRRSTITRNTNLNT
VTLKMTSLTAADTATYFCA
SEQ ID NO:VLQPVLTQSPSASAALGSSAKLTCTLSSAHKTYYIEWYQQ
62TBIfabP19QQGEAPRYLMQVKSDGSYTKGTGVPDRFSGSSSGADR
YLIIPSVQADDEADYYCGADYSGGYVLGGGTQLTVT
SEQ ID NO:VHQSLEESGGGLFKPADTLTLACTVSGFSLSTYGVIWVRQ
63TBIfabT114APGKGLEYIAYINYSGSPYYASWAKSRSTITRNTNEKT
VTLKMTSLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSASAALGSSAKLTCTLSSAHKTYYIDWYQ
64TBIfabT114QQQGEAPRYLMQLGSDGSYTKGTGVPDRFSGSSSGAD
RYLIIPSVQADDEADYYCGSDYSGGYVFGGGTQLTVT
SEQ ID NO:VHSRWRSPGGGLFKPTDTLTLTCTVSGFSLSGYGVSWVR
65TBIfabT116QAPGNGLEWIGAISSGGSAYYARWAKSRSTITRNTNL
NTVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSVSAALGASAKLTCTLSSAHKTYTIDWYQ
66TBIfabT116QQQGEAPRYLMQLKSDGSYTKGTGVPDRFSGSSSGAD
RYLIIPSVQADDEADYYCGADYSGGYVFGGGTQLTVT
SEQ ID NO:VHQSVKESEGGLFKPTDTLTLTCTVSGFSLSNYGVSWVR
67TBIfabT124QAPGNGLEYIGFISNGGATFYATWARSRATITRNTGLN
TVALTMTSLTAADTATYFCVR
SEQ ID NO:VLELDMTQTPPSLSASVGETVRIRCLASEDIGSAISWYQQ
68TBIfabT124KPGKPPTLLIYGVFNLESGVPPRFSGSGSGTDYTLTIGG
VQAEDAATYYCLGGASDSSTGLTFGAGTNVEIK
SEQ ID NO:VHQSVKESEGGLFKPTDTQTLTCTVSGFSLSSNAISWVRQ
69TBIfabT129APGNGLKSIGFINSGGGAYYATWAKSRSTITRNTNENT
VTLKMTSLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSLSASLGTTARLTCTLSTGYSVGEYPLVWL
70TBIfabT129QQVPGRPPRYLLSFTSDEDKHHDSWGPTRFSGSKDTSE
NTFILSISGLQPEDEADYYCATAHGSDNSLHYVFGGRT
QLTVT
SEQ ID NO:VHQSLEESGGGLFKPTDTLTLTCTVSGFALNNYNIHWVR
71TBIfabT134QAPGNGLEWIGAIGSSGSAYYASWAKSRSTITRNTNLN
TVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLQPVLTQSPSASAALGSSAKLTCTLSSAHKTYYIEWYQQ
72TBIfabT134QQGEAPRYLIQLKSDGSYTKGTGVPDRFSGSSSGTDRY
LIISSVQAEDEADYSCGADYSGGFVFGGGTQLTVT
SEQ ID NO:VHVGGGVQGGGLVKPGDTLTLTCTVSGFPLSSYDMNWV
73TBIfabP212RQAPGEGLEWIGWITYDGYNHYASWANGRSTITRNTN
ENAVTLKMTSLTAADTATYFCAR
SEQ ID NO:VLELVLTQTPPSLSASVGGTVTINCLASENVYSAVAWYQ
74TBIfabP212QKPGKPPTLLISGTSNLEAGVPPRFSGSGSGTDYTLTIG
GVQAEDAATYFCQGYSSYPLTFGAGTNVEIK

TABLE 2
TEGS-specific antibody region nucleic acid sequences
CloneNucleic Acid Sequence
SEQ ID NONumber(VL-linker-VH; linker underlined)
SEQ ID NO:TBIfabT203CCCAGCCGGCCATGGCTGAGCTCGTGATGACCCAGA
75CTGAATCGCCCGTGTCTGCAGCTGTGGGAAGCACAG
TCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATA
GTAACAACAACTTAGCCTGGTTTCAGAAGAAACCAG
GGCAGCCTCCCAAGCGCCTGATCCATTCTGCATCCA
CTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCA
GTGGATCTGGGACACAGTTCACTCTCACCATCAGCG
ACCTGGAGTGTGACGATGCTGCCACTTACTACTGTG
CAGGCGTTTTTAGTGGTAGTATTAGTGTTTTCGGCG
GAGGGACCGAGGTGGTCGTCAAGGTGGTTCCTCTAG
ATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGG
TGGGCAGTCGCTGGAGGAGTCCGGGGGAGGTCTCTT
CAAGCCAACGGATACCCTGACACTCACCTGCACAGT
CTCTGGATTCTCCCTCAGTAACTATGGAGTGGTCTG
GGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGA
TCGGAATCATTGATCATCATGGTATCCCATACTACG
CAACCTGGGCGAAAAGCCGATCCACCATCACCAGA
AACACCAACCTGGACACGGTGACTCTGAAAATGAC
CAGTCTGACAGCCGCGGACACGGCCACCTATTTCTG
TGCGAGAGCTTACGTTAATTTTGGCTGGGATTATGC
TCTTAACATCTGGGGTCCAGGCACCCTGGTCACCGT
CTCCTCAGGGCAACCTAAGGCTCCGTCAGTCACTAG
TGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT205CCGGCCATGGCTCAGCCTGTGCTGACTCAGTCGCCC
76TCTGTGTCTGCTGCCCTGGGATCCTCGGCCAAGCTC
ACCTGCACTCTGAGCAGTGCTCACAAGACCTACTAT
ATTGAATGGTATCAGCAACAACAAGGGGAGGCCCC
TCGGTACCTGATGCAACTTGAGAGTGATGGAAGCTA
CACCAAGGGGACCGGGGTCCCTGATCGCTTCTCGGG
CTCCAGCTCTGGGGCTGACCGCTACTTGATCATCTC
CAGCGTCCAGGCTGAGGACGAAGCCGACTACTATTG
TGGTGCAGATTATAGTGGTGGGTTTGTGTTCGGCGG
AGGGACCCAGCTGACCGTCACAGGTGGTGGTTCCTC
TAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGG
TGGTGGGCAGTCGGTGAAGGAGTCCGGGGAGGTCT
CCTCAAGCCAACGGATACCCTGACACTCACCTGCAC
AGTCTCTGGATTCTCCCTCAATAGCTATGCAGTATTC
TGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATG
GATCGGAACCGTTAGTAGTGTTGGTGACACATACTT
CGCGACCTGGGCGAAAAGCCGATCCACCATCACCA
GAAACACCAACCTGAACACGGTGACTCTGAAAATG
ACCAGTCTGACAGCCGCGGACACGGCCACCTATTTT
TGTGCGAGGGGGGTTGGTGTTAGTTATTATCTTGAT
GCTTTTGATTCTTGGGGCCCAGGCACCCTGGTCACC
GTCTCCTCAGGGCAACCAGGCTCCATCAGTCACTAG
TGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT206CCGGCCATGGCTGAGCTCGTGATGACCCAGACTGAA
77CCCCCCGTGTCTGCACCTGTGGGAGGCACAGTCACC
ATCAATTGCCAGGCCAGTCAGAACATTGGTAGTAGC
TACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCT
CCCAAGCTCCTGATCTACCAGGCTTCCACTCTGGCA
TCTGGGGTCCCATCGCGGTTCAAAGGCGGTGGATCT
GGGACAGACTACAGTCTCACCATCAGCGGCGTGCA
GTGTGCCGATGCCGCCACTTATTACTGTCAAAGTAC
TTTTTATAGTAGTGGTACTGGTTATGCTTTCGGCGGA
GGGACCGAGCTGGAGATCCTGGTGGTTCCTCTAGAT
CTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTG
GGCAGTCGGTGGAGGAGTCCGGGGGAGGTCTCTTC
AAGCCAACGGATACCCTGACACTCACCTGCACAGTC
TCTGGATTCTCCCTCAGTGGCTATGGAGTGAGCTGG
GTCCGCCAGGCTGCAGGGAACGGGCTGGAATGGAT
CGGAGCCATTAGTAGTGGTGGTAGCGCATACTACGC
GAGATGGGCGAAAAGCCGATCCACCATCACCAGAA
ACACCAACCTGAACACGGTGACTCTGAAAATGACC
AGTCTGACAGCCGCGGACACGGCCACCTATTTCTGT
GCGAGAGGTTACTATACTGCTATTGGTGGTACTTAT
GACAATGCTTTTGATCCCTGGGGCCCAGGCACCCTG
GTCACCGTCTCCTCAGGGCAACCTAAGGCCATCAGT
CACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT208GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
78GTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGC
CAAGCTCACCTGCACCCTGAGCAGTGGCCACAAGAC
CTACACCATTGACTGGTATCAGCAGCAGCAGCAAGG
GGAGGCCCCTCGGTACCTGATGCAGATTGGGAGTGA
TGGAAGCTACACCAAGGGGACCGGGGTCCCTGATC
GCTTCTCGGGCTCCAGCTCTGGGACTGACCGCTACT
TGATCATCTCCAGCGTCCAGGCTGAGGACGAAGCCG
ACTACTATTGTGGTGCAGATTATAGTGGTGGGTTTG
TGTTCGGCGGAGGGACCCAGCTGACCGTCACAGGTG
GTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGG
CTCGGGCGGTGGTGGGCAGTCGGTGAAGGAGTCCG
AGGGAGGTCTCTTCAAGCCAACGGATACCCTGACAC
TCACCTGCACAGCCTCCGAATTCACCATCGGTAGTT
ATAGTAGTGGCTGGGTCCGCCAGGCTCCAGGGAAG
GAGCTGGAGTGGATCGGAACCCTTAGTTCTACTGGT
AGCGCACACTACGCGAACTGGGCGAAAGGCCGTTC
CACCATCACCAGAAACACCAACGAGAACACGGTGA
CTCTGAAGATGGCCAGTCTGACAGCCGCGGACACG
GCCACCTATTTCTGTGCGAGAGCTGATTATGGGCCC
TGTTATTTTGACATCTGGGGCCCAGGCACCCTGGNC
ACCGTTTTCTCNGGNAACCTNANNCTCCATCAGTCA
CTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT209CCGGCCATGGCTGAGCTCGTGATGACCCAGACTCCA
79TCCTCTGTGTCTGCAGCTGTGGGAGGCACAGTCACC
ATCAATTGCCAGGCCAGTCAGACTATTAACAACCTC
TTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCC
AAGCTCCTGATTTATGGTGCATCCACTCTGGCATCT
GGGGTCCCATCGCGTTTCAGCGGCAGTGGATCTGGG
ACACAGTTCATTCTCACCATCAGTGGCATGAAGGCT
GAAGATGCTGCCACTTATTACTGTCAAAGTGCTTAT
TATAATGCTGGTGCGACTTTTGGAGCTGGCACCAAT
GTGGAAATCAAGGTGGTTCCTCTAGATCTTCCTCCT
CTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGTCG
GTGGAGGAGTCCGGGGGAGGTCTCTTCAAGCCAAC
GGATACCCTGACACTCACCTGCACAGTCTCTGGAAT
CGACCTCAGTAGAAATGGAGTGACCTGGGTCCGCCA
GGCTCCAGGGAGCGGGCTGGAATGGATCGGAGTCA
TTAATAGTCATGGTGACAGTGATTACGCGACCTGGG
CGAACAGCCGATCCACCATCACCAGAAACACCAAC
CTGAACACGGTGACTCTGAAAATGACCAGTCTGACA
GCCGCGGACACGGCCACCTATTTCTGTGCGAGTACT
TATGATAGTTATTATGATTATGCTTGGCCTAATTTTG
GCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCT
CAGGGCAACCTAAGGCCAGTCACTAGTGGCCCGGG
AGGCCA
SEQ ID NO:TBIfabT210GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
80GTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGC
CAAGTTCACCTGCACCCTGAGCAGTGGCCACAAGAC
CTACACCATTGACTGGTATCAGCAGCAGCAGCAAGG
GGAGGCCCCTCGGTACCTGATGCAGATTGGGAGTGA
TGGAAGCTACACCAAGGGGACCGGGGTCCCTGATC
GCTTCTCGGGCTCCAGCTCTGGGACTGACCGCTACT
TGATCATCTCCAGCGTCCAGGCTGAGGACGAAGCTG
ACTACATCTGTGGTGTAACTGGTAGTAATGTTTATG
CACAGGACCCAGCTGACCGTCACAGGTGGTGGTTCC
TCTAGATCTTCCCCTGGTGGCGGTGGCCGGGCGGTG
GTGGGCAGTCGCTGGAGGAGTCCGGGGGAGGTCTC
TTCAAGCCAACGGATACCCTGACACTCACCTGCACA
GTCTCTGGATTCTCCCTCAGTAACAGTGCAATGAGC
TGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATG
GATCGGAGACATTGATAGTAGTGGTAGCGCATACTA
CGCGAGCTGGGCGAAAAGCCGATCCACCATCACCA
GAAACACCAACCTGAACACGGTGACTCTGAAAATG
ACCAGTCTGACAGCCGCGGACACGGCCACCTATTTC
TGTGCGAGAGGGGGTTATGGTAATGCTGGTACTCCT
TACTATGGCATGGACCTCTGGGGCCCAGGGACCCTC
GTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCA
GTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT216CCGGCCATGGCTGAGCTCGTGATGACCCAGACTCCA
81TCCTCTGTGTCTGCAGCTGTGGGAGGCACAGTCACC
ATCAATTGCCAGGCCAGTGAAAGCATTAGCAACTAC
TTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCC
AAGCTCCTGACCTATGATGCATCTGATCTGGCATCT
GGGGTCCCATCGCGGTTCAGCGGCAGTGGATATGGG
ACAGAGTTCACTCTCACCATTAGTGGCGTGAAGGCT
GAAGATGCTGCCACTTATTATTGTCAAAGTGGTTAT
GTTAGTGCTGGGACTTTTGGAGCTGGCACCAATGTG
GAAATCAAGGTGGTTCCTCTAGATCTTCCTCCTCTG
GTGGGGCGGTGGTGGGCAGTCGTTGGAGGAGTCCG
GGGGAGGTCTCTTCAAGCCAACGGATACCCTGACAC
TCACCTGCACAGTCTCTGGATTCGACATTAGTGGCG
TTTACATGAGCTGGGTCCGCCAGGCTCCAGGGAACG
GGCTGGAGTGGATCGGAGCCATTGATCGTGGTGGTG
GCACTTACTACGCGAGCTGGGCGATAGGCCGATCCA
CCATCACCAGAAACACCAACGACAACACGGTGACT
CTGGAAATGACCAGTCTGACAGCCGCGGACACGGC
CACCTATTTCTGTGCGAAAGGATATAGTGTTCTTGA
TCCCTGGGGCCCAGGCACCCTGGNCACCGTCTCCTC
AGGGCAACCTAAGGCTCCATCAGTCACTAGTGGCCC
GGGAGGCCA
SEQ ID NO:TBIfabT222GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
82GTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGC
CAAGCTCACCTGCACCCTGAGCAGTGCCCACAAGAC
CTACACCATTGACTGGTATCAGCAGCAGCAAGGGG
AGGCCCCTCGATACCTGATGCATCTTAAGAGTGATG
GAACCTACACCAAGGGGACCGGGGTCCCTGATCGCT
TCTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGA
TCATCCCCAGCGTCCGAACTGATGACGAAGCCGACT
ACTATTGTGGTACAGATTACAGCGGTGGGTATGTAT
TCGGCGGAGGGACCCAGCTGACCGTCACAGGTGGT
GGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCT
CGGGCGGTGGTGGGCAGTCGCTGGAGGAGTCCGGG
GGAGGCCTGATCAAGCCAACGGATATGTTGACACTC
ACCTGCACAGTCTCTGGATTCTCCCTCAGTAACTAT
GGAGTGATGTGGGTCCGCCAGGCTCCAGGGAACGG
ACTGGAGTCGATCGGATATATTGGTAGTGGTGGTGA
CACATCCTACGCGAGCTGGGCGAAAAGCCGATCCA
CCATCGCCAGAAACACCAACGAGAACACGGTGTCT
CTGCTCATGAATGGTCTGACAGCCGCGGACACGGCC
ACCTATTTCTGTGCGAGAGATCCTGGTTATAGTGCT
GGTAGTGCTTTTGATCCCTGGGGCCCAGGCACCCTG
GTCACCGTCTTCTCAGGGCAACCTAAGGCTCCATCA
GTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT226GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
83GTCGCCCTCTGCATCTGCTGCCCTGGGATCCTCGGC
CAAGCTCACCTGCACTCTGAGCAGTGCTCACAAGAC
CTACTATATTGACTGGTATCAGCAGCAGCAAGGGGA
GGCCCCTCGGTACCTGATGCAGGTTAAGAGTGATGG
AAGCTACACCAGGGGGACCGGGGTCCCTGATCGCTT
CTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGAT
CATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTA
CTATTGTGGTTCAGATTATAGCGGTGGGTATGTGTT
CGGCGGAGGGACCCAGCTGACCGTCACAGGTGGTG
GTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTC
GGGCGGTGGTGGGCAGTCGTTGGAGGAGTCCGGGG
GAGGTCTCTTCAAGCCAACGGATACCCTGACACTCA
CCTGCACAGTCTCTGGATTCTCCCTCAGTATCTATGG
AGTGAGCTGGGTCCGCCAGGCTCCGGGGAATGGGC
TGGAATGGGTCGGAGCCATTGGTAGTGGTGGTAGCG
CATACTACGCGACCTGGGCGAAAAGCCGATCCACC
ATCACCAGAAACACCAACCTGAACACGGTGACTCTG
AAAATGGCCAGTCTGACAGCCGCGGACACGGCCAC
CTATTTCTGTGCGAGAGGTTACTATACTGCTATTGGT
GGTACTTATGACAATGCTTTTGATCCCTGGGGCCCA
GGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAG
TCCATCAGTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT229CCGGCCATGGCTGAGCTCGATCTGACCCAGACTCCA
84TCCTCTGTGTCTGCAGCTGTGGGAGGCACAGTCACC
ATCAATTGCCAGGCCAGTCAGAGTGTTAGCAACCTC
TTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCC
AAGCTCCTGATTTATGGTGCATCCAATCTGGAATCT
GGGGTCCCATCGCGTTTCCGTGGCAGTGGATCTGGG
ACAGAGTTCACTCTCACCATCAGCGATGTGGTGTGT
GACGATGCTGCCACTTACTACTGTGCAGGACATAAA
AGTAGTAGTACTGATGGTACTGCTTTCGGCGGAGGG
ACCGAGCTGGAGATCCTGGTGGTTCCTCTAGATCTT
CCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGC
AGTCGGTGGAGGAGTCCAGGGGAGGTCTCTTCAAG
CCAACGGATACCCTGACACTCACCTGTACAGTCTCT
GGATTCTCCCTTAGTACCTACAACATACAATGGGTC
CGCCAGGCTCCAGGGAACGGGCTGGAATATATCGG
AACCATTGGTAGTAGTGGTAGCGCATACTACGCGAG
CCGGGCGAAAAGCCGATCCACCATCACCAGAAACA
CCGCCCTGAACACGGTGTCTCTGCAAGTGGACAGTC
TGACAGACGCGGACACGGCCACCTATTTCTGTGCGA
GAGGAGGGACTTGGTATACAGATGGTCTTGCTTATG
TTGATGCTTTTGATCTCTGGGGCCCAGGCACCCTGG
NCACCGCCTNCTCNGGCAACCTAGTCACTAGTGGNC
CGGGAGGCCA
SEQ ID NO:TBIfabT232GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
85GTCGCCCTCTGCATCTGCTGCCCTGGGATCCTCGGC
CAAGCTCACCTGCACTCTGAGCAGTGCTCACAAGAC
CTACTATATTGACTGGTATCAGCAGCAGCAAGGGGA
GGCCCCTCGGTATCTGATGCAGGTTAAGAGTGATGG
AAGCTACACCAAGGGGACCGGGGTCCCTGATCGCTT
CTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGAT
CATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTA
CTATTGTGGTTCAGATTATAGCGGTGGGTATGTGTT
CGGCGGAGGGGCCCAGCTGACCGTCACAGGTGGTG
GTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTC
GGGCGGTGGTGGGCAGTCGGTGAAGGAGTCCGAGG
GAGGTCTCTTCAAGCCAACGGATACCCTGACACTCA
CCTGCACAGTCTCTGGATTCACCGTCAGTAACAATG
CAATAAGCTGGGTCCGCCAGGCTCCAGGGAATGGG
CTGGAATGGATCGGAGCCATTAGTTACGGTGGTAAC
ACATACTACGCGAACTGGCCGAAAAGCCGATCCAC
CATCACCAGAAACACCAACCTGAACACGGTGACTCT
GAAAATGACCAGTCTGACAGCCGCGGACACGGCCA
CCTATTTCTGTGCGAGATTCTACTATGGTGCTGGTTA
TGCCTATGACATCTGGGGCCCAGGCACCCTGGTCAC
CGTCTTCTCAGGGCAACCTAAGGCTCCGTCAGTCAC
TAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT234CCGGCCATGGCTGAGCTCGATATGACCCAGACTCCA
86TCCTCTGTGTCTGCAGCTGTGGGAGACACAGTCACC
ATCAATTGTCAGGCCAGTCAGAGTGTTACCAACCTC
TTAGCCTGGTATCAGCAGAAACCAAGGCAGCCTCCC
AAACTCCTGATTTATGATGCATCCAATCTAGAATCT
GGAGTCCCATCGCGTTTCCGTGGCAGTGGATCTGGG
ACAGAGTTCACTCTCACCATCAGTGGCATGAAGGCT
GAAGATGCTGCCACTTATTACTGTCAAAGTGGTTAT
TATAGTGCTGGTGCGACTTTTGGAGCTGGCACCAAT
GTGGAAATCAAGGTGGTTCCTCTAGATCTTCCTCCT
CTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGTCG
GTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAAC
GGATACCCTGACACTCACCTGCACAGTCTCTGGATT
CTCCCTCAGTAACTATGGAGTGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGAGGTGGAGTGGATCGGATACA
TTAACAGTGGTGGTAGTACTAATTACGCGAGCTGGG
CGAAAAGCCGATCCACCATCACCAGAAACACCAAT
TTGAACACGGTGACTCTGAAAATGACCAGCCTGACA
GCCGCGGACACGGCCACCTATTTCTGTGCGAGAGGT
TACCGTGGTTATAATGTTGGTATGGATGCTTTTGATG
TCTGGGGCCCANGCAATCTGGTCACCGTCTCCTCAG
GNAACCTANNCTCTCAGTCACTAGTGGCCCGGGAGG
CCA
SEQ ID NO:TBIfabT236CCGGCCATGGCTGAGCTCGTGCTGACCCAGACTCCA
87TCCCCAGTGTCTGCGGCTGTTGGAGGCACAGTCACC
ATCAATTGCCAGTCCAGTCAGAGTGTTTATAGTAAC
AACCGCTTAGCCTGGTATCAGCAGAAACCAGGGCA
GCCTCCCAAGCAACTGATCTATTATGCATCCACTCT
GGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGG
ATCTGGGACACAGTTCACTCTCACCATCAGCGATGT
GGTGTGTGACGATGCTGCCACTTACTACTGTGCAGG
ATATAAAAATAGTGGTATTGATGAACATGCTTTCGG
CGGAGGGACCGAGCTGGAGATCCTGGTGGTTCCTCT
AGACTTCTCCTCTGGTGGCGGTGGCTCGGGCGGTGG
TGGGCAGTCGGTGAAGGAGTCCGAGGGAGGTCTCTT
CAAGCCAATGGATAGCATGACACTCACCTGCACAGT
CTCTGGATTCTCCCTCAGTAGCTATGGAGTGAGCTG
GGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGA
TCGGAGCCATTAGTAGTGGTGGTAGCGCATACTACG
CGAGATGGGCGAAAAGCCGAGCCACCATCACCAGA
AACACCAACCTGAACACGGTGACTCTGAAAATGGC
CAGTCTGACAGCCGCGGACACGGCCACCTATTTCTG
TGCGAGAGGTTACTATACTGCTATTGGTGGTACTTA
TGACAATGCTTTTGATCCCTGGGGCCCAGGCACCCT
GGTCACCGTCTCCTCAGGGCAACCTAACAGTCACTA
GTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT239GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
88GTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGC
CAAGCTCACCTGCACCCTGAGCAGTGGCCACAAGAC
CTACACCATTGACTGGTATCAGCAGCAGCAAGGGG
AGGCCCCTCGGTACCTGATGCAGCTTGGGAGTGATG
GAAGCTACACCAAGCAGACCGGGGTCCCTGATCGCT
TCTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGA
TCATCTCCAGCGTCCAGGCTGATGACGAAGCCGACT
ACTATTGTGGTGCGGATTATAGTGGTGGGTTTGTGT
TCGGCGGAGGGACCCAGCTGACCGTCACAGGTGGT
GGCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTC
GGGCGGTGGTGGGCAGTCGTTGGAGGAGTCCGGGG
GAGGTCTCTTCAAGCCAACGGATACCCTGACACTCA
CCTGCACAGTCTCTGGATTCTCCCTCAACAGGTACG
ACATGAGTTGGGTCCGCCAGGCTCCAGGGAACGGG
CTGGAATGGATCGGAGTCATTAATAGTGGTGGGTTC
ACATACTACGCGAGCTGGGCGAAAAGCCGATCCAC
CATCACCAGAAACACCAACGAGAACACGGTGACTC
TGAAAATGACCAGTCTGACAGCCGCGGACACGGCC
ACCTATTTCTGTGCGAGAGGTTACCGTGGTTATAAT
GTTGGTATGGATGCTTTTGATGTCTGGGGCCCAGGC
AATCTGGTCACCGTCTCCTCAGGGCAACCTAAGTCC
GTCAGTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT240GGCCCAGCCGGCCATGGCTGAGCTCGTGCTGACCCA
89GACTCCATCGTCCGTGTCTGCAGCTGTGGGAGGCAC
AGTCAGCATCAGTTGCCAGTCCAGTCAGAGTGTTTA
TAGTAACTACTTAGCCTGGTATCAGCAGAAACCAGG
GCAGCCTCCCAAGCTCCTGATCTATTATGCATCCAC
TCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAG
TGGATCTGGGACACAGTTCACTCTCACCATCAACGG
CGTGCAGTGTGACGATGCTGCCACTTACTACTGTCA
AGGCACTTTTGATGATGGTTTGTACAAGGCTTTCGG
CGGAGGGACCGAGCTGGAGATCCTGGTGGTTCCTCT
AGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGT
GTGGGCAGTCGGTGAAGGAGTCCGAGGGAGGTCTC
TTCAAGCCAACGGATACCCTGACACTCACCTGCACA
GTCTCTGGATTCTCCCTCAGTAACAATGCAATAAAC
TGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAGTG
GATCGGAGCCGTTGGTAGTGGTGGTAGGGCATACTA
CGCGGGCTGGGCGAAAAGCCGATCCACCATCACCA
GAAACACCAACCTGAACACGGTGACTCTGAAAATG
ACGAATCTGACAGCCGCGGACACGGCCACCTATTTC
TGTGCGAGAGTCGGATTTTACTATGGCAATGGTCTT
TCTTATGGTATTGGCGCTTTTGATCCCTGGGGCCCAG
GCACCCTGGTCACCGTCTCCTCAGGGCAACCAGGCT
CCGTCAGTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT241CCGGCCATGGCTGAGCTCGTGATGACCCAGACTCCA
90TCCTCTGTGTCTGCAGCTGTGGGAGGCACAGTCACC
ATCAATTGCCAGGCCAGTCAGAGTGTTTACAACCTC
TTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCC
AAGCTCCTGACTCATGGTACATCCAATCTGGAATCT
GGGGTCCCATCGCGTTTCCGTGGCAGTGGATCTGGG
ACAGAGTTCACTCTCACCATCAGTGGCATGAAGGCT
GAAGATGCTGCCACTTATTACTGTCAAAGTGGTTAT
TATAGTACTGGTGCGACTTTTGGAGCTGGCACCAAT
GTGGAAATCAAGGTGGTTCCTCTAGATCTTCCTCCT
CCGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGTCG
CTGGAGGAGTCCGGGGGAGGTCTCTTCAAGCCAAC
GGATACCCTGACACTCACCTGCACAGTCTCTGGATT
CTCCCTCAGTAACTATGGAATGGGCTGGGTCCGCCA
GGCTCCAGGGAACGGGCTGGAATACATCGGATTCAT
TAGTAGTGGTGGTAATACATACTACGCGAGCTGGGC
GAAAAGCCGATCCACCATCACCAGAGACACCAACC
TGAACACGGTGACTCTGAAAATGAGCAGTCTGACA
GCCGCGGACACGGCCACCTATTTCTGTGCGAGAGGT
TACCGTGGTTATAATGTTGGTATGGATGCTTTTGATG
TCTGGGGCCCAGGCAATCTGGTCACCGTCTCCTCAG
GGCAACAAGGCTCCATCAGTCACTAGTGGCCCGGG
AGGCCA
SEQ ID NO:TBIfabT247CCGGCCATGGCTGAGCTCGATCTGACCCAGACTCCA
91TCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACC
ATCAATTGCCAGTCCAGTCAGAGTGTTGATAGTAAC
AATTACTTATCCTGGTATCAGCAGAAACCAGGGCAG
CCTCCCAAGCTCCTGATCTATGATGCATCCACTCTG
GCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGA
TCTGGGACACAGTTCACTCTCACCATCAGCGAAGTA
CAGTGTGACGATGCTGCCACTTACTACTGTCAAGGC
AGTTATTATAGTGGTGATTGGTATGGGGCTTTCGGC
GGAGGGACCGAGCTGGAGATCCTGGTGGTTCCTCTA
GATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTG
GTGGGCAGTCGTTGGAGGAGTCCGGGGGAGGTCTCT
TCAAGCCAACGGATCCCCTGACACTCACCTGCACAG
TCTCTGGATTCTCCATCAATGACTACAACATGCAAT
GGGTCCGCCAGGCTCCAGGGATCGGGCTGGAATGG
ATCGGAGCCATTAATGCTTGGGGTGATACATACTAT
ACGAGCTGGGCGAAAAGCCGATCCACCATCACCAG
AGACACCAACCTGAACACGGTGACTCTGAAAATGA
CCAGTCTGACAGCCGCGGACACGGCCACCTATTTCT
GTGCNAGAGGTTACAGTCTTGACATCTGGGGCCCAG
GCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGG
CTCCGTCAGTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT249CCCAGCCGGCCATGGCTGAGCTCGTGATGACCCAGA
92CTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAG
TCACCATCAGTTGCCAGGCCAGTCAGGGTGTTTATA
GCGACCGCCTAGCCTGGTATCAACAGAAACCAGGG
CAGCCTCCCAAGCTCCTGATGTATTATGCATCCGAT
CTGTCATCTGGGGTCCCATCGCGGTTCAAAGGCAGT
GGATCTGGGACAGAGTTCACTCTCACCATCAGCGAC
CTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAA
AGCAATTATGGTAGTCTTAGTAGTAGTTATACTTTC
GGCGGAGGGACCGAGGTGGTCGTCAAGGTGGTTCC
TCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGC
GGTGGTGGGCAGTCGTTGGAGGAGTCCGGGGGAGG
TCTCTTCAAGCCAACGGATACCCTGACACTCACCTG
CACAGCCTCCGGATTCACCGTCACTAGCAACGCAAT
AAGCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGG
AATATATCGGATTCATTGGTGCTGCTGGTAATGCAA
ACTACGCGAGCTGGGCGAAAAGCCGCTCCACCATC
ACCAGAAACACCAACCTGAACACGGTGACTCTGAA
AATGACCAGTCTGACAGCCGCGGACACGGCCACCT
ATTTCTGTGCGAGAGAGGGTGGTTGGGGTACATTGT
TTGGTGCTTTTGATTCCTGGGGCCCAGGCACCCTGG
TCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAG
TCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabP202GGCCCAGCCGGCCATGGCTGAGCTCGTGATGACCCA
93GACTCCACCCTCCCTGTCTGCATCTGTGGGAGAAAC
TGTCAGGATTAGGTGCCTGGCGAGTGAGAACGTTTA
CAGTGCTGTAGCCTGGTATCAACAGAAGCCAGGGA
AACCTCCTACACTCCTGATCTCTGGTGCATCCAATTT
AGAATCTGGGGTCCCACCACGGTTCAGTGGCAGTGG
ATCTGGGACAGATTACACCCTCACCATCGGCGGCGT
GCAGGCTGAAGATGCTGCCACTTACTTCTGTCAAGG
GTATAGCAGTTACCTGACTTTTGGAGCTGGCACCAA
TGTGGAAATCAAGGTGGTTCCTCTAGATCTTCCTCCT
CTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGTCG
GTGGAGGAGTCCAGGGGAGGTCTCTTCAAGCCAAC
GGATACCCTGACACTCACCTGCACAGTCTCTGGATT
TACCATCGATACCTATGGAGTGACCTGGGTCCGCCA
GGCTCCAGGGAACGGGCTGGAATATATCGGATTCAT
TAGTAGTGGTGGTGCCGCATACTACGCGAGCTGGGC
GAAAAGCCGATCCACCATCACCAGAAACACCAATC
TGAACACGGTGACTCTGAAAATGACCAGTCTGACAG
CCGCGGACACGGCCACCTATTTCTGTGCGAGAGATC
GTGGCTATGTTTATGGTTATGGTGATGGTACTGACA
TCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAG
GGCAACCTAAGGCTCCATCAGTCACTAGTGGCCCGG
GAGGCCA
SEQ ID NO:TBIfabP210CCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCAGT
94CGCCCTCTGCGTCTGCTGCCCTGAGATCCTCGGCCA
AGCTCACCTGCACCCTGAGCAGTGCCCACAAGAGTT
ACGACATTGACTGGTATCAGCAGCAGTCAGGGGAG
GCCCCTCGGTACCTAATGCGTCTTAGGAGTGATGGA
AAGTACACCAAGGGGACCGGGGTCCCTGATCGCTTC
TCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGATC
ATCCCCAGCGTCCAGGCTGATGACGGAGCCGACTAT
TATTGTGGTACAGATTATAGCGGTGGATATGTGTTC
GGCGGAGGGACCCAGCTGACCGTCACAGGTGGTGG
TTCCTCTAGATCTTCCTCCTGGTGGCGGTGGCTCGGG
CGGTGGTGGGCAGTCGCTGGAGGAGTCCGGGGGAG
GCCTGATCAAGCCAACGGATACCCTGACACTCACCT
GCACAGTCTCTGGATTCTCCCTCAGTATCTACGACA
TAAGCTGGGTCCGCCAGGCTCCAGGGAACGGGCTG
GAATGGATCGGAGCCATTGGTAGTGGTGATACCACA
TACTACGCGAGCTGGGCGAAAAGCCGATCCACCATC
ACCAGAAACACCTACCTGAACACGGTGACTCTGAA
AATGACCAGTCTGACAGCCGCGGACACGGCCACCT
ATTTCTGTGCGAGAGGGGGGTTATCCTGGTTCAACA
TCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAG
GGCAACCTAAGGCTCCGTCAGTCACTAGTGGCCCGG
GAGGCCA
SEQ ID NO:TBIfabP214CCGGCCATGGCTGAGCTCGTGATGACCCAGACTGAA
95TCCCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACC
ATCAATTGCCAGGCCAGTCAGAGCATTAGCAGTTGG
TTGGCCTGGTATCAACAGAAGCCAGGGAAACCTCCT
ACACTCCTGATCTCTGGTGCATCCAATTTAGAATCT
GGGGTCCCACCACGGTTCAGTGGCAGTGGATCTGGG
ACAGATTACACCCTCACCATTGGCGGCGTGCAGGCT
GAAGATGCTGCCACCTACTATTGTCTAGGCGGTTAT
AGTTACAGTAGTATCGGTACGACTTTTGGAGCTGGC
ACCAATGTGGAAATCAAGGTGGTTCCTCTAGATCTT
CCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGC
AGGAGCAGCTGGTGGAGTCCGAGGGAGGTCTCTTC
AAGCCAACGGATACCCTGACACTCACCTGCACAGTG
TCTGGATTCTCCCTCAATAACTATGGAGTGACCTGG
GTCCGCCAGGCTCCAGGGAGGGGGCTGGAATGGAT
CGGAGCCGTTTGGAGTGGTGCTACCACAGACTATGC
GAGCTGGGCGAAAAGCCGATCCACCATCACCAGAA
ACACCAACGAGAACACGGTGACTCTGAAAATGTCC
AGTCTGACAGCCGCGGACACGGCCACCTATTTCTGT
GCGAATTTTCCTGGTTATACTTCTGGTACCGACATCT
GGGGCCCTGGCACCCTGGTCACCGTCTCCTCAGGGC
AACCTAAGGCTCCGTCAGTCACTAGTGGCCCGGGAG
GCCA
SEQ ID NO:TBIfabP217CCGGCCATGGCTGAGCTCGTGATGACCCAGACTCCA
96TCCTCTGTGTCTGCAGCTGTGGGAGGCACAGTCACC
ATCAATTGTCAGGCCAGTCAGAGTGTTAACAACCTC
TTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCC
AAGCTCCTGACTTATGGTACATCCAATCTGGAATCT
GGGGTCCCATCGCGTTTCCGTGGCAGTGGATCTGGG
ACAGAGTTCACTCTCACCATCAGTGGCATGAAGGCT
GAAGATGCTGCCACTTATTACTGTCAAAGTGGTTAT
TATAGTGCTGGTTTGACTTTTGGAGCTGGCACCAAT
GTGGAAATCAAGGTGGTTCCTCTAGATCTTCCTCCT
CTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGAG
CAGCTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCC
TGGGGGATCCCTGAAACTCTCCTGCAAAGCCTCTGG
ATTCGACTTCATTAACTATGGAGTAATCTGGGTCCG
CCAGGCTCCTGGGAAGGGGCTGGAGTGGATCGGAT
ACATTGATCCTATTTTTGGTAACACAATCTACGCGA
GCTGGGTGAATGACCGATTCACCATCTCCAGCCACA
ACGCCCAGAACACGCTGTATCTGCAACTGAACAGTC
TGACAGCCGCGGACACGGCCACCTATTTCTGTGCGA
GATGCGGGGATAGACGTGGCTGGGGTTATGGTTTTG
ATCCCTGGGGCCCAGGCACCCTGGTCACCGTCTCCT
CAGGGCAACCTAAGGCTCCGTCAGTCACTAGTGGCC
CGGGAGGCCA
SEQ ID NO:TBIfabP219CCGGCCATGGCTGAGCTCGTGCTGACCCAGACTCCA
97GCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACC
ATCAAGTGCCAGGCCAGTCAGAACATTTACAGTGGT
ATATCCTGGTACCAACAGAAGCCAGAGAAACCTCCT
ACACTCCTGATCTCTGGTGCATCCAATTTAGAACCT
GGGGTCCCACCACGGTTCAGTGGCAGTGGATCTGGG
ACAGATTACACCCTCACCATTGGCGGCGTGCAGGCT
GGAGATGCTGCCACCTACTACTGTCTAGGCGTTTAT
AGTTTCGGTAGTACCGATTTGACTTTTGGAGCTGGC
ACCAATGTGGAAATCAAGGTGGTTCCTCTAGATCTT
CCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGC
AGTCGCTGGAGGAGTCCGGGGGAGGCCTCATCAAG
CCAACGGATACCCTGACACTCACCTGCACGGTCTCT
GGATTCTCCCTCAGTACCAATGGAGTGAGTTGGGTC
CGCCAGGCTCCAGGGAGCGGGCTGGAATGGATCGG
AGCCATTGATCTTTATGGTGCCACATATTACGCGAC
CTGGGCGAAAAGCCGATCCACCATCACCAGAAACA
CCAACCTAAACACGGTGACTCTGAAAATGACCAGTC
TGACAGCCGCGGACACGGCCACCTATTTCTGTGCGA
GAGAGGGATATGGTTATCCAAACATCTGGGGCCCA
GGCACCCTGGTCACCGTCTCCTCNGGCAACCTNANN
NTCCGTCAGTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabP220GGCCCAGCCGGCCATGGCTGAGCTCGATCTGACCCA
98GACTCCATCGTCCGTGTCTGCAGCTGTGGGAGGCAC
AGTCAGCATCAGTTGCCAGTCCAGTCAGAGTGTTTA
TAATAACTACTTAGCCTGGTATCAGCAGAAACCAGG
GCAGCCTCCCAAGCTCCTGATCTATTATGCATCCAA
ACTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAG
TGGATCTGGGACACAGTTCACTCTCACCATCAGCGA
CGTGCAGTGTGACGATGCTGCCACTTACTACTGTCA
AGGCACTTTTGATAATGGTTTGTACAAGGCTTTCGG
CGGAGGGACCGAGCTGGAGATCCTGGTGGTTCCTCT
AGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGT
GGTGGGCAGTCGTTGGAGGAGTCCGGGGGAGGTCT
CTTCAAGCCAACGGATACCCTGACACTCACCTGCAC
AGTCTCTGGATTCTCCCTCAGTAGCTATGCAATAAG
CTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAAT
GGATCGGATACATTAATTACGATGGTATCGCATACT
ACGCGAGCTGGGCGAAAAGCCGATCCACCATCACC
AGAAACACCAACCTGAACACGGTGACTCTGAAAAT
GACCGGTCTGACAGCCGCGGACACGGCCACCTATTT
CTGTGCGAGAGATGACTATACTACTCCTTATGGTTA
TCGATTGGNTCTCTGGGGCCAGGGCACCCTGGTCAC
CGTCTCCTCAGGGCAACCTAAGGCTCCGTCAGTCAC
TAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabP221GGCCCAGCCGGCCATGGCTGAGCTCGATATGACCCA
99GACTCCATCCTCTGTGTCTGCAGCTGTGGGAGGCAC
AGTCACCATCAATTGCCAGGCCAGTGAAAGCATTAG
CAACCTCTTAGCCTGGTATCAGCAGAAACCAGGGCA
GCCTCCCAAGCTCCTGATCTATTCTGCATCCACTCTG
GCATCTGGGGTCCCATCGCGTTTCCGTGGCAGTGGA
TCTGGGACAGAGTTCACTCTCACCATCAGTGGCATG
AAGGCTGAAGATGCTGCCACTTATTACTGTCAAAGT
GGTTATTATAGTACTGGTGCGACTTTTGGAGCTGGC
ACCAATGTGGAAATCAAGGTGGTTCCTCTAGATCTT
CCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGC
AGTCGCTGGAGGAGTCCGGAGGAGGCCTGGTCCAG
CCGGGGGGATCCCTGAAACTCTCCTGCAAAGGCTCT
GGGTTCGACCTCGATAGCAATGCAATGTGCTGGGTC
CGCCAGGCTCCAGGGAGCGGGCTGGAATGGATCGG
AACCATTACTAGTGGTGGTAGCGCATACTACGCGAG
CTGGGCGAAAAGCCGATCCACCATCACCAGAAACA
CCAACCTAAACACGGTGACTCTGAAAATGACCAGTC
TGACAGCCGCGGACACGGCCACCTATTTCTGTGCGA
GAGGACCTACTTGGGGAATTGGAGATACTTTTCATC
CCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCGG
GGCAACCTAAGGCTCCGTCAGTCACTAGTGGCCCGG
GAGGCCA
SEQ ID NO:TBIfabT7GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
100GTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGC
CAAGCTCACCTGCACCCTGAGCAGTGCCCACAAGAC
CTACACCATTGACTGGTATCAACAGCAGTCAGGGGA
GGCCCCTCGATACCTGATGCAGCTTAAGAGTGATGG
AAACTACACCAAGGGGACCGGGGTCCCTGATCGCTT
CTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGAT
CATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTA
CTATTGTGGTGCAGATTATAGCGGTGGGTATGTGTT
TGGCGGAGGGACCCAGCTGACCGTCACAGGTGGTG
GTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTC
GGGCGGTGGTGGGCAGGAGCAGCTGGAGGAGTCCG
GGGGTCGCCTGGTCAAGCCTGACGAAACCCTGACAC
TCACCTGCACAGTCTCTGGACTCTCCCTGAATAATTT
TGGAGTGAGCTGGGTCCGCCAGGCCCCAGGAAACG
GGCTGGAATGGATCAGAGCCATTGATTTTGGTAGTG
GTAGCGCATACTACGCGAACTGGGCGAAAAGTCGG
TCCACCATCACCAGCAACACTCGCCTGAACACGGTG
ACTCTGAAAATGATTAGTCTGACAGCCGCGGACACG
GCCACCTATTTTTGTTCGAGAGGAGACATCTGGGGC
CCAGGCACCCTGGTCACCGTCTTCTCAGGGCAACCT
AAGGCTCCGTCAGTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT14GCCCAGCCGGCCATGGCTGAGCTCGATATGACCCAG
102ACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACA
GTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTTAT
AATAACAACAATTTATCCTGGTATCAGCAAAAACCA
GGGCAGCCTCCCAAGCTCTTGATCTACGATGCATCC
AAATTGGCATCTGGGGTCCCATCGCGGTTCAAAGGC
AGTGGATCTGGGACACAGTTCACTCTCACCATTAGC
GACCTGGAGTGTGACAATGCTGCCACTTACTTCTGT
CAACAGGGTTATGATGGTAGTGATGTTGATAATGTT
TTCGGCGGAGGGACCGAGGTGGTGGTCAAGGTGGT
TCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGG
GCGGTGGTGGGCAGTCGGTGAAGGAGTCCGAGGGA
GGTCTCTTCAAGCCAACGGATACCCTGACACTCACC
TGCACAGTCTCTGGATTCTCCCTCAGTAACTATGCA
ATAAACTGGGTCCGCCAGGCTCCAGGGGAGGGGCT
GGAGTGGATCGGCTATATTGATCCTACTTTTGGTAG
CACGTACTACGCGAGCTGGGTGAATGACCGATTCAC
CATCTCCAGCCACAACGCCCAGAACACGCTGTATCT
GCAACTGAACAGTCTGACACCTGCGGACACGGCCA
CCTATTTCTGTGCGAGAGATGATATTAGTATTAGTG
GTTATTCATTTGACATCTGGGGCCCAGGCACCCTGG
TCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAG
TCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabP9GGCCCAGCCGGCCATGGCTGAGCTCGTGCTGACCCA
103GACTCCACCCTCCCTATCTGCATCTGTGGGAGAAAC
TGTCAGGATTAGGTGCCTGGCCAGTGAGTTCCTTTTT
AATGCTGTATCCTGGTATCAACAGAAGCCAGAGAA
ACCTCCTACACTCCCGATCTATGGTGCATCCAATTTA
GAATCTGGGGTCCCACCACGGTTCAGTGGCAGTGGA
TCTGGGACACAGTTCACTCTCACCATCAGCGACCTG
GAGTGTGACGATGCTGCCACTTACTACTGTGCAGGC
GATTATAGTGATTGGATTTATGCTTTTGGCGGGGGG
ACCGAGGTGGTGGTCAAGGTGGTTCCTCTAGATCTT
CCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGC
AGTCGTTGGAGGAGTCCGGGGGAGGTCTCTTCAAGC
CTGGAGGATCCCTGACACTCACCTGCACAGTCTCTG
GATTCACCATCACTAGCTACCACATGTGCTGGGTCC
GCCAGGCTCCAGGGAACGGGCTGGGATGGATCGGA
GCCGTTAGTGCTAGCGGACACACATATTACGCGAAC
TGGGCGAAAAGCCGATCCACCATCACCAGAGACAC
CAACTTAAACACCATGACTCTGAAAATGACCAGTCT
GACAGCCGCGGACACGGCCACCTATTTCTGTGCGAC
ACCATATCCTGGTTATGATATTGATCCCTTTGACATC
TGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGG
CAACCTAAGGCTCCGTCAGTCACTAGTGGCCCGGGA
GGCCA
SEQ ID NO:TBIfabP12GGCCCAGCCGGCCATGGCTGAGCTCGTGCTGACCCA
104GACTCCATCCTCCGTGCCTGCAGCTGTGGGAGGCAC
AGTCACCATCGATTGCCAGTCCAGTGAGAGTGTTTA
TAATAACAACAACTTAGCCTGGTATCAGCAGAAACC
AGGGCAGCCTCCCAAGCTCCTGATCTACGGGGCATC
CACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGG
CAGTGGATCTGGGACAGAGTTCACTCTCACCATCAG
CGACCTGGAGTGTGCCGATGCTGCCACTTACTACTG
TCAACAGGGTTATAGTATTGGTAATGTAGATAATGC
TTTCGGCGGAGGGACCGAGCTGGAGATCCTGGTGGT
TCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGG
GCGGTGGTGGGCAGTCGCTGGAGGAGTCCGGGGGA
GGTCTCTTCAAGCCAACGGATACCCTGACACTCACC
TGCACGGTCTCTGGATTCTCCCTCAGTAGCTATGGA
ATTACTTGGGTCCGCCAGGCTCCAGGGAACGGGCTG
GAATGGATCGGCGCCATTGGTAGTGATGCTAAAACA
TACTACGCGAGCTGGGCGAAAGGCCGATCCACCATC
ACCGGAGACACCAACCTGAACACGGTGACTCTGAG
AATGACCAGTCTGACAGCCGCGGACACGGCCACCT
ATTTCTGTGCGAGATATTTTGATTGGTTTAATGTGGA
CATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTC
AGGGCAACCTAAGGCTCCATCAGTCACTAGTGGCCC
GGGAGGCCA
SEQ ID NO:TBIfabP18GGCCCAGCCGGCCATGGCTGAGCTCGTGATGACCCA
105GACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCAC
TGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAG
TAGCTACTTAGCCTGGTATCAACAGAAACCAGGGCA
GCCTCCCAAGCTCCTGATCTACAAGGCTTCCACTCT
GGCATCTGGGGTCCCGTCGCGGTTCAAAGGCAGTGG
ATCTGGGACACAGTTCACTCTCACCATCAGCGATGT
GGTGTGTGACGATGCTGCCACTTACTACTGTGCAGG
ATATAAAGGTGGTAGTAGTGATGGTAGTGCTTTCGG
CGGAGGGACCGAGCTGGAGATCCTGGTGGTTCCTCT
AGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGT
GGTGGGCAGTCGGTGAAGGAGTCCGAGGGAGGTCT
CTTCAAGCCAACGGATACCCTGACACTCACCTGCAC
AGTCTCTGGATTCTCCCTCAGTAGCTATGGAGTGAG
CTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAAT
GGATCGGAGCCATTAGTAGTGGTGGTGACGCATACT
ACGCGAGCTGGGCGACCAGCCGATCCACCATCACC
AGAAACACCAACCTGAACACGGTGACTCTGAAAAT
GACCAGTCTGACAGCCGCGGACACGGCCACCTATTT
CTGTGCGAGACATTTTGGTTATGGTACTGCTGGGGG
CATCTGGGGCCCAGGCACCCTCGTCACCGTCTCTTC
AGGGCAACCTAAGGCTCCGTCAGTCACTAGTGGCCC
GGGAGGCCA
SEQ ID NO:TBIfabP19GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
106GTCGCCCTCTGCATCTGCTGCCCTGGGATCCTCGGC
CAAGCTCACCTGCACTCTGAGCAGTGCTCACAAGAC
CTACTATATTGAATGGTATCAGCAGCAGCAAGGGGA
GGCCCCTCGGTACCTGATGCAGGTTAAGAGTGATGG
AAGCTACACCAAGGGGACCGGGGTCCCTGATCGCTT
CTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGAT
CATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTA
CTATTGTGGTGCAGATTATAGCGGTGGGTATGTGCT
CGGCGGAGGGACCCAGCTGACCGTCACAGGTGGTG
GTTCCTCTAGATCTTCCTCCTCTGGTGGCTCGGGCGG
TGGTGGGCAGTCGGTGGAGGAGTCCGGGGGAGGTC
TCTTCAAGCCAGCGGATACCCTGACACTCACCTGCA
CAGTCTCTGGATTTTCCCTCAGTTACCCTGGAGTGA
GCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAA
TACATCGGATTCATTAATGCTGATGGTGACTCATAC
TACCCGACCTGGGCGAAACGCCGATCCACCATCACC
AGAAACACCAACCTGAACACGGTGACTCTGAAAAT
GACCAGTCTCACAGCCGCGGACACGGCCACCTATTT
CTGTGCGAGAGAGGGTGGCTGGGGTACATTGTTTGG
TGCTTTTGATTCCTGGGGCCCAGGCACCCTAGTCAC
CGTCTTCTCAGGGCAACCTAAGGCTCCGTCAGTCAC
TAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT114GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
107GTCGCCCTCTGCATCTGCTGCCCTGGGATCCTCGGC
CAAGCTCACCTGCACTCTGAGCAGTGCTCACAAGAC
CTACTATATTGACTGGTATCAGCAGCAGCAAGGGGA
GGCCCCTCGGTACCTGATGCAGCTTGGGAGTGATGG
AAGTTACACCAAGGGGACCGGGGTCCCTGATCGCTT
CTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGAT
CATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTA
CTATTGTGGTTCAGATTATAGCGGTGGGTATGTGTT
CGGCGGAGGGACCCAGCTGACCGTCACAGGTGGTG
GTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTC
GGGCGGTGGTGGGCAGTCGCTGGAGGAGTCCGGGG
GAGGTCTCTTCAAGCCAGCGGATACCCTGACACTCG
CCTGCACAGTCTCTGGATTCTCCCTCAGTACTTATGG
AGTGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGC
TGGAATACATCGCATACATTAATTATAGTGGTAGTC
CATACTACGCGAGCTGGGCGAAAAGCCGATCCACC
ATCACCAGAAACACCAACGAGAAAACGGTGACTCT
GAAAATGACCAGTCTGACAGCCGCGGACACGGCCA
CCTATTTCTGTGCGAGGGGTGTTCCTGGTTACAATG
CGGATATGGGGGACATCTGGGGCCCAGGCACCCTG
GTCACCGTCTTCTCAGGGCAACCTAAGGCTCCATCA
GTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT116GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
108GTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGC
CAAGCTCACCTGCACCCTGAGCAGTGCCCACAAGAC
CTACACCATTGATTGGTATCAGCAGCAGCAAGGGGA
GGCCCCTCGGTACCTGATGCAGCTTAAGAGTGATGG
AAGCTACACCAAGGGGACCGGGGTCCCTGATCGCTT
CTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGAT
CATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTA
CTATTGTGGTGCAGATTATAGCGGTGGGTATGTGTT
CGGCGGAGGGACCCAGCTGACCGTCACAGGTGGTG
GTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTC
GGGCGGTGGTGGGCAGTCGGTGGAGGAGTCCAGGG
GGAGGTCTCTTCAAGCCAACGGATACCCTGACACTC
ACCTGCACAGTCTCTGGATTCTCCCTCAGTGGCTAT
GGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAACGG
GCTGGAATGGATCGGAGCCATTAGTAGTGGTGGTAG
CGCATACTACGCGAGATGGGCGAAAAGCCGCTCCA
CCATCACCAGAAACACCAACCTGAACACGGTGACTC
TGAAAATGACCAGTCTGACAGCCGCGGACACGGCC
ACCTATTTCTGTGCGAGAGGTTACTATACTGCTATTG
GTGGTACTTATGACAATGCTTTTGATCCCTGGGGCC
CAGGCACCCTGGNCACCGTCTCCTCAGGGCAACCTA
AGGCCAGTCACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT124GGCCCAGCCGGCCATGGCTGAGCTCGATATGACCCA
109GACTCCACCCTCCCTGTCTGCATCTGTGGGAGAAAC
TGTCAGGATTAGGTGCCTGGCCAGTGAGGACATTGG
CAGTGCTATATCCTGGTACCAACAGAAGCCAGGGA
AACCTCCTACACTCCTGATCTATGGTGTATTTAATTT
AGAATCTGGGGTCCCACCACGATTCAGTGGCAGTGG
ATCTGGGACAGATTACACCCTCACCATTGGCGGCGT
GCAGGCTGAAGATGCTGCCACCTACTACTGTCTAGG
CGGTGCTAGTGACAGTAGTACCGGTTTGACTTTTGG
AGCTGGCACCAATGTGGAAATCAAGGTGGTTCCTCT
AGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGT
GGTGGGCAGTCGGTGAAGGAGTCCGAGGGAGGTCT
CTTCAAGCCAACGGATACCCTGACACTCACCTGCAC
AGTCTCTGGATTCTCCCTCAGTAACTATGGAGTGAG
CTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAAT
ACATCGGCTTCATTAGTAACGGTGGTGCCACATTCT
ACGCGACCTGGGCGAGAAGCCGAGCCACCATCACC
AGAAACACCGGCCTGAACACGGTGGCTCTGACAAT
GACCAGTCTGACAGCCGCGGACACGGCCACCTATTT
CTGTGTGAGGGATTCTGTTGCTACTTATGCTACTGAT
GTTGCTGCTTTTGATCCCTGGGGCCCAGGCACCCTG
GTCACCGTCTCCTCAGGGCAACCTAACTCCGTCAGT
CACTAGTGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT129GCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCAG
110TCGCCCTCCCTGTCTGCGTCTCTGGGCACAACGGCC
AGACTCACCTGCACCCTGAGCACTGGCTACAGTGTT
GGCGAGTACCCTTTAGTGTGGCTCCAGCAGGTGCCA
GGGAGGCCTCCCAGGTATCTCCTGAGCTTCACCTCA
GATGAAGACAAACACCATGACTCTTGGGGCCCCACC
CGCTTTTCTGGATCCAAAGACACCTCAGAGAATACC
TTTATCCTGAGCATCTCTGGGCTGCAGCCCGAGGAC
GAGGCCGACTATTACTGTGCTACAGCTCATGGTAGT
GATAACAGCCTCCATTATGTCTTCGGCGGAAGGACC
CAGCTGACCGTCACAGGTGGTGGTTCCTCTAGATCT
TCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGG
CAGTCGGTGAAGGAGTCCGAGGGAGGTCTCTTCAA
GCCAACGGATACCCAGACACTCACCTGCACAGTCTC
TGGATTCTCCCTCAGTAGCAATGCAATAAGCTGGGT
CCGCCAGGCTCCAGGGAACGGGCTGAAAAGCATCG
GATTCATTAATAGTGGTGGTGGCGCATATTACGCGA
CCTGGGCGAAAAGCCGATCCACCATCACCAGAAAC
ACCAACGAGAACACGGTGACTCTGAAAATGACCAG
TCTGACAGCCGCGGATACGGCCACCTATTTCTGTGC
GAGAACACCCCATTATTATGATACTTATGATACCTC
ATTTAACATATGGGGCCCAGGCACCCTGGTCACCGT
CTTCTCAGGGCAACCTAAGGCTCCGTCAGTCACTAG
TGGCCCGGGAGGCCA
SEQ ID NO:TBIfabT134GGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTCA
111GTCGCCCTCTGCATCTGCTGCCCTGGGATCCTCGGC
CAAGCTCACCTGCACTCTGAGCAGTGCTCACAAGAC
CTACTATATTGAATGGTATCAGCAGCAGCAAGGGGA
GGCCCCTCGGTACCTGATACAACTTAAGAGTGATGG
AAGCTACACCAAGGGGACCGGGGTCCCTGATCGCTT
CTCGGGCTCCAGCTCTGGGACTGACCGCTACTTGAT
CATCTCCAGCGTCCAGGCTGAGGACGAAGCCGACTA
CTCTTGTGGTGCAGATTATAGTGGTGGGTTTGTGTTC
GGCGGAGGGACCCAGCTGACCGTCACAGGTGGTGG
TTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCG
GGCGGTGGTGGGCAGTCGCTGGAGGAGTCCGGGGG
AGGTCTCTTCAAGCCAACGGATACCCTGACACTCAC
CTGCACAGTCTCTGGATTCGCCCTCAATAACTACAA
CATACACTGGGTCCGCCAGGCTCCAGGGAACGGGCT
GGAATGGATCGGAGCCATTGGTAGTAGTGGTAGCG
CATACTACGCGAGCTGGGCGAAAAGCCGATCCACC
ATCACCAGAAACACCAACCTGAACACGGTGACTCTG
AAAATGACCAGTCTGACAGCCGCGGACACGGCCAC
CTATTTCTGTGCGAGAGGTTATAATTCTGATGATTCT
TATCTCTGGGGCCCAGGCACCCTGGTCACCGTCTCC
TCAGGGCAACCTAAGGCTCCATCAGTCACTAGTGGC
CCGGGAGGCCA
SEQ ID NO:TBIfabP212CCGGCCATGGCTGAGCTCGTGCTGACCCAGACTCCA
112CCCTCCCTGTCTGCATCTGTGGGAGGCACAGTCACC
ATAAACTGTCTGGCGAGTGAGAACGTCTACAGTGCT
GTAGCCTGGTATCAACAGAAGCCAGGGAAACCTCCT
ACACTCCTGATCTCTGGTACATCCAATTTAGAGGCT
GGGGTCCCACCACGGTTCAGTGGCAGTGGATCTGGG
ACAGATTACACCCTCACCATCGGCGGCGTGCAGGCT
GAAGATGCTGCCACTTACTTCTGTCAAGGGTATAGC
AGTTACCCTTTGACTTTTGGAGCTGGCACCAATGTG
GAAATCAAGGTGGTTCCTCTAGATCTTCCTCCTCGG
TGGCGGTGGCTCGGGCGGTGGTGGGCAGTCGGTGG
AGGAGTCCAGGGGGGAGGCCTGGTCAAGCCTGGGG
ATACCCTCACACTCACCTGCACAGTCTCTGGATTCC
CCCTCAGCAGCTACGACATGAACTGGGTCCGCCAGG
CTCCAGGGGAGGGGCTGGAATGGATCGGATGGATT
ACTTATGATGGTTACAATCACTACGCGAGCTGGGCG
AATGGCCGATCCACCATCACCAGAAACACCAACGA
GAACGCGGTGACTCTGAAAATGACCAGTCTGACAG
CCGCGGACACGGCCACCTATTTCTGTGCGCGAGATT
ACTATAATGGTGCTTATGTTTATGCCTTTAACATCTG
GGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCA
ACCTAAGGCTCCATCAGTCACTAGTGGCCCGGGAGG
CCA

TABLE 3
PCR Primer Sequences
SEQ ID NOSEQ NAMENUCLEIC ACID SEQUENCE
SEQ ID NO:SKVHF01GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
113GCTCGGGCGGTGGTGGGCAGTCGGTGGAGGAGTCCR
GG
SEQ ID NO:SKVHF02GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
114GCTCGGGCGGTGGTGGGCAGTCGGTGAAGGAGTCCG
AG
SEQ ID NO:SKVHF03GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
115GCTCGGGCGGTGGTGGGCAGTCGYTGGAGGAGTCCG
GG
SEQ ID NO:SKVHF04GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
116GCTCGGGCGGTGGTGGGCAGSAGCAGCTGGWGGAG
TCC
SEQ ID NO:SKCHR01TGGTGTTGGCCTCCCGGGCCACTAGTGACTGAYGGA
117GCCTTAGGTTGC
SEQ ID NO:SKOVERPATGGTGTTGGCCTCCCGGGCCACTA
118DR1
SEQ ID NO:SKVLKF01GGGCCCAGCCGGCCATGGCTGAGCTCGTGMTGACCC
119AGACTCCA
SEQ ID NO:SKVLKF02GGGCCCAGCCGGCCATGGCTGAGCTCGATMTGACCC
120AGACTCCA
SEQ ID NO:SKVLKF03GGGCCCAGCCGGCCATGGCTGAGCTCGTGATGACCC
121AGACTGAA
SEQ ID NO:SKVLLF01GGGCCCAGCCGGCCATGGCTGAGCTCGTGCTGACTC
122AGTCGCCCTC
SEQ ID NO:SKVLLF02GGGCCCAGCCGGCCATGGCTCAGCCTGTGCTGACTC
123AGTCG
SEQ ID NO:SKOVERPATACTCAGGGCCCAGCCGGCCATGGCTGA
124DF1
SEQ ID NO:SKVLKR01GGAAGATCTAGAGGAACCACCAGGATCTCCAGCTCG
125GTCCC
SEQ ID NO:SKVLKR02GGAAGATCTAGAGGAACCACCTTGATTTCCACATTG
126GTGCC
SEQ ID NO:SKVLKR03GGAAGATCTAGAGGAACCACCTTGACSACCACCTCG
127GTCCC
SEQ ID NO:SKVLLR01GGAAGATCTAGAGGAACCACCACCTGTGACGGTCAG
128CTGGGTCC
SEQ ID NO:SKpf41F01-2TAAGCAGAATTCGGCCCAGCCGGCCGCGGTGGGAAC
129ACTAGGA
SEQ ID NO:SKpf41F02-3AGCAGAATTCTGGCCCAGCCGGCCGCGGTGGGAACA
130CTAG
SEQ ID NO:SKpf41F03-4AGCAGAATTCTAGGCCCAGCCGGCCGCGGTGGGAAC
131ACTA
SEQ ID NO:SKpf41R01-2CTCTAGATCTGGCCTCCCGGGCCTTTTTATATACCAC
132AGCCAGTTTG
SEQ ID NO:SKpf41R01-3CTCTAGATCTAGGCCTCCCGGGCCTTTTTATATACCA
133CAG
SEQ ID NO:SKpf41R01-4CTCTAGATCTATGGCCTCCCGGGCCTTTTTATATACC
134ACA
SEQ ID NO:SKRTlucF01AACATAAAGAAAGGCCCGGC
135
SEQ ID NO:SKRTlucR01GCCTTATGCAGTTGCTCTCCA
136

SEQ ID NOs 113-128 are used for amplification of rabbit VL and VH cDNA for production of the phagemid inserts.

SEQ ID NOs 129-134 are used for amplification of gp41, ligation into the pFUSE hIgG1-2 vector, and excision to generate sfiI restriction sites in multiple frames.

SEQ ID NOs 135-136 are used for amplification of firefly luciferase reverse transcripts produced in the Reverse Transcriptase assay.

In one embodiment, a gene encoding an anti-virus particle product antibody is chemically synthesized. Nucleotide sequences encoding the amino acid sequences shown in Table 1 are selected, broken into multiple oligonucleotides, chemically synthesized, and successively ligated together, to produce a gene encoding an anti-virus particle product antibody.

Once an antibody or ABP sequence is known, particularly the CDRs, any suitable method of making an ABP therefrom can be employed, including both cell-based and cell-free methods. Various methods of cell-free antibody synthesis are found in Carlson, E et al., Biotechnology Advances, 2012, 30: 1185-94. In cell-based methods, an anti-virus particle product monoclonal antibody or ABP gene is inserted into an expression vector for construction of a recombinant expression vector. An expression vector generally has a promoter region upstream of a translation start codon (ATG), as well as a polyA signal region downstream of a translation stop codon (TAA, TGA, or TAG). A recombinant expression vector is introduced into a host cell, and the transformed host cells are cultured to express the anti-virus particle product monoclonal antibody. Escherichia coli, yeast, or other animal cells can be used as host cells.

An immunoassay using an anti-virus particle product monoclonal antibody (or fragment thereof) can be used to detect the presence of HIV. In one embodiment, the immunoassay procedure involves allowing an anti-virus particle product monoclonal antibody to react with a sample including an antigen, and allowing any resulting antigen-antibody complex to further react with an antibody having a detectable label. In a further embodiment, detection or quantification of the antibody may be performed by a method such as enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay, radioimmunoassay (RIA), luminescent immunoassay, enzyme antibody technique, fluorescence antibody technique, immunonephelometry, latex agglutination reaction, latex turbidimetry method, hemagglutination, particle agglutination, Western blotting method, competition method, or sandwich method. In a further embodiment, the sandwich method uses a solid-phased antibody or a labeled antibody as the anti-virus particle product monoclonal antibody.

In some embodiments, the monoclonal antibody is sensitized/immobilized to a solid phase carrier, such as polystyrene, styrene-butadiene copolymer, (meth)acrylic acid ester polymer, latex, gelatin, a liposome, a microcapsule, erythrocyte, silica, alumina, carbon black, a metallic comipound, metal, ceramic, or a magnetic body. Sensitization methods include physical adsorption method, chemical bond method, or a combined physical adsorption and chemical bond method. In an embodiment, measurement is performed using an optical method, such that the sample reacts with an antibody (sensitized to a solid phase carrier or unsensitized), and a transmitted or scattering light is measured by an end point method or a rate method. In another embodiment, measurement is performed by visual observation, such that the sample reacts with an antibody that has been sensitized to a solid phase carrier in a vessel (for example, a plate or microtiter plate), and the agglutinated reaction product is visually observed. A microplate reader is further used in some embodiments to observe the agglutinated reaction product.

In a like manner, TEGS can be used in any suitable immunoassay format, such as those described above, to detect and/or quantify HIV antibodies or ABPs in a sample. TEGS can be immobilized on an immunoassay support in, for example, a standard sandwhich format, or used in solution in, for example, a standard competition assay.

FIG. 1 provides an illustrated flowchart that summarizes a method for producing TEGS, according to an embodiment. First, the HIV virus is isolated from multiple sources of acute infection virus (i.e., from antibody low/negative subjects) by using specialized coated beads (102). Next, the virus is propagated in a purified and homogenous cell substrate (104). In an embodiment, the virus is propagated in serum-free medium. The number of passages of virus is minimized, and the capacity of the cell substrate to produce type 1 interferons (e.g., interferon-alpha), type 2 interferons (e.g., interferon-gamma), and beta-chemokines (e.g., RANTES, MIP1 alpha, and MIP1 beta) is reduced. The cytokines can inhibit virus replication and add complexity to the TEGS composition. Next, the fluid containing the virus is concentrated by using molecular weight cutoff (MWCO) filters (106). Use of serum-free medium is required in an embodiment to use the MWCO filters, to prevent clogging the filter pores. Thus, ultracentrifugation, which potentially disrupts any non-covalently attached envelope proteins, is not required. Next, the fluid containing virus is treated with agents to inactivate virus (108). These agents include a cyclodextrin and Benzonase. In an embodiment, the fluid containing the virus is additionally exposed to heat (e.g., 56° C. or less). The fluid containing the virus is then processed to remove any inactivating agents (110). In one embodiment, a smaller MWCO filter (such as a 10 KDa filter) is used for this step to prevent the loss of free gp41. Finally, the inactivated virus composition is reconstituted in final buffer (112). In some embodiments, the final buffer is dPBS with no calcium or magnesium ions. Additives (such as azide or PEG) are used in some embodiments to stabilize proteins and prevent microbial contamination. In other embodiments, other additives such as CpG-rich DNA are used to boost localized immune response to TEGS.

Pharmaceutical Compositions of the Invention

Methods for treatment of diseases are also encompassed by the present invention. Said methods of the invention include administering a therapeutically effective amount of anti-virus particle product antibody (for example, a TEGS-specific antibody) for preventing or treating HIV infection. These antibodies can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the anti-virus particle product antibody, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives can be included, as required.

Other routes of administration of the pharmaceutical composition are in accord with known methods, e.g. orally, through injection by intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, transdermal, or intraperitoneal; as well as intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems or by implantation devices. Administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

The pharmaceutical compositions of the present invention are preferably made under GMP conditions. It is also preferred that the compositions are packaged in a suitable sterile container, typically in a unit dosage form. The composition can be administered for therapeutic or prophylactic reasons. For example, therapeutic TEGS-specific antibodies can be administered as a substitute for an anti-retroviral drug. Therapeutic antibodies can also be administered in addition to an anti-retroviral drug, and/or added to a combination of antiretroviral drugs in Highly Active Anti-Retroviral Therapy (HAART). In an embodiment, this combination of antibody and antiretroviral drug is administered to a patient who is failing antiretroviral therapy. In another embodiment, the composition is administered prophylactically for protection of a fetus from an infected mother.

An effective amount of a pharmaceutical composition to be employed therapeutically will depend on the therapeutic context and objectives. One of skill in the art will appreciate that appropriate dosage levels for treatment will vary depending on the indication for which the therapeutic antibody is being used, the route of administration, and the size and condition of the patient. A typical dosage may range from about 0.1 mg/kg to up to about 100 mg/kg or more. Therapeutic antibodies may be preferably injected or administered intravenously. In some embodiments, the antibodies are lyophilized.

In some embodiments, therapeutic antibodies are administered at a dosage of from about 1 ng of antibody per kg of subject's weight per dose, to about 10 mg/kg/dose, or preferably from about 500 ng/kg/dose to about 5 mg/kg/dose. In other embodiments, a dosage for subcutaneous or intravenous administration of a dose of 0.5, 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 milligrams of therapeutic antibody is administered per kilogram body mass of the subject (mg/kg) (Flego et al, BMC Medicine 2013, 11:4). The dose can be administered once to the subject, or more than once at a certain interval, for example, once a day, three times a week, twice a week, once a week, three times a month, twice a month, once a month, once every two months, once every three months, once every six months, or once a year. The duration of the treatment, and any changes to the dose and/or frequency of treatment, can be altered or varied during the course of treatment in order to meet the particular needs of the subject.

The term “pharmaceutical agent composition” (or agent or drug) as used herein refers to a chemical compound, composition, agent or drug capable of inducing a desired therapeutic effect when properly administered to a patient. It does not necessarily require more than one type of ingredient.

The term “therapeutically effective amount” refers to the amount of an agent determined to produce a therapeutic response in a mammal Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.

The term “modulator,” as used herein, is a compound that changes or alters the activity or function of a molecule. For example, a modulator can cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Certain exemplary activities and functions of a molecule include, but are not limited to, binding affinity, enzymatic activity, and signal transduction. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described in, e.g., U.S. Pat. No. 6,660,843 (corresponding to PCT Application No. WO 01/83525).

The terms “patient” and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc. In some embodiments, the patient is a human subject with a homozygous deletion of 32 base pairs in the CCR5 gene, which encodes a major HIV-1 coreceptor (Buseyne F et al., J Infect Dis 1998, 178: 1019-1023). In an embodiment, the patient is a human subject who is failing antiretroviral therapy.

The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.

In an embodiment, an anti-virus particle product monoclonal antibody is formulated into a pharmaceutical composition and administered to a human patient. In a further embodiment, the dosage for the treatment is dependent on the degree of symptoms, age, and body weight of the patient, as well as the administration method. The antibody weight for the pharmaceutical composition ranges from 10 ng to 100 mg/kg of body weight, in an embodiment.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B(1992).

Example 1

Manufacture of the Virus Particle Product

Those of ordinary skill in the art can appreciate that there are many processes employed in the production of the non-infectious virus particle product that retains trimeric envelope glycoprotein subunits (TEGS) in their natural state. While TEGS is the virus particle product in the following examples, other virus particle products may also be produced and used. In a preferred embodiment, infectious virus stocks were prepared from plasma in mitogen-stimulated PBMC or CD4+ blood cells (Vyas G N. Human peripheral blood mononuclear cell substrate for propagating wild type HIV-1, Dev Biol (Basel) 2001 106:345-356), purified and concentrated, and then inactivated by treatment to deplete membrane cholesterol, capsid proteins, and RNA (FIG. 1).

Example 2

Production of High-Titer HIV-1 Stocks

The production of the viral particle product requires appreciable amounts of infectious virus. In a preferred embodiment, high-titer stocks of HIV were produced using HIV isolated from HIV-infected individuals who were in the earliest stage of infection prior to antibody detection. Such viruses have been called ‘Fiebig I/II’ isolates or ‘founder’ viruses (Fiebig E W, et al., AIDS 2003, 17:1871-1879; Keele B F et al. Proc Nat'l Acad Sci 2008, 105: 7552-7557). The expansion of virus was performed using primary blood cells that can be obtained from blood banks while preserving the anonymity of the blood donors. Primary blood cells were used to expand the virus in its most natural form, as opposed to the use of cell lines that produce variants of the original virus.

Peripheral blood mononuclear cells (PBMCs) were isolated by routine Ficoll density gradient separation procedures (Vyas G N et al., Biologicals, 2012 40: 15-20). To further purify blood cells for use in the expansion of virus, CD4+ cells were isolated from the PBMC by positive selection using immunomagnetic beads or other cell sorting methods. The PBMC or PBMC subpopulation was stimulated with PHA (1-3 micrograms per ml) for 2-4 days in medium A or B; medium A is the traditional growth medium that is comprised of RPMI 1640, 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 ng/ml recombinant human IL-2; medium B is a preferred growth medium that is comprised of F12/DMEM, 1× insulin, transferrin, selenium (ITS), and 10 ng/ml recombinant human IL-2. The use of medium B has advantages including that viral particle products can be produced in the absence of serum. The mitogen-stimulated primary cells were acutely infected for 2 hr (37° C. with periodic mixing) with infectious virus (e.g., 10e4 TCID50).

Five days later, the virus-infected cells were collected, washed, and mixed (1:10) with fresh mitogen-stimulated cells to allow for virus expansion. These cells (10e9/device) were then placed into a gas permeable rapid cell expansion device (e.g., G-Rex bioreactor, Wilson Wolf, New Brighton, Minn.). The cell culture fluids were collected and exchanged with fresh complete medium every 3 days for a period of 9-21 days. Peak virus concentrations (˜1 ug/ml p24) were observed at days 6-12 in many cases. HIV-1 levels were measured by p24 ELISA or assays for reverse transcriptase activity.

In FIG. 1A, it is demonstrated that the preferred procedures allow for substantial gains in the amount of virus produced in cell culture. In this case, the use of a bioreactor for the culture of virus-infected cells was found to be superior to the use of a conventional flask.

In another method of producing high-titer HIV-1 stocks, peripheral blood mononuclear cells (PBMCs) were isolated by routine Ficoll density gradient separation procedures (Vyas G N et al., Biologicals, 2012 40: 15-20). To further purify blood cells for use in the expansion of virus, CD4+ cells were isolated from the PBMC by positive selection using immunomagnetic beads or other cell sorting methods. The PBMC or PBMC subpopulation was stimulated with PHA (1-3 micrograms per ml) for 2-4 days in medium A or B; medium A is the traditional growth medium that is comprised of RPMI 1640, 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 ng/ml recombinant human IL-2; medium B is a preferred growth medium that is comprised of F12/DMEM, 1× insulin, transferrin, selenium (ITS), and 10 ng/ml recombinant human IL-2. The use of medium B has advantages including that viral particle products can be produced in the absence of serum. The mitogen-stimulated primary cells were acutely infected for 2 hr (37° C. with periodic mixing) with infectious virus (e.g., 10e4 TCID50). Next, the virus-infected cells were collected and expanded as described above.

Example 3

Measuring Viruses and Virus Particle Products

The assay most widely used in research laboratories for measuring HIV levels is an ELISA that detects the p24 capsid protein. The p24 protein is the most abundantly produced virus protein in infected cells and is the most abundant protein in the virus. Thus, the p24 ELISA is a useful assay for detecting the presence of HIV. However, because the majority of p24 is not incorporated into the virus and because p24 is present in defective viruses, the p24 ELISA does not reliably measure infectious viruses. An alternative assay measures levels of the virus reverse transcriptase protein. The present application embodies a preferred method for measuring virus reverse transcriptase: a one-step quantitative non-radioactive RT-PCR method. The method involved pelleting virus from an aliquot of the sample to be tested, lysis of the pelleted virions, the addition of a reaction mixture, incubations of varying lengths of time at various temperatures, and the acquisition and analysis of the data. This method is applied to the measurement of HIV, other viruses, and virus-like constructs.

In the case of FIG. 2, it is demonstrated that a quantitative RT-PCR assay has been validated and optimized for the measurement of virus. The left panel shows the performance of serial dilutions (10-fold) of a purified virus reverse transcriptase enzyme. The right panel shows the assay applied to the measurement of HIV at days 4, 7, and 10 of cell culture.

Example 4

Refinement of the Virus Particle Product

The preferred method for the production of a virus particle product from an infectious virus stock employs gentle virus inactivation procedures collectively termed “cholesterol extraction with nucleic acid depletion” or “CENAD”. Briefly, fluids containing infectious virions were concentrated using centrifugal filtration devices (e.g., 100 kDa molecular weight cut off, MWCO) and/or ultracentrifugation. The retentate or pellet was treated with beta-cyclodextrin to remove membrane-bound cholesterol, permeabilize the virions, and to expel capsid proteins, reverse transcriptase and viral RNA (Graham D R et al., J Virol 2003, 77: 8237-8248). The resulting viral particle product was further washed and concentrated using centrifugal filtration devices (e.g., 100 kDa MWCO, Millipore). This method for producing the virus particle product is a modified (streamlined and optimized) version of the one reported by Vyas et al (Vyas G N et al., Biologicals 2012, 40: 15-20).

Example 5

Coupling Multiple Virus Particle Products

The virus particle products derived from distinct infectious viruses can vary in protein sequence and structure. Therefore, collections of pooled virus particle products can be more diverse in protein composition than individual virus particle products (Vyas G N. Dev Biol (Basel) 2001, 106: 345-356; Vyas G N et al., Biologicals 2012, 40: 15-20). The simplest procedure for creating diverse virus particle products is to combine equal or non-equal amounts of individual virus particle products in a single vessel. The surface of a microsphere is coated with a variety of virus particle products. This is achieved by co-incubating virus particle products with DC-SIGN-expressing lipoparticles in PBS (pH7, 30 min, 4° C.), followed by washing and concentrating steps using 100 kDa MWCO filters. Alternatively, specialized microparticles such as Virobeads (AdemTech) can be employed to capture virus particle products. We have termed microparticles that are coated with pooled virus particle products “iTEGS”. In one example, iTEGS is used as an antigen that presents multiple virus particle products in a concentrated format. A product that displays multiple virus particle products is highly useful for vaccines that present multiple antigens from one or several different types of viruses.

In the case of FIG. 3, a diagram of one type of iTEGS is presented. In this case, viral particle products are produced from multiple clades of HIV-1. Then the viral particle products are pooled and captured with a microparticle (e.g., DC-SIGN-expressing lipoparticles or Virobeads)

Example 6

Metrics of ABPs

TEGS is useful for the capture, analysis, and/or measurement of ABPs. An example is the use of the virus particle product as a means to capture anti-virus antibodies on a solid platform. The method involved coating a surface, such as high-binding plastic, with the virus particle product, followed by the input of a source that potentially contained anti-viral antibodies. After an incubation period of 1-2 hours, the surface was washed, and then bathed in a detection antibody that broadly bound antibodies from the species used to generate the anti-viral antibody. Emphasized is that the virus particle product, where displaying natural envelope proteins, binds to antibodies that would otherwise remain unbound by viral proteins that have been subjected to procedures that disrupt the natural envelope structure. Thus the present virus particle product has advantages over commonly used virus lysates that are prepared using procedures that disrupt the natural structure of envelope proteins. The use of the virus particle product can be applied to a wide variety of immunodetection platforms including ELISA, ELISpot assays, bead-based assays, flow cytometry, mass spectrometry, microscopy-based assays, and Western and other blot-based assays.

In the case of FIG. 4, it is demonstrated that the virus particle product can be used for the capture, detection and measurement of anti-virus particle product ABPs. The left panel shows raw RT-PCR data from an experiment where a virus-particle-product-specific antibody (J3) (McCoy et al., J Exp Med 2012: 209(6) 1091-103) was incubated with a virus particle product and then evaluated for infectivity in cell culture. The right panel is a bar graph that shows the data in terms of the fold-reduction in HIV replication.

Example 7

TEGS Production

In a separate experiment, a TEGS composition was generated. HIV-1 was isolated from a 10-year old plasma specimen (stored at −80° C.) obtained from an individual at the time of acute HIV infection, when the nucleic acid amplification test for HIV was positive and the antibody test was negative. Specialized beads (Virobeads; Accurate Chemical & Scientific Corp., Westbury, N.Y.) were used to capture the virus from plasma. This method allows for successful isolation of infectious HIV where other procedures fail, perhaps due to the separation of infectious virus from inhibitory factors commonly found in plasma by using other procedures. Using the specialized beads also acts to concentrate the virus.

The virobead-captured HIV was then propagated in vitro using primary blood cells that were obtained from blood banks while preserving the anonymity of the blood donors. Primary blood cells were used to expand the virus in its most natural form, as opposed to the use of cell lines that could produce variants of the original virus. The HIV was used for in vitro infection of primary CD4+ cells that were isolated from a leukoreduction chamber (LRC) obtained from a healthy blood bank donor. In some cases, a PCR test for CCR5 genotyping was performed and used to screen blood from a healthy donor. R5-TEGS was produced in CCR5 positive cells. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the total blood in the LRC using a routine ficoll gradient separation procedure. From the PBMCs, CD4+ cells were isolated using immunomagnetic beads (Miltenyi Biotec, Auburn, Calif.; Stemcell Technologies, Vancouver BC, Canada). The CD4+ cells were mitogen stimulated in vitro with PHA (2 μl/mL phytohemagglutinin, Sigma-Aldrich, St. Louis, Mo.) and then acutely infected with the virobead-captured HIV. The acutely HIV-infected CD4+ cells were cultured in serum-free medium: F12/DMEM (Thermo Fisher Scientific, Life Technologies), lx ITS (Thermo Fisher Scientific, Life Technologies), 3 μl/mL human IL-2 (Thermo Fisher Scientific, Invitrogen), and 1× pen/strep (UCSF Cell Culture Facility). The cells were cultured in the serum-free medium and cell culture supernatants were collected at routine intervals for up to 14 days. In some cases, freshly stimulated CD4+ was added to increase HIV yields. A portion (half) of the cell culture supernatant was collected and replaced with fresh serum-free medium every 2-3 days. HIV levels in the cell culture supernatants were measured using a p24 ELISA assay and a reverse transcriptase (RT) assay.

PCR amplicons were cloned into a per2.1-TOPO vector (Invitrogen) that was used to transform competent cells. Colonies from the transformed E. coli preparations were selected and cultured. Plasmids from the cultures were isolated (minipreps; Qiagen) and sequenced (ElimBio, Hayward, Calif.). The resulting sequences were compiled and analyzed using tools in the LANL online database.

HIV virions in the cell culture supernatants were concentrated using molecular weight cutoff (MWCO) filtration. The supernatants containing an appreciable amount of HIV were centrifuged (e.g., 2000×g for 15 minutes) and filtered through 0.2μ filters to remove large particulate matter. The supernatants were then concentrated using 100 kDa molecular weight cutoff filters (MWCO; e.g., Amicon filters; Millipore Corp., Billerica, Mass.).

Concentrated HIV virions were treated with agents to render them non-infectious. The HIV virions were treated with beta-cyclodextrin (BCD, Trappsol, CTD Inc., High Spring, Fla.) to remove cholesterol from virion membranes. As a third orthogonal inactivation procedure, fluids can be briefly heated (e.g. up to 56° C. for 5 min) The benefit of this method for inactivating viruses is that the inactivation procedures are not known to modify the native envelope structure of the virus. Also, BCD can act to release virus proteins that are not attached to the surface of the virus. Notably, BCD can be increasingly effective in the reduced presence of serum. The inactivated virus material was washed with PBS, using MWCO filters. This procedures removed BCD and washed away smaller proteins and debris. Notably, this procedure involving MWCO filters was facilitated by the production of serum-free fluids. The retentate was resuspended in a buffer that varied with the intended use of the resulting TEGS composition. Additives were sometimes used to stabilize the proteins (e.g., PEG) and prevent microbial contamination (e.g., azide). Other additives (e.g. CpG-rich DNA) could be used to boost the localized immune response to the TEGS composition. The resulting material was then analyzed for its HIV Env composition using ELISA and Western blot procedures.

Example 8

TEGS Elicit Production of TEGS-Specific Antibodies in Immunized Animals

New Zealand white rabbits were immunized with the TEGS generated in Example 7 (Pacific Immunology, San Diego, Calif.). The immunization protocol included 4 injections and 4 production bleeds, with injections taking place at weeks 0, 3, 6, and 10, and bleeds taking place at weeks 7, 9, 11, and 13. At week 14, the rabbit was euthanized, followed by an exsanguinations bleed and a harvesting of the bone marrow and spleen.

FIG. 5 illustrates SDS-PAGE gels showing that rabbits immunized with TEGS produce TEGS-specific antibodies. The LEFT panel shows the immunoreactivity of the final bleed sera from two rabbits (#79 and #81) against TEGS separated by SDS-PAGE under denaturing and reducing conditions. Arrows denote bands of appreciable intensity above background. The MIDDLE panel shows the immunoreactivity of pooled sera from the pre- and post-immunization (final) bleeds of rabbits #79 and #81 against recombinant gp41 protein separated by SDS-PAGE under denaturing and reducing conditions. The RIGHT panel shows the immunoreactivity of pooled post-immune sera from rabbits #57 and #58 against various recombinant gp120 (lanes 1-4) and gp140 proteins (lanes 5-7) separated by SDS-PAGE under denaturing and reducing conditions. Together, the data in FIG. 5 demonstrate that rabbits immunized with TEGS produce antibodies against TEGS and recombinant gp41 and gp120 proteins. Thus, it can be concluded that TEGS prepared from an acute HIV infection isolate elicit a pronounced anti-HIV antibody response.

FIG. 6 is a graph demonstrating that the antibody response elicited by immunizing rabbits with TEGS is dose dependent, and serum dependent. A microtiter plate was coated with 100 ul of a virus particle product (1:5 dilution of TBI01280 stock in PBS). Pre- and post-immune sera from rabbits immunized with serum-free TEGS or serum-grown TEGS were diluted 1:10000 (grey bars) or 1:1000 (black bars) in PBS and added to the coated plates. Shown are the resulting O.D.s (450 nm). Respectively, rabbits #79, #81, and #83 received TEGS doses of 300×, 30×, and 3× volumetric equivalents based on the volume of virus particle product concentrated using MWCO filters. The TEGS in these cases were grown in serum-free medium (medium B). Rabbits #57 and #58 were immunized with equivalent TEGS doses, the TEGS in these cases grown in serum-grown medium (medium A). A strong dose-response relationship is observed. Based on the frequencies of virus-particle-specific hybridoma fluids, a similar dose-response relationship was observed in mice immunized with TEGS (see Example 10). These data are consistent with the elevated frequency of bands and the heightened band intensities seen with rabbit #79 as compared to #81 in FIG. 5 (LEFT). Altogether, these data help establish a TEGS optimal immunizing dose.

FIG. 7 illustrates that the TEGS anti-serum inhibits HIV infection (i.e., neutralizes HIV). The pre-immune serum and the final bleed serum from rabbit #79 were tested in a neutralization assay. Briefly, the sera were pre-adsorbed with human PBMC to remove antibodies capable of binding human cells. Next, the sera were added to aliquots (1000 TCID50) of two distinct HIV-1 isolates (TBI01280 (R5) and HIV-1 SF33 (X4)), resulting in the dilution of the serum. RPMI was used as a control. After a 1-hour incubation period (37° C.), the entire 200 μl serum/virus mixture was added to 1×106 PHA-stimulated human CD4 cells. Following a 2 hour incubation (37° C. with periodic mixing), the cells were washed with RPMI and plated in duplicate in a 96-well plate (BD Falcon; 1×105 cells per well) in complete growth medium. The medium was changed at 3-day intervals and aliquots of the supernatants were stored. HIV levels in the supernatants were measured by an RT-PCR assay designed to measure reverse transcriptase (RT) levels. FIG. 7 shows a representative amplification profile (LEFT) and neutralization results (RIGHT), which reveal that the TEGS antiserum from rabbit #79 markedly reduces the replication of two distinct HIV-1 isolates in vitro, presumably by binding virus envelope proteins. Notably, anti-sera from rabbits immunized with serum-grown TEGS (i.e., using medium A), did not neutralize HIV-1 in repeated experiments. Therefore, serum-free TEGS (i.e., using medium B) is distinguished by the specificities and/or titers of the antibodies elicited in immunized rabbits.

FIG. 8 is a chart showing that select recombinant HIV-1 envelope proteins do not block the neutralizing activity of the TEGS antiserum from rabbit #79. Briefly, the specificity of the neutralizing anti-serum was evaluated by pre-absorbing the anti-serum with recombinant gp41 and gp120 proteins (1 μl/ml; NIH AIDS Reagents Program) prior to performing neutralization assays with TBI01280. FIG. 8 shows that the reductions in HIV-1 replication result from treatment of the virus particle product with TEGS antiserum alone, TEGS antiserum pre-absorbed with recombinant gp41, TEGS antiserum pre-absorbed with a pool of recombinant gp120 proteins, and TEGS antiserum pre-absorbed with recombinant gp41 and pooled gp120 proteins. The pre-absorption procedure did not lower the antiviral activity of the TEGS antiserum. Thus, it can be concluded that the neutralizing activity of the TEGS antiserum is attributable to the recognition of an epitope that is not present in recombinant gp41 and gp120 monomeric proteins. These results provide evidence in support of the premise that TEGS can elicit antibodies against a discontinuous determinant(s) present in the quaternary structure of the native HIV-1 envelope.

FIG. 9 shows a substantial improvement of the virus particle product. Various samples (i-vii) of medium compositions and virus particle products were mixed with lithium dodecyl sulfate buffer (Invitrogen) and reducing agent (Invitrogen), heated (70° C., 10 min), loaded into the lanes of a 12% Bis-Tris gel (Invitrogen), and electrophoresed (200V, 40 min) in MES buffer (Invitrogen). The LEFT panel shows an image of a gel stained with G-250 coomassie (BioRad). The samples shown are: i) serum containing medium only, ii) serum-grown TEGS, iii) serum-free medium, iv) serum-free TEGS. The results in the left panel demonstrate the superiority of TEGS produced using serum-free medium, such as medium B in Example 2. The serum-free medium has markedly reduced levels of more than 8 different proteins. In comparison to a composition similar to mHIVenv, serum-free TEGS have greatly reduced levels of proteins in the 175 kDa and 60 kDa range and the overall composition is distinguished. The RIGHT panel shows the Western blot results of the immunoreactivity of a high positive control (BioRad; pooled sera from HIV-infected individuals). The samples shown are: v) concentrated serum-free TBI01280, vi) serum-free TEGS, and vii) serum-free medium. The results in the right panel demonstrate that HIV proteins are retained through the various procedures (i.e., FIG. 1 elements 106 thru 112) involved in producing TEGS.

Example 9

Cloning of Envelope Genes from the Virus Particle Product

It will be apparent to those skilled in the art that the HIV envelope gene can be cloned and expressed in prokaryotic and eukaryotic cells and even cell-free systems. The expression of HIV envelope proteins or peptides can facilitate a wide variety of downstream applications related to the production and/or characterization of anti-envelope antibodies.

In the case of FIG. 10, it is demonstrated that an envelope gene from a virus particle product has been cloned into a plasmid and sequenced, as shown below. HIV-1 was isolated from the plasma of a healthy blood bank donor who tested negative for anti-HIV antibodies. After propagation of the virus in primary human mononuclear blood cells, RNA was extracted from the virus particle product and amplified by RT-PCR using consensus primers for HIV-1 clade B isolates. The PCR amplicons were cloned into a per2.1-TOPO vector (Invitrogen) that was then used to transform competent cells (Invitrogen). Colonies from the transformed E. coli preparations were selected and cultured. Plasmids from the cultures were isolated (minipreps; Qiagen) and sequenced (ElimBio). The resulting sequences were compiled and analyzed using tools in the LANL online database (http://www.hiv.lanl.gov/).

The sequence encoding gp41 (SEQ ID NO: 137) in the per2.1-TOPO vector was amplified and ligated into an expression vector, in-frame, between an IL-2 secretion peptide sequence and a sequence encoding a human IgG-Fc tag. After routine preparation of a plasmid stock, gp41 expression vector was transfected into 293 cells. Dot blot analysis of the cell lysate and cell culture supernatant confirmed the expression and secretion of gp41 (data not shown). The resulting secreted protein, derived from the virus particle product, can be useful for analysis of ABPs elicited by immunization with the virus particle product or otherwise.

SEQ ID NO: 137:
ATGGGGCACCATGCTCCTTGGGATGTTGATGATCTGTAGTGCTGCAGATA
AATTGTGGGTCACAGTCTACTATGGGGTACCTGTGTGGAAAGAAGCAACC
ACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACA
TAATGTTTGGGCCACGCATGCCTGTGTACCCACAGACCCCAACCCACAAG
AAGTAGTATTGGAAAATGTGACAGAAAATTTTAACATGTGGAAAAATGAC
ATGGTAGAACAAATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCT
AAAGCCATGTGTAAAATTAACCCCACTCTGTGTCACTTTAAATTGCAATG
ATGTCAATAATAATAGTACTATCAACAATGGAACTACTAATGCCACTAGT
CATAGTGGGGGAAAAATAGAGAGAGGAGAAATAAAAAATTGCTCTTTCAA
TGTCACCACAAACATAAAAAATAAGCTGCAGAAAGAATATGCACTGTTTT
ATAAGCTTGATCTAGTACCAACAGATGATAATAATTCTAGATATAGGTTG
ATACATTGTAATACCTTAGTCATTACACAAGCCTGTCCAAAGGTATCCTT
TGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAA
AGTGCAAAGATAGGAATTTCACAGGAAAAGGACAATGTAAAAATGTCAGC
ACAGTACAATGTACACATGGCATTAGGCCAGTAGTGTCAACTCAACTGCT
GTTAAATGGCAGTCTAGCAGAAGATGGGGTAGTAATTAGATCTGCCAATA
TCACAGACAATACTAAAACCATAATAGTACAGTTGAAGGAAGCTGTAGAA
ATTAATTGTACAAGACCCAATAACAATACAAGGAAAAGTATAACTATAGG
ACCAGGGAGAGCATTTTGGACAACAGGAGGAATAATAGGAGATATAAGAC
AAGCACATTGTAACCTTAGTAGCACAAAATGGAATAACACTTTAAGACAG
ATAGCTACAAAATTAAGAGAACAATTTGGTAACAAAACAATAGTTTTTAA
TCAATCCTCAGGAGGGGACCAAGAAATTGTGATGCACACTTTTAATTGTG
GAGGGGAATTTTTCTACTGTAGTACAACACAACTGTTTAATAGTACTTGG
ATTGCAAATAAGACTGGGAATGATACTGGAGGATCAAATGGAACTATTAC
ACTTCCATGCAGAATAAAACAAATTGTAAACATGTGGCAGGAAGTAGGAA
AAGCAATGTATGCCCCTCCCATCAAAGGACAAATTAGATGTTCATCAAAC
ATTACAGGACTGCTATTATTAAGAGATGGTGGTAAGAATAACGGGACAGG
AAACATGACAGAAATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATT
GGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGA
GTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGC
GGTGGGAACACTAGGAGCCTTGTTCCTTGGGTTCTTGGGAACAGCAGGAA
GCACTATGGGCGCAGCATCACTAACGCTGACGGTACAGGCCAGACTATTA
TTGCCTGGTATAGTGCAACAGCAAAACAATTTGCTGAGAGCTATTGAGGC
GCAACAGCATTTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGG
CAAGAGTCCTGGCTATGGAAAGATACCTACAGGATCAACAGCTCCTAGGG
ATTTGGGGTTGCGCTGGAAAACTCATTTGCACCACAGCTGTGCCTTGGAA
TACTAGTTGGAGTAATAAATCTCTGGATCAGATTTGGAATAACATGACCT
GGATGCAATGGGAAAGAGAAATTGACAATTACACACACACAATATACAGC
TTAATTGAAGAATCGCAGAACCAACAAGAAAAAAATGAACAAGAATTATT
GGAACTAGACAAGTGGGCAAGTTTGTGGAATTGGTTTGACATAACAAACT
GGCTGTGGTATATAAAAATATTCATAATGGTAGTAGGAGGCTTAGTAGGT
TTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGG
ATACTCACCATTATCGTTGCAGACCCGATTCCCAGTCCAGAGGGGACCCG
ACAGGCCCGAAGGAATCGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGA
TCCGGTCGATTAGTGACCGGATTCTTACCTCTTATCTGGGACGACCTGCG
GAGCCTGTGCCTCTTCAGCTACCGCCGCTTGAGAGACTTACTCTTGATTG
CAGCGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAACTCCTCAAA
TATTGGTGGAATCTCCTAAAATATTGGAGTCAGGAACTAAAGAATAGTGC
TGTCAGCTTGTACAACGCCACAGCTATAGCAGTAGCTGAGGGAACAGATA
GGGTTATAGAAATAGTAAGAAGAACCTTTAGAGCTATTATCCACATACCT
AGAAGAATAAGACAGGGCTTGGAAAGGGCTTTGCTATAAGATGGGTGGCA
AGTGGTCAAAAAGTAGTGTGGTTGGATGGCCTGAGATAAGAGAAAGAATG
AGACGAACCGAGCCACGAACCGAGCCAGCAGCAGAGGGGGTGGGAGCAGC
ATCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAG
CTACTAATGCTGCTTGTGCCTGGCTAGAAGCACAAGAAGAAGAGGAAGTG
GGTTTTCCAGTCAGACCTCAGGTACCTTTAAGACCAATGACCT

Example 10

Production of Cell Lines Producing TEGS-Specific Antibodies

FIG. 11 shows that the supernatants from mouse hybriboma cells produce virus-particle-product-specific antibodies. Mice were repeatedly injected with TEGS at biweekly intervals. With splenocytes from the mice, hybridomas were constructed. The supernatants from the hybridoma cultures were screened in ELISA assays for immunoreactivity to TEGS and to recombinant HIV proteins. The ELISA and Western blot data show that the hybridoma fluid is reactive with recombinant gp41, but not pooled recombinant gp120 proteins. While pre-immune serum from the mouse was not available for testing, normal mouse serum generally lacks immunoreactivity with recombinant HIV proteins. Thus, the anti-gp41 response likely resulted from immunization with TEGS. Therefore, the anti-gp41 immunoreactivity is likely TEGS-specific. It will be apparent to one skilled in the art that mouse B-cell hybridomas comprise immortalized cell lines that produce monoclonal antibodies.

Example 11

Cloning of TEGS-Specific ABPs from Rabbits Immunized with TEGS

FIG. 12 illustrates an antibody-phage display approach that was used with splenocytes and bone marrow derived from rabbits immunized with TEGS. Cells binding TEGS were selected, and total RNA was extracted. The RNA was converted to cDNA, and the antibody-encoding DNA was PCR-amplified using custom sequence-specific oligonucleotides (SSOs). The DNA was cloned into a phagemid vector (PADL-10b; Ab Design Labs; San Diego, Calif.) to create a library, the phagemid library was transfected into bacteria, the resulting bacteriophages were isolated, antigen-specific bacteriophages were captured (i.e., panning with TEGS), and the candidate antibody genes were subcloned into custom expression vectors. Unique SSOs were designed and validated. The resulting phagemids expressed novel single-chain variable fragment (scFV) proteins based on the unique combination of SSOs and the linker sequence used. Panning was performed using TEGS. For subcloning of the scFVs, a plasmid containing an IL-2 secretion signal and human Fc gene was used (Invivogen; San Diego, Calif.). The multiple cloning site (MCS) of the plasmid was uniquely modified to enable the subcloning of antibody sequences from the phagemids. Specifically, new restriction sites were introduced and a portion of the MCS was deleted.

Phagemids captured by panning with TEGS were used to infect E. coli. Phagemid DNA from the E. coli cultures was sequenced. The DNA sequences were analyzed using web-based tools at the international imMunoGeneTics information system (IMGT). These analyses were used in the identification of the rabbit variable chain genes and constructing alignments of the complementarity determining regions or CDRs. Amino acid sequences and nucleic acid sequences of antibody regions are provided in Tables 1 and 2, respectively.

Example 12

Production and Screening of TEGS-Specific ABPs

FIG. 13 illustrates a TEGS-specific ABP that was produced and validated. (LEFT) Briefly, the multiple cloning site (MCS) of an expression vector encoding an IL-2 secretion peptide and a human IgG Fc chain (Invivogen, San Diego, Calif.) was modified to enable the in-frame and directional ligation of the scFV constructs; unique SfiI sites were added and part of the MCS was deleted. The scFV insert (TBIfabT7; SEQ ID No: 151) from a phagemid captured with immobilized TEGS was excised with SfiI and then subcloned into an expression vector in-frame between sequences encoding an IL-2 secretion peptide and a human IgG Fc protein. Following the transfection of 293 cells, the cell culture supernatants were collected and analyzed for immunoreactivity in dot blot assays. (RIGHT) Shown are the results from a blot that was spotted with a) dPBS, e) FBS, and the concentrated supernatants from cultures of human primary mononuclear cells infected with HIV-1 virus particle products b) TBI01280-5F2, c) 1292-GNV, d) 1362-GNV, and TEGS-prepared from those isolates respectively (f-h). The supernatant from 293 cells transfected with the TBIfabT7 expression vector was used as a primary antibody, followed by the use of an anti-human IgG-HRP secondary antibody. The results show that TBIfabT7 is immunoreactive with each of the acute HIV infection virus isolates and, to a similar degree, with each of the TEGS compositions. TBIfabT7 is not immunoreactive with the multitude of proteins present in fetal bovine serum. In addition to demonstrating the physical production of a TEGS-specific ABP, these results establish the utility of TEGS for three distinct applications: immunization, phagemid capture, and ABP screening.

Example 13

Analytics of TEGS-Specific ABPs

The preceding Examples demonstrate the ability to produce virus particle product proteins and virus particle specific ABPs. This combined ability enables the exquisite analysis of the relationships among simple to complex TEGS epitopes and TEGS-specific ABPs. One analytic goal is to identify a highly conserved TEGS epitope(s) that is targeted by a broadly neutralizing TEGS-specific ABP(s). One skilled in the art will appreciate that a variety of analytic and experimental approaches can be used to map TEGS-specific ABPs to distinct TEGS epitopes.

In the case of FIG. 14, an analytic approach to determining the relatedness among TEGS-specific and recombinant HIV protein-specific ABPs is demonstrated. Briefly, a similarity matrix between all of the heavy chain variable region amino acid sequences in Table 2 was computed using the BioEdit software package. A separate similarity matrix was computed for the light chain variable region amino acid sequences. Then, the two matrices were compiled in a spreadsheet. Shown are the two similarity matrices separated by a dark diagonal line. Shaded boxes indicate amino acid sequences having a high degree of homology (e.g. >90%). The results reveal the presence of highly homologous amino acid sequences; the majority are among light chains. One skilled in the art will appreciate that similar analyses can be performed on the complementarity determining regions and other subsets of antibody sequences. When combined with protein or peptide binding data, the approach demonstrated can provide insight for the fine specifities of ABPs.

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.