MODIFIED ENVELOPE PROTEIN OF HUMAN IMMUNODEFICIENCY VIRUS AND USE THEREOF

Abstract

Provided are a newly designed HIV-1 Env trimer protein, and an HIV-1 pseudovirus and virus expressing the Env trimer protein, and the use thereof for the prevention and/or treatment of HIV infection.

Claims

1. A recombinant protein, which comprises gp120 and gp41 ectodomain (gp41ECTO), wherein the gp120 is located between 27 and 8 of the gp41ECTO; preferably, the recombinant protein comprises: 6, 7, 27 of gp41ECTO; gp120; 8, 9 of gp41ECTO, from its N-terminal to C-terminal.

2. The recombinant protein according to claim 1, wherein the gp41ECTO comprises a substitution of one or more (e.g., 1-12, 5-12, 5-10; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) consecutive amino acids in a linkage region between 27 and 8with gp120; preferably, the linkage region corresponds to amino acid positions 607-618 of a gp160 sequence of isolate HXB2.

3. The recombinant protein according to claim 2, wherein the gp41ECTO comprises a substitution of one or more (e.g., 1-7, 5-7; e.g., 1, 2, 3, 4, 5, 6, or 7) consecutive amino acids in a region corresponding to amino acid positions 610-616 of the gp160 sequence of isolate HXB2 with gp120; preferably, the gp120 is located between amino acid positions corresponding to positions 606 and 619 of the gp160 sequence of isolate HXB2.

4. The recombinant protein according to claim 1, wherein the gp120 is inserted between adjacent amino acids in a linkage region between 27 and 8 of gp41ECTO; preferably, the linkage region corresponds to amino acid positions 607-618 of a gp160 sequence of isolate HXB2; preferably, the gp120 is located between amino acid positions corresponding to positions 609 and 610 of the gp160 sequence of isolate HXB2; preferably, the gp120 is located between amino acid positions corresponding to positions 616 and 617 of the gp160 sequence of isolate HXB2.

5. The recombinant protein according to any one of claims 1-4, wherein the gp120 is a modified gp120 that has a furin cleavage site containing a mutation to prevent being cleaved compared to a natural gp120; preferably, the mutation is selected from amino acid substitution, insertion or deletion; preferably, the furin cleavage site corresponds to amino acid positions 508-511 of a gp160 sequence of isolate HXB2; preferably, the furin cleavage site is deleted in the modified gp120 compared to the natural gp120.

6. The recombinant protein according to any one of claims 1-4, wherein the gp120 is a modified gp120, that has C-terminal truncation of 1-11 (e.g., 4-11; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids compared with a natural gp120; preferably, the modified gp120 has a deletion of one or more (e.g., 1-11, 4-11; e.g.,1, 2, 3, 4, 6, 7, 8, 9, 10, or 11) consecutive amino acids in a region corresponding to amino acid positions 501-511 of a gp160 sequence of isolate HXB2; preferably, the modified gp120 has a deletion of the region corresponding to amino acid positions 501-511 of the gp160 sequence of isolate HXB2.

7. The recombinant protein according to any one of claims 1-6, wherein, a disulfide bond is contained between gp120 and gp41ECTO of the recombinant protein; preferably, the recombinant protein comprises a disulfide bond between amino acid positions corresponding to positions 37 and 605 of a gp160 sequence of isolate HXB2; preferably, the recombinant protein comprises residue Cys at amino acid positions corresponding to positions 37 and 605 of the gp160 sequence of isolate HXB2.

8. The recombinant protein according to any one of claims 1-4, wherein the gp120 is a natural gp120.

9. The recombinant protein according to any one of claims 1-8, wherein the N-terminal and/or C-terminal of the gp120 is linked to the gp41ECTO optionally via a peptide linker; preferably, the peptide linker is (GmS)n, wherein m is an integer selected from 1-4, and n is an integer selected from 1-3.

10. The recombinant protein according to any one of claims 1-9, which further possesses one or more of the following features: (1) the gp41ECTO comprises the following amino acid substitution: I559P; (2) the gp120 comprises the following amino acid substitution: T332N; (3) the gp120 comprises the following amino acid substitutions: E64K and H66R; (4) the gp120 comprises the following amino acid substitution: A316W; (5) the N-linked glycosylation site (PNGS) near the CD4bs epitope of the gp120 is replaced to prevent glycosylation; preferably, the PNGS is selected from the group consisting of N276, N301, N360, N463; (6) the gp120 comprises an internal disulfide bond; preferably, the gp120 comprises an internal disulfide bond between I201C and A433C; (7) a disulfide bond is further contained between the gp120 and gp41ECTO; for example, the recombinant protein comprises a disulfide bond between E49C and L555C; the numbering of the above positions is according to the numbering in gp160 of HIV-1 isolate HXB2.

11. The recombinant protein according to any one of claims 1-10, wherein the gp41ECTO and gp120 are derived from the same or different HIV-1 strains; preferably, the gp41ECTO and gp120 are derived from the same HIV-1 strain.

12. The recombinant protein according to any one of claims 1-11, comprising an amino acid sequence selected from: (1) an amino acid sequence consisting of amino acid residues at positions 40 to 651 of the sequence set forth in SEQ ID NO: 1; (2) an amino acid sequence consisting of amino acid residues at positions 40 to 652 of the sequence set forth in SEQ ID NO:2; (3) an amino acid sequence consisting of amino acid residues at positions 40 to 651 of the sequence set forth in SEQ ID NO:3; (4) an amino acid sequence consisting of amino acid residues at positions 40 to 652 of the sequence set forth in SEQ ID NO:4; (5) an amino acid sequence consisting of amino acid residues at positions 40 to 665 of the sequence set forth in SEQ ID NO:5; (6) an amino acid sequence consisting of amino acid residues at positions 40 to 678 of the sequence set forth in SEQ ID NO:6; (7) an amino acid sequence consisting of amino acid residues at positions 40 to 661 of the sequence set forth in SEQ ID NO:7; (8) an amino acid sequence consisting of amino acid residues at positions 40 to 666 of the sequence set forth in SEQ ID NO:8; (9) an amino acid sequence consisting of amino acid residues at positions 40 to 671 of the sequence set forth in SEQ ID NO:9; (10) an amino acid sequence consisting of amino acid residues at positions 40 to 676 of the sequence set forth in SEQ ID NO:10; (11) an amino acid sequence consisting of amino acid residues at positions 36 to 607 of the sequence set forth in SEQ ID NO:11; (12) an amino acid sequence consisting of amino acid residues at positions 36 to 646 of the sequence set forth in SEQ ID NO:12; (13) an amino acid sequence consisting of amino acid residues at positions 36 to 648 of the sequence set forth in SEQ ID NO:13; (14) an amino acid sequence consisting of amino acid residues at positions 36 to 638 of the sequence set forth in SEQ ID NO:14; (15) an amino acid sequence consisting of amino acid residues at positions 36 to 639 of the sequence set forth in SEQ ID NO:15; (16) an amino acid sequence consisting of amino acid residues at positions 36 to 836 of the sequence set forth in SEQ ID NO:16; (17) an amino acid sequence consisting of amino acid residues at positions 36 to 673 of the sequence set forth in SEQ ID NO:30; (18) an amino acid sequence consisting of amino acid residues at positions 36 to 678 of the sequence set forth in SEQ ID NO:31; (19) an amino acid sequence consisting of amino acid residues at positions 36 to 683 of the sequence set forth in SEQ ID NO:32; (20) an amino acid sequence consisting of amino acid residues at positions 36 to 678 of the sequence set forth in SEQ ID NO:33; (21) an amino acid sequence consisting of amino acid residues at positions 36 to 688 of the sequence set forth in SEQ ID NO:34; (22) an amino acid sequence consisting of amino acid residues at positions 36 to 670 of the sequence set forth in SEQ ID NO:35; (23) an amino acid sequence consisting of amino acid residues at positions 36 to 676 of the sequence set forth in SEQ ID NO:36; (24) an amino acid sequence consisting of amino acid residues at positions 36 to 680 of the sequence set forth in SEQ ID NO:37; (25) an amino acid sequence consisting of amino acid residues at positions 36 to 673 of the sequence set forth in SEQ ID NO:38; (26) an amino acid sequence consisting of amino acid residues at positions 36 to 678 of the sequence set forth in SEQ ID NO:39; (27) an amino acid sequence consisting of amino acid residues at positions 36 to 683 of the sequence set forth in SEQ ID NO:40; or (28) a variant of the sequence described in any one of (1) to (27), in which the variant has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) or has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the sequence from which it is derived, and the variant retains the properties of the sequence from which it is derived.

13. The recombinant protein according to any one of claims 1-12, which optionally comprises one or more sequences selected from the following at its N-terminal or C-terminal: a signal peptide, a translation initiation sequence (e.g., a Kozak consensus sequence), a tag sequence; preferably, the recombinant protein optionally comprises a signal peptide and/or a translation initiation sequence (e.g., a Kozak consensus sequence) at its N-terminal; preferably, the recombinant protein optionally comprises a tag sequence at its C-terminal.

14. A fusion protein, which comprises the recombinant protein according to any one of claim 1-13, and transmembrane region and intracellular region sequences of gp41 linked to its C-terminal; preferably, the transmembrane region and intracellular region sequences of gp41 and the gp4 lECTO in the recombinant protein are derived from the same HIV-1 strain.

15. The fusion protein according to claim 14, comprising an amino acid sequence selected from: (1) an amino acid sequence consisting of amino acid residues at positions 33 to 836 of the sequence set forth in SEQ ID NO: 16; (2) an amino acid sequence consisting of amino acid residues at positions 34 to 837 of the sequence set forth in SEQ ID NO:17; (3) a variant of the sequence described in any one of (1) to (2), in which the variant has a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) or has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the sequence from which it is derived, and the variant retains the properties of the sequence from which it is derived.

16. A multimer comprising a plurality of monomers, wherein each monomer is independently selected from the recombinant protein according to any one of claims 1-13, or independently selected from the fusion protein according to in claim 14 or 15; preferably, the monomers are identical to each other; preferably, the multimer is a trimer or a dimer.

17. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15, or the multimer according to claim 16.

18. A vector, which comprises the isolated nucleic acid molecule according to claim 17.

19. A host cell, which comprises the isolated nucleic acid molecule according to claim 17 or the vector according to claim 18; preferably, the host cell is a mammalian cell.

20. A method for preparing the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15 or the multimer according to claim 16, which comprises cultivating the host cell according to claim 19 under appropriate conditions, and recovering the recombinant protein, the fusion protein or the multimer from a cell culture; preferably, the recombinant protein or the fusion protein exists in the form of a multimer (e.g., trimer or dimer).

21. A particle, displaying on its surface the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15, or the multimer according to claim 16; preferably, the particle is a liposome or a nanoparticle.

22. A pseudoviral particle, comprising on its surface the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15, or the multimer according to claim 16; preferably, the pseudoviral particle is obtained by co-expressing (i) a vector comprising the nucleic acid molecule according to claim 17, and (ii) a packaging vector (e.g., a backbone plasmid) in a host cell.

23. A packaging system for producing the pseudoviral particle according to claim 22, which comprises: (i) an expression vector comprising the nucleic acid molecule according to claim 17, and (ii) a packaging vector (e.g., a backbone plasmid).

24. A modified HIV virus, which expresses the fusion protein according to claim 14 or 15 as its envelope protein; preferably, the modified HIV virus has a genome comprising the following modifications: substitution of wild-type env gene with a nucleotide sequence encoding the fusion protein according to claim 14 or 15; preferably, the HIV is HIV-1.

25. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the genome of the modified HIV virus according to claim 24.

26. A vector, which comprises the isolated nucleic acid molecule according to claim 25.

27. A composition, which comprises the recombinant protein according to any one of claim 1-13, the fusion protein according to claim 14 or 15 or the multimer according to claim 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the host cell according to claim 19, the particle according to claim 21, the pseudoviral particle according to claim 22, the packaging system according to claim 23, the modified HIV virus according to claim 24, the isolated nucleic acid molecule according to claim 25, or the vector according to claim 26; preferably, the composition further comprises a pharmaceutically acceptable carrier and/or excipient.

28. The composition according to claim 27, wherein the composition is an immunogenic composition or a vaccine; preferably, the composition comprises an adjuvant.

29. The composition according to claim 28, wherein the composition is a protein vaccine, which comprises the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15, or the multimer according to claim 16, or the particle according to claim 21.

30. The composition according to claim 28, wherein the composition is a virus-based vaccine, which comprises the pseudoviral particle according to claim 22 or the modified HIV virus according to claim 23.

31. The composition according to claim 28, wherein the composition is a nucleic acid vaccine, which comprises the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the isolated nucleic acid molecule according to claim 25, or the vector according to claim 26; preferably, the nucleic acid vaccine comprises DNA or RNA; preferably, the DNA or RNA may be naked or encapsulated in a shell with delivery and/or protection functions.

32. The composition according to any one of claims 27-31, which optionally comprises an antiretroviral agent, such as a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, or a fusion protein inhibitor.

33. Use of the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15 or the multimer according to claim 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the host cell according to claim 19, the particle according to claim 21, the pseudoviral particle according to claim 22, the packaging system according to claim 23, the modified HIV virus according to claim 24, the isolated nucleic acid molecule according to claim 25, the vector according to claim 26, or the composition according to any one of claims 27-32, for inducing an immune response against HIV in a subject and/or for preventing and/or treating an HIV infection in a subject, or in the manufacture of a medicament for inducing an immune response against HIV in a subject and/or for preventing and/or treating an HIV-infection in a subject; preferably, the medicament is a vaccine; preferably, the subject is a human; preferably, the HIV is HIV-1; optionally, the recombinant protein, the fusion protein, the multimer, the isolated nucleic acid molecule, the vector, the host cell, the particle, the pseudoviral particle, the packaging system, the modified HIV virus, the composition is administrated, for example simultaneously, separately or sequentially in combination with an antiretroviral agent; preferably, the antiretroviral agent is selected from a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, or a fusion protein inhibitor.

34. A method for inducing an immune response against HIV in a subject or for preventing and/or treating an HIV infection in a subject, which comprises administering to a subject in need thereof an immunologically effective amount of the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15 or the multimer according to claim 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the host cell according to claim 19, the particle according to claim 21, the pseudoviral particle according to claim 22, the packaging system according to claim 23, the modified HIV virus according to claim 24, the isolated nucleic acid molecule according to claim 25, the vector according to claim 26, or the composition according to any one of claims 27-32; preferably, the method comprises administering an immunogenic composition or vaccine (e.g., a protein vaccine) comprising the recombinant protein according to any one of claims 1-13, the fusion protein according to claim 14 or 15 or the multimer according to claim 16, or the particle according to claim 21; preferably, the method comprises administering an immunogenic composition or vaccine (e.g., a virus-based vaccine) comprising the pseudoviral particle according to claim 22 or the modified HIV virus according to claim 23; preferably, the method comprises administering an immunogenic composition or vaccine (e.g., a nucleic acid vaccine) comprising the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the isolated nucleic acid molecule according to claim 25, or the vector according to claim 26; preferably, the subject is a human; preferably, the HIV is HIV-1; optionally, the recombinant protein, the fusion protein, the multimer, the isolated nucleic acid molecule, the vector, the host cell, the particle, the pseudoviral particle, the packaging system, the modified HIV virus, the composition is administrated, for example simultaneously, separately or sequentially in combination with an antiretroviral agent; preferably, the antiretroviral agent is selected from a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, or a fusion protein inhibitor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0165] FIGS. 1A to 1B show the hypothetical spatial structure (1A) and secondary structure topology (1B) of monomeric protein of the gp140 trimer before modification (BG505 SOSIP, PDB No. 4TVP) and the trimer after modification (BG505 TSTIP).

[0166] FIGS. 2A to 2B show the results of reducing SDS polyacrylamide gel electrophoresis of the modified gp140 trimer after one-step Ni Sepharose excel purification. FIG. 2A shows the BG505 strain, and FIG. 2B shows the NL4-3 strain. 1 represents TSTIP protein, 2 represents TST protein, 3 represents SOSIP protein. Both TSTIP and TST showed a single band under reducing conditions, with a molecular weight of about 140 KD, while SOSIP showed two bands under reducing conditions, which were gp120 and gp41 extracellular segment. This shows that the protein of the present invention contains a stable covalent linkage between subunits as expected.

[0167] FIG. 3 shows the molecular sieve superdex200-16/600 purification results of the modified gp140 trimer, in which A represents BG505 strain, B represents NL4-3 strain. The molecular sieve chromatography results of TSTIP protein and TST protein showed a single elution peak, while the chromatographic result of SOSIP protein showed three elution peaks, indicating that the components of the proteins TSTIP and TST of the present invention were more uniform than those of SOSIP.

[0168] FIGS. 4A to 4C show the results of non-reducing polyacrylamide gel electrophoresis of the modified gp140 trimer after molecular sieve purification. A represents BG505 TSTIP, B represents NL4-3 TSTIP, C represents BG505 SOISP. The results showed that after molecular sieve purification, the main component of the protein TSTIP of the present invention was trimer, and had a trimer content higher than that of SOSIP.

[0169] FIG. 5 shows the Tm measurement results of the modified gp140 trimer by differential scanning calorimetry (DSC). The results showed that the Tm of the protein BG505 TSTIP of the present invention was 74.5 C., and the Tm of NL4-3 TSTIP was 63.41 C., which was higher than the Tm of the control protein BG505 SOSIP: 68.2 C. and the Tm of the control protein NL4-3 SOSIP: 62 C. This shows that the thermal stability of the modified protein is higher than that of the corresponding control protein SOSIP.

[0170] FIG. 6 shows the enzyme-linked immunosorbent assay (ELISA) results of the modified gp140 trimer with various reported human monoclonal antibodies. The antibodies used include broad-spectrum neutralizing antibodies VRC01, B12, PGT121, PGT125, 2G12, non-broad-spectrum neutralizing antibodies F105, F240, 17b, and HIV-1 receptor molecule CD4. The results showed that the proteins BGTSTIP and NL4-3 TSTIP of the present invention had a binding ability to various reported broad-spectrum neutralizing antibodies and non-broad-spectrum neutralizing antibodies that was basically equivalent to that of the control proteins BG505 SOSIP and NL4-3 SOSIP, and could recognize and bind well to CD4 receptor molecule. In addition, like SOSIP, the recognition of the modified protein by CD4 would induce the conformational change of the protein of the present invention, thereby exposing the 17b binding site.

[0171] FIG. 7 shows the results of the virus neutralization experiment of the immune serum samples obtained by immunizing BALB/C mice with the modified gp140 trimer on the B subtype NL4-3 and B subtype 2626 strain viruses; wherein, M1 to M6 referred to different mice, respectively. The results showed that the modified protein TSTIP and the control protein could induce a strong neutralizing response against the corresponding strain after immunizing mice, but could not induce neutralizing response against the Tier2 strain of the same subtype. The modified protein TSTIP and the control protein SOSIP showed comparable immunogenicity.

[0172] FIG. 8A shows the results of western blot of the pseudovirus based on the modified gp140 trimer. FIG. 8B shows the quantitative results of p24 of the pseudovirus based on the modified gp140 trimer. The results showed that both the transfected cells and the collected virus supernatant had the expression of full-length TSTIP protein, and the content of p24 in the supernatant of the pseudovirus of the present invention was decreased in some extent compared with the wild-type pseudovirus, but certain amounts of p24 and Env could still be detected, indicating that the pseudovirus of the present invention could be normally packaged to form a complete pseudoviral particle, but the yield might be reduced to a certain extent.

[0173] FIG. 9 shows the results of negative staining electron microscopy of the pseudoviral particles based on the modified gp140 trimer. It showed that the pseudoviral particles similar to the wild-type pseudovirus could be seen under the negative staining electron microscope.

[0174] FIG. 10 shows the infectivity detection results of the pseudovirus based on the modified gp140 trimer by using the Elispot method with LacZ as reporter gene. It showed that the wild-type pseudovirus maintained normal infectivity, while the pseudovirus of the present invention had lost the infectivity, which was in line with the inventor's expectation.

[0175] FIG. 11A shows the results of reducing SDS polyacrylamide gel electrophoresis of the modified gp140 trimers BG505/NL4-3 TSTIP G1/G2/Full. It showed that the molecular weights of BG505 TSTIP-G2 and NL4-3 TSTIP-G1 were larger than expected, the two proteins could still be dimers under reducing conditions, while no target proteins with high purity were available for BGTSTIP-Full, NL4-3-G1 and NL4-3-Full after purification. In contrast, the protein TSTIP of the present invention had a broader range of application.

[0176] FIG. 11B shows the AUC results of the modified gp140 trimers BG505/NL4-3 TSTIP G1/G2/Full, which were consistent with the results of SDS -PAGE.

[0177] FIG. 12 shows the results of enzyme-linked immunosorbent assay of the modified gp140 trimers BG505/NL4-3 TSTIP G1/G2/Full with various reported antibodies. It showed that the six proteins could all react with various antibodies and had certain activities, but the reactivity of some proteins was weaker than that of the protein TSTIP of the present invention.

[0178] FIG. 13 shows the virus neutralization experiment of the immune serum samples obtained by immunizing BALB/C mice with various modified gp140 trimers on B subtype NL4-3 strain. The results showed that all six proteins could induce neutralizing antibody responses in mice.

[0179] FIG. 14A shows the SDS polyacrylamide gel electrophoresis results of the modified gp140 trimers of various strains. The results showed that the modified proteins of the various strains were well expressed, and could be purified to reach a higher purity. Under the reducing SDS-PAGE, the proteins of the present invention showed a single band, which proved that the subunits thereof were linked by a stable covalent linkage.

[0180] FIG. 14B shows the molecular sieve superdex200 16/600 purification results of TSTIP protein of the five strains, showing that the molecular sieve peaks of the five proteins of the present invention were relatively single, indicating better protein uniformity, and the proteins obtained by applying the strategy of the present invention to different strains formed trimers as main component.

[0181] FIG. 14C shows the results of enzyme-linked immunosorbent assay of the proteins of the five strains with various antibodies, showing that the five proteins of the present invention had certain reactivity with various reported antibodies, proving their good activities.

[0182] FIG. 14D shows the neutralization test results of pseudovirus by the serum samples obtained by immunization with TSTIP proteins of the five strains, wherein the strains detected by immune serum samples of CH119 TSTIP and X1632 TSTIP were the corresponding CH119 and X1632 strains respectively; the strain detected by immune serum samples of 25710 and 246F3 TSTIP was BJOX200; the strain detected by immune serum sample of CNE8 TSTIP was TR011. The results showed that the immune serum samples of the fourth injection of TSTIP protein of the five strains all detected virus neutralization to some extent, indicating that the immunization of TSTIP protein of different strains could stimulate neutralization responses in mice.

[0183] FIGS. 15A to 15K show the experimental results of electrophoresis (I), molecular sieve purification (II), analytical ultracentrifugation (III), and ELISA (IV) of each TSTIP protein construct in Example 11. FIG. 15A: BG-B 1(1/1); FIG. 15B: BG-B 1(1/2); FIG. 15C: BG-B1 (2/2); FIG. 15D: BG-B1 (2/1); FIG. 15E: BG-B1(2/3); FIG. 15F: BG-B2(1-1); FIG. 15G: BG-B2(1-2); FIG. 15H: BG-B2(1-3); FIG. 15L BG-C1 (1/1); FIG. 15J: BG-C1 (1/2); FIG. 15K: BG-C1 (1/3).

SEQUENCE INFORMATION

[0184] Information on some sequences involved in the present invention is provided in Table 1 below.

TABLE-US-00001 TABLE1 Descriptionofsequences SEQ ID NO Description Sequenceinformation 1 BG505TST MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLAVGIGA VFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAIEAQQ HLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVP NLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVP TDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVK LTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSL FYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPI HYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLN GSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIG PGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNN TIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGS NSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLIL TRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTR RNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLA LDHHHHHHHH* 2 NL4-3TST MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLAVGIGA LFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAIEAQQ HLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVP NLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVP TDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCV KLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRDK VQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEPIPIHYCA PAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLA EEDVVIRSANFTDNAKTIIVQLNTSVEINCTRPNNNTRKSIRIQRGPG RAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLREQFGNNKTIIF KQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNT EGSDTITLPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTR DGGNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKK SLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKHHHHHHHH* 3 BG505TSTIP MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLAVGIGA VFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQ QHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTN VPNLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHAC VPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCV KLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVY SLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPI PIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLL LNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIR IGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGN NTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQG SNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLI LTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT RRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLL ALDHHHHHHHH* 4 NL4-3TSTIP MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLAVGIGA LFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAPEAQ QHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAV PNLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACV PTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCV KLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRDK VQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEPIPIHYCA PAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLA EEDVVIRSANFTDNAKTIIVQLNTSVEINCTRPNNNTRKSIRIQRGPG RAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLREQFGNNKTIIF KQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNT EGSDTITLPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTR DGGNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKK SLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKHHHHHHHH* 5 BG505TSTIPfull MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLAVGIGA VFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQ QHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTN VPWNSSWSNNLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHN VWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLW DQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELR DKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIK PVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPN NNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVK QLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNST WISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVI RCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVV KIEPLGVAPTRAKRRVVGRNLSEIWDNMTWLQWDKEISNYTQIIYG LLEESQNQQEKNEQDLLALDHHHHHHHH* 6 NL4-3TSTIPfull MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLREKRAV GIGALFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAP EAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICT TAVPWNASWSNNLWVTVYYGVPVWKEATTTLFCASDAKAYDTEV HNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDII SLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIK NCSFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPV VSTQLLLNGSLAEEDVVIRSANFTDNAKTIIVQLNTSVEINCTRPNNN TRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKL REQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFN STWSTEGSNNTEGSDTITLPCRIKQFINMWQEVGKAMYAPPISGQIR CSSNITGLLLTRDGGNNNNGSEIFRPGGGDMRDNWRSELYKYKVV KIEPLGVAPTKAKRRVVQKSLEQIWNNMTWMEWDREINNYTSLIHS LIEESQNQQEKNEQELLELDKGSGSGGSGHHHHHHHH* 7 BG505TSTIPG1 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLAVGIGA VFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQ QHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTN VPGGGGSNLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVW ATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQ SLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDK KQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACP KVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPV VSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNN NTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQL RKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWIS NTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCV SNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP LGVAPTRGGGGSRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQ NQQEKNEQDLLALDHHHHHHHH* 8 NL4-3TSTIPG1 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLREKRAV GIGALFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAP EAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICT TAVPGGGGSNLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHN VWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISL WDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNC SFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKV SFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVS TQLLLNGSLAEEDVVIRSANFTDNAKTIIVQLNTSVEINCTRPNNNTR KSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLRE QFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNST WSTEGSNNTEGSDTITLPCRIKQFINMWQEVGKAMYAPPISGQIRCS SNITGLLLTRDGGNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIE PLGVAPTKGGGGSKSLEQIWNNMTWMEWDREINNYTSLIHSLIEES QNQQEKNEQELLELDKGSGSGGSGHHHHHHHH* 9 BG505TSTIPG2 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLAVGIGA VFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQ QHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTN VPGGGGSGGGGSNLWVTVYYGVPVWKDAETTLFCASDAKAYETE KHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTT ELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAI TQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTH GIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCT RPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKV VKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFN STWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQG VIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYK VVKIEPLGVAPTRGGGGSGGGGSRNLSEIWDNMTWLQWDKEISNY TQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH* 10 NL4-3TSTIPG2 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLREKRAV GIGALFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAP EAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICT TAVPGGGGSGGGGSNLWVTVYYGVPVWKEATTTLFCASDAKAYD TEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMH EDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKG EIKNCSFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQ ACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEDVVIRSANFTDNAKTIIVQLNTSVEINCTRP NNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIA SKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNST WFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQEVGKAMYAPPIS GQIRCSSNITGLLLTRDGGNNNNGSEIFRPGGGDMRDNWRSELYKY KVVKIEPLGVAPTKGGGGSGGGGSKSLEQIWNNMTWMEWDREINN YTSLIHSLIEESQNQQEKNEQELLELDKGSGSGGSGHHHHHHHH* 11 25710-TSTIP AVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLR APEAQQHLLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLI CTTAVPNLWVTVYYGVPVWKEATTTLFCASDAKAYDKEVHNVWA THACVPTDPNPQEMVLGNVTENFNMWKNEMVNQMHEDVISLWD QSLKPCVKLTPLCVTLECSNVTYNESMKEVKNCSFNLTTELRDKKQ KVHALFYRLDIVPLNDTEKKNSSRPYRLINCNTSAITQACPKVTFDPI PIHYCTPAGYAILKCNDKKFNGTGPCHKVSTVQCTHGIKPVVSTQLL LNGSLAEGEIIIRSENLTNNAKTIIVHLNQSVEIVCARPSNNTRTSIRIG PGQTFYATGAITGDIRQAHCNISKDKWNETLQRVGEKLAEHFPNKTI KFNSSSGGDLEITTHSFNCRGEFFYCNTSGLFNGTFNGTYVSPNSTDS NSSSIITIPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLVRD GGTGSESNKTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTKR SQDDIWDNMTWMQWDKEISNYTNTIYKLLEDSQIQQEKNEKDLLA LDSHHHHHHHH 12 X1632-TSTIP MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAIGLGTVLLGF LGTAGSTMGAASITLTVQVRQLLSGIVQQQSNLLRAPEAQQHLLQLT VWGIKQLQARVLAVERYLKDQQILGIWGCSGKLICTTNVPNLWVTV YYGVPVWEDADTTLFCASDAKAYSTESHNVWATHACVPTDPNPQEI YLENVTEDFNMWENNMVEQMQEDIISLWDESLKPCVKLTPLCVTLT CTNVTNVTDSVGTNSRLKGYKEELKNCSFNTTTEIRDKKKQEYALF YKLDIVPINDNSNNSNGYRLINCNVSTIKQACPKVSFDPIPIHYCAPA GFAILKCRDKEFNGTGTCRNVSTVQCTHGIKPVVSTQLLLNGSLAEG DIVIRSENITDNAKTIIVHLNKTVSITCTRPNNNTRKSIRIGPGQALYAT GAIIGDTRQAHCNINGSEWYEMIQNVKNKLNETFKKNITFNPSSGGD LEITTHSFNCRGEFFYCNTSELFNSSHLFNGSTLSTNGTITLPCRIKQI VRMWQRVGQAMYAPPIAGNITCRSNITGLLLTRDGGTNKDTNEAET FRPGGGDMRDNWRSELYKYKVVKIKPLGVAPTRKSYSDIWDNLTWI QWEREISNYTQQIYTLLEESQNQQEKNEQELLALDKHHHHHHHH 13 CH119-TSTIP(BC) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGVAGSTMGAASMTLTVQARQLLSGIVQQQSNLLRAPEAQQHLLQ LTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLICTTAVPNLWV TVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPSP QELVLENVTENFNMWKNEMVNQMHEDVISLWDQSLKPCVKLTPL CVTLECSKVSNNETDKYNGTEEMKNCSFNATTVVRDRQQKVYALF YRLDIVPLTEKNSSENSSKYYRLINCNTSAITQACPKVSFEPIPIHYCT PAGYAILKCNDKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSL AEGEIIIRSENLTNNVKTILVHLNQSVEIVCTRPNNNTRKSIRIGPGQT FYATGDIIGDIRQAHCNISKWHETLKRVSEKLAEHFPNKTINFTSSSG GDLEITTHSFTCRGEFFYCNTSGLFNSTYMPNGTYLHGDTNSNSSITI PCRIKQIINMWQEVGRAMYAPPIEGNITCKSNITGLLLVRDGGTESN NTETNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTAKSQK EIWDNMTWMQWDKEISNYTNTIYKLLEDSQNQQESNEKDLLALDH HHHHHHH 14 CNE8-TSTIP(AE) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAMIFGF LGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAPEAQQHLLQL TVWGIKQLQARVLAVERYLKDQKFLGLWGCSGKIICTTAVPNLWV TVYYGVPVWRDADTTLFCASDAKAYDTEVHNVWATHACVPTDPN PQEIHLENVTENFNMWKNKMAEQMQEDVISLWDESLKPCVQLTPL CVTLNCTNANLNATVNASTTIGNITDEVRNCSFNTTTELRDKKQNV YALFYKLDIVPINNNSEYRLINCNTSVIKQACPKVSFDPIPIHYCAPA GYAILRCNDKNFNGTGPCKNVSSVQCTHGIKPVVSTQLLLNGSLAE DEIIIRSENLTDNVKTIIVHLNKSVEINCTRPSNNTRTSVRIGPGQVFY RTGDIIGDIRKAYCEINRTKWHETLKQVATKLREHFNKTIIFQPPSGG DIEITMHHFNCRGEFFYCNTTKLFNSTWGENTTMEGHNDTIVLPCRI KQIVNMWQGVGQAMYAPPIRGSINCVSNITGILLTRDGGTNMSNET FRPGGGNIKDNWRSELYKYKVVEIEPLGIAPTKRSYEEIWDNMTWIE WEREISNYTSQIYEILTESQNQQDRNEKDLLELDKHHHHHHHH 15 p246F3-TSTIP MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFIGF LGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAPEAQQHLLKL TVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTNVDLWVT VYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNP QEIVMANVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLC VTLDCKDYNYSITNNSTGMEGEIKNCSYNITTELRDKRQKVYSLFY RLDVVQINDSNDRNNSQYRLINCNTTTMTQACPKVTFDPIPIHYCAP AGFAILKCNNKTFNGKGPCNNVSSVQCTHGIKPVVSTQLLINGSLA EKEIVIRSENLTDNVKTIIVHLNESVEINCTRPNNNTRKSVRIGPGQTF YATGDIIGNIRQAHCNVNKTEWNTALTRVSKKLKEYFPNKTIAFQPS SGGDLEITTFSFNCRGEFFYCNTSDLFNGTFNETSGQFNSTFNSTLQC RIKQIINMWQEVGQAMYAPPIAGSITCISNITGLILTRDGGNTNSTKE TFRPGGGNMRDNWRSELYKYKVVKIEPLGVAPTKKSQDEIWDNMT WLQWDKEISNYTQIIYNLIEESQTQQELNERDLLALDHHHHHHHH 16 Bal.26-TSTIP-gp160 MRVTEIRKSYQHWWRWGIMLLGMLMICNAEEKAVGIGAVLLGFL (AA:646-836are GAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQL transmembrane TVWGIKQLQARVLAVERYLRDQQLLGPWGCSGKLICTTAVPNLWV region TVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPN andintracellular PQEVKMENVTENFNMWKNNVVEQMHEDIISLWDQSLKPCVKLTPL region) CVTLNCTDLKNATNGNNTNTTSSSGGMMGGGEMKNCSFNITTNIR GKVQKEYALFYELDIVPIDNKIDSYRLISCNTSVITQACPKVSFEPIPI HYCAPAGFAILKCKDKKFNGKGPCSNVSTVQCTHGIRPVVSTQLLL NGSLAEEEVVIRSENFTNNAKIIVVQLNESVEINCTRPNNNTRKSIHI GPGRAFYTTGEIIGDIRQAHCNLSRAKWNDTLNKIVIKLREQFGNKT IVFKHSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWNVTEESNNTV ENNTITLPCRIKQIINMWQEVGRAMYAPPIRGQIRCSSNITGLLLTRD GGPEDDKTEVFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKKS LNKIWDNMTWMEWDREINNYTSIIYSLIEESQNQQEKNEQELLELD KWASLWNWFDITKWLWYIKIFIMIVGGLIGLRIVESVLSIVNRVRQG YSPLSFQTHLPASRGPDRPGGIEEEGGERDRDRSGPLVNGFLTLIWV DLRSLFLFSYHRLRDLLLIVTRIVELLGRRGWEVLKYWWNLLQYWS QELKNSAVSLLNTIAIAVAEGTDRVIEVVQRAVRAILHIPRRIRQGLE RALL 17 NL4-3TSTIP-gp160 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKAVGIGALFLGF (aa646-837are LGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAPEAQQHLLQ transmembrane LTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPNLWV region TVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPN andintracellular PQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPL region) CVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRDKVQKE YAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEPIPIHYCAPAGF AILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEDV VIRSANFTDNAKTIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFV TIGKIGNMRQAHCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSS GGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSD TITLPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGN NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWA SLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSP LSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRS LCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQEL KNSAVNLLNATAIAVAEGTDRVIEVLQAAYRAIRHIPRRIRQGLERIL L 18 BG505gp160 MRVMGIQRNCQHLFRWGTMILGMIIICSAAENLWVTVYYGVPVWK (aa662-859are DAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEE transmembrane FNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNN region ITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGN andintracellular RSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKD region) KKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENIT NNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIR QAHCTVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTT HSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQ IINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRP GGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRVVGREKRAVGI GAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAIEA QQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTT NVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQN QQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRI VFAVLSVIHRVRQGYSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGR STRLVSGFLALAWDDLRSLCLFCYHRLRDFILIAARIVELLGHSSLKG LRLGWEGLKYLWNLLAYWGRELKISAINLFDTIAIAVAEWTDRVIEI GQRLCRAFLHIPRRIRQGLERAL 19 NL4-3gp160 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYYGVP (aa663-854are VWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLV transmembrane NVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCT region DLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRDKVQKEYAFFYKL andintracellular DIVPIDNTSYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNN region) KTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEDVVIRSANF TDNAKTIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGN MRQAHCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRI KQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEI FRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKR AVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKL ICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEE SQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLV GLRIVFAVLSIVNRVRQGYSPLSFQTHLPIPRGPDRPEGIEEEGGERD RDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRG WEALKYWWNLLQYWSQELKNSAVNLLNATAIAVAEGTDRVIEVL QAAYRAIRHIPRRIRQGLERILL* 20 25710gp160 MRVRGTLRNYQQWWIWGVLGFWMLMICNVGGNLWVTVYYGVP (aa657-855are VWKEATTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEMVLG transmembrane NVTENFNMWKNEMVNQMHEDVISLWDQSLKPCVKLTPLCVTLECS region NVTYNESMKEVKNCSFNLTTELRDKKQKVHALFYRLDIVPLNDTEK andintracellular KNSSRPYRLINCNTSAITQACPKVTFDPIPIHYCTPAGYAILKCNDKK region) FNGTGPCHKVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENLTNN AKTIIVHLNQSVEIVCARPSNNTRTSIRIGPGQTFYATGAITGDIRQAH CNISKDKWNETLQRVGEKLAEHFPNKTIKFNSSSGGDLEITTHSFNC RGEFFYCNTSGLFNGTFNGTYVSPNSTDSNSSSIITIPCRIKQIINMWQ EVGRAMYAPPIAGNITCKSNITGLLLVRDGGTGSESNKTEIFRPGGG DMRDNWRSELYKYKVVEIKPLGVAPTKAKRRVVEREKRAVGIGAV FLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHL LQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLICTTAVPWN YSWSNRSQDDIWDNMTWMQWDKEISNYTNTIYKLLEDSQIQQEKN EKDLLALDSWENLWNWFNITNWLWYIKIFIIIVGGLIGLRIIFAVLPIV NRVRQGYSPLSFQTHTPTPGGPDRLGRIEEEGGEQDNVRSIRLVNGF LALAWDDLRNLCLFSYHRLRDFILVAARVVELLGRNSLRGLQKGW EALKYLGSLVQYWGLELKRSAISLLDTIAIAVAEGTDRIIQLGQGICR AICNIPRRIRQGLEAALQ 21 X1632gp160 MKVKGTQRDWHSLWNWGILILGLVIICSASNNLWVTVYYGVPVWE (aa660-858are DADTTLFCASDAKAYSTESHNVWATHACVPTDPNPQEIYLENVTED transmembrane FNMWENNMVEQMQEDIISLWDESLKPCVKLTPLCVTLTCTNVTNV region TDSVGTNSRLKGYKEELKNCSFNTTTEIRDKKKQEYALFYKLDIVPI andintracellular NDNSNNSNGYRLINCNVSTIKQACPKVSFDPIPIHYCAPAGFAILKCR region) DKEFNGTGTCRNVSTVQCTHGIKPVVSTQLLLNGSLAEGDIVIRSENI TDNAKTIIVHLNKTVSITCTRPNNNTRKSIRIGPGQALYATGAIIGDT RQAHCNINGSEWYEMIQNVKNKLNETFKKNITFNPSSGGDLEITTHS FNCRGEFFYCNTSELFNSSHLFNGSTLSTNGTITLPCRIKQIVRMWQR VGQAMYAPPIAGNITCRSNITGLLLTRDGGTNKDTNEAETFRPGGG DMRDNWRSELYKYKVVKIKPLGVAPTRARRRVVEREKRAIGLGTV LLGFLGTAGSTMGAASITLTVQVRQLLSGIVQQQSNLLRAIEAQQHL LQLTVWGIKQLQARVLAVERYLKDQQILGIWGCSGKLICTTNVPWN SSWSNKSYSDIWDNLTWIQWEREISNYTQQIYTLLEESQNQQEKNE QELLALDKWASLWNWFDITNWLWYIKIFIMIVGGLIGLRIVFAVLSII NRVRKGYSPLSFQTLTRHQREPDRPGGIEEEDGEQDRDKSVRFVSGF LSPVWDDLRSLCLFSYRRLRDFILVAARTVELLGRSSLKGLRLGWE GLKYLWNLLLYWGRELKSSAINLLDTTAIAVANWTDRVIEVGQRIV RAFLHIPVRIRQGLERALL 22 CH119gp160 MRVTGIRKNYRHLWRWGTMLLGMLMICSAVGNLWVTVYYGVPV (aa663-862are WKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPSPQELVLENVT transmembrane ENFNMWKNEMVNQMHEDVISLWDQSLKPCVKLTPLCVTLECSKVS region NNETDKYNGTEEMKNCSFNATTVVRDRQQKVYALFYRLDIVPLTEK andintracellular NSSENSSKYYRLINCNTSAITQACPKVSFEPIPIHYCTPAGYAILKCND region) KTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENLT NNVKTILVHLNQSVEIVCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQ AHCNISKWHETLKRVSEKLAEHFPNKTINFTSSSGGDLEITTHSFTCR GEFFYCNTSGLFNSTYMPNGTYLHGDTNSNSSITIPCRIKQIINMWQE VGRAMYAPPIEGNITCKSNITGLLLVRDGGTESNNTETNNTEIFRPGG GDMRDNWRSELYKYKVVEIKPLGVAPTAAKRRVVEREKRAVGIGA VFLGFLGVAGSTMGAASMTLTVQARQLLSGIVQQQSNLLRAIEAQQ HLLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLICTTAVPW NSSWSNKSQKEIWDNMTWMQWDKEISNYTNTIYKLLEDSQNQQES NEKDLLALDSWNNLWNWFNITQWLWYIKIFIIIVGGLIGLRIIFAVLSI VNRVRQGYSPLSFQTLTPTSGGRPDRLERIEEEGGEQDRDRSIRLVNG FLALAWDDLRNLCLFSYHRLRDFILVAARVVELLGRTSLRGLQRGW EALKYLGSLVQYWGQELKKSAISLVDTIAIVVAEGTDRIIDIVQAFCR AIYNIPRRIRQGFEAALQ 23 CNE8gp160 MRVKETQMNWPNLWKWGTLILGLVIICSASDNLWVTVYYGVPVW (aa652-850are RDADTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVT transmembrane ENFNMWKNKMAEQMQEDVISLWDESLKPCVQLTPLCVTLNCTNA region NLNATVNASTTIGNITDEVRNCSFNTTTELRDKKQNVYALFYKLDIV andintracellular PINNNSEYRLINCNTSVIKQACPKVSFDPIPIHYCAPAGYAILRCNDK region) NFNGTGPCKNVSSVQCTHGIKPVVSTQLLLNGSLAEDEIIIRSENLTD NVKTIIVHLNKSVEINCTRPSNNTRTSVRIGPGQVFYRTGDIIGDIRKA YCEINRTKWHETLKQVATKLREHFNKTIIFQPPSGGDIEITMHHFNC RGEFFYCNTTKLFNSTWGENTTMEGHNDTIVLPCRIKQIVNMWQGV GQAMYAPPIRGSINCVSNITGILLTRDGGTNMSNETFRPGGGNIKDN WRSELYKYKVVEIEPLGIAPTKAKRRVVEREKRAVGIGAMIFGFLG AAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAPEAQQHLLQLTV WGIKQLQARVLAVERYLKDQKFLGLWGCSGKIICTTAVPWNSTWS NRSYEEIWDNMTWIEWEREISNYTSQIYEILTESQNQQDRNEKDLLE LDKWASLWNWFDITRWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVR QGYSPLSFQTPTHHQREPDRPERIEEGGGEQDRDRSVRLVSGFLALA WDDLRSLCLFSYHRLRDLILIAVRTVELLGHGGLKGLRRGWEGLKY LGNLLLYWGQELKISAISLLDATAIAVAGWTDRIIEVAQRAWRAILH IPRRIRQGLERSLL 24 p246F3gp160 MRARGMLRTWQHWWIWGILGFWMLMICNMQDLWVTVYYGVPV (aa654-852are WKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVMAN transmembrane VTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLDCKD region YNYSITNNSTGMEGEIKNCSYNITTELRDKRQKVYSLFYRLDVVQIN andintracellular DSNDRNNSQYRLINCNTTTMTQACPKVTFDPIPIHYCAPAGFAILKC region) NNKTFNGKGPCNNVSSVQCTHGIKPVVSTQLLLNGSLAEKEIVIRSE NLTDNVKTIIVHLNESVEINCTRPNNNTRKSVRIGPGQTFYATGDIIG NIRQAHCTVNKTEWNTALTRVSKKLKEYFPNKTIAFQPSSGGDLEIT TFSFNCRGEFFYCNTSDLFNGTFNETSGQFNSTFNSTLQCRIKQIINM WQEVGQAMYAPPIAGSITCISNITGLILTRDGGNTNSTKETFRPGGGN MRDNWRSELYKYKVVKIEPLGVAPTKARRRVVEREKRAVGIGAVFI GFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHLL KLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTNVPWNS SWSNKSQDEIWDNMTWLQWDKEISNYTQIIYNLIEESQTQQELNER DLLALDKWANLWNWFDITKWLWYIKIFIMIVGGLIGLRIIFAVLSIVN RVRQGYSPLSFQTLTPNPRGPDRPGGIEEEGGEQGRNSYTRLVSGFLP LAWDDLRSLCLFSYHLLRDFILIAARAAELLGRSSLRGLQRGWETLK YLGSLVQYWGLELKKSAISLLDTIAIQVAEGTDRIIELIQGIYRAIRNIP RRIRQGAETALV 25 Bal.26gp160 MRVTEIRKSYQHWWRWGIMLLGMLMICNAEEKLWVTVYYGVPV (aa662-853are WKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVKMEN transmembrane VTENFNMWKNNVVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTD region LKNATNGNNTNTTSSSGGMMGGGEMKNCSFNITTNIRGKVQKEYA andintracellular LFYELDIVPIDNKIDSYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFA region) ILKCKDKKFNGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVV IRSENFTNNAKIIVVQLNESVEINCTRPNNNTRKSIHIGPGRAFYTTGE IIGDIRQAHCNLSRAKWNDTLNKIVIKLREQFGNKTIVFKHSSGGDPE IVTHSFNCGGEFFYCNSTQLFNSTWNVTEESNNTVENNTITLPCRIKQ IINMWQEVGRAMYAPPIRGQIRCSSNITGLLLTRDGGPEDDKTEVFR PGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGAVLLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAI EAQQHLLQLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLIC TTAVPWNASWSNKSLNKIWDNMTWMEWDREINNYTSIIYSLIEESQ NQQEKNEQELLELDKWASLWNWFDITKWLWYIKIFIMIVGGLIGLR IVFSVLSIVNRVRQGYSPLSFQTHLPASRGPDRPGGIEEEGGERDRDR SGPLVNGFLTLIWVDLRSLFLFSYHRLRDLLLIVTRIVELLGRRGWE VLKYWWNLLQYWSQELKNSAVSLLNTIAIAVAEGTDRVIEVVQRA VRAILHIPRRIRQGLERALL* 26 BG505-deleted WNSSWSN sequencefromgp41 27 BG505-deleted AKRRVVGREKR sequencefromgp120 28 Linker-1 GGGGS 29 Linker-2 GGGGSGGGGS 30 BG-B1(1/1) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATH ACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKP CVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQK VYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSF EPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQ LLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRK SIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHF GNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSV QGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGV APTRAKRRVVGGGGGSWNSSWSNRNLSEIWDNMTWLQWDKEISN YTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH 31 BG-B1(1/2) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATH ACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKP CVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQK VYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSF EPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQ LLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRK SIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHF GNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSV QGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNIT GLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGV APTRAKRRVVGGGGGSGGGGSWNSSWSNRNLSEIWDNMTWLQW DKEISNYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH 32 BG-B1(2/2) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSGGGGSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHN VWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLW DQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELR DKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIK PVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPN NNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVK QLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNST WISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVI RCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVV KIEPLGVAPTRAKRRVVGGGGGSGGGGSWNSSWSNRNLSEIWDNM TWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHH H 33 BG-B1(2/1) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSGGGGSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHN VWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLW DQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELR DKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIK PVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPN NNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVK QLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNST WISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVI RCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVV KIEPLGVAPTRAKRRVVGGGGGSWNSSWSNRNLSEIWDNMTWLQ WDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH 34 BG-B1(2/3) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSGGGGSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHN VWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLW DQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELR DKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQA CPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIK PVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPN NNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVK QLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNST WISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVI RCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVV KIEPLGVAPTRAKRRVVGGGGGSGGGGSGGGGSWNSSWSNRNLSE IWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDHH HHHHHH 35 BG-B2(1-1) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACV PTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVK LTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSL FYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPI HYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLN GSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIG PGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNN TIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGS NSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLIL TRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTR AKRRVVGGGGGSWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQII YGLLEESQNQQEKNEQDLLALDHHHHHHHH 36 BG-B2(1-2) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACV PTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVK LTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSL FYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPI HYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLN GSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIG PGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNN TIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGS NSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLIL TRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTR AKRRVVGGGGGSGGGGGSWNSSWSNRNLSEIWDNMTWLQWDKEI SNYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH 37 BG-B2(1-3) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPGGG GSLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACV PTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVK LTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSL FYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPI HYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLN GSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIG PGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNN TIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGS NSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLIL TRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTR AKRRVVGGGGGSGGGGSGGGGSWNSSWSNRNLSEIWDNMTWLQ WDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH 38 BG-C1(1/1) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSS WSNGGGGSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKH NVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISL WDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTE LRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAIT QACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTH GIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCT RPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKV VKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFN STWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQG VIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYK VVKIEPLGVAPTRAKRRVVGGGGGSRNLSEIWDNMTWLQWDKEIS NYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH 39 BG-C1(1/2) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSS WSNGGGGSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKH NVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISL WDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTE LRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAIT QACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTH GIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCT RPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKV VKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFN STWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQG VIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYK VVKIEPLGVAPTRAKRRVVGGGGGSGGGGSRNLSEIWDNMTWLQ WDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHHH 40 BG-C1(1/3) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAVGIGAVFLGF LGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLK LTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSS WSNGGGGSAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKH NVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISL WDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTE LRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAIT QACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTH GIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCT RPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKV VKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLEN STWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQG VIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYK VVKIEPLGVAPTRAKRRVVGGGGGSGGGGSGGGGSRNLSEIWDNM TWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDHHHHHHH H 41 gp160protein-furin REKR cleavagesite

EXAMPLES

[0185] The present invention will now be described with reference to the following examples, which are intended to illustrate the present invention, but not to limit it.

[0186] Unless otherwise specified, the molecular biology experiment methods and immunoassay methods used in the present invention were basically referred to the methods described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes were used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention.

Example 1: Expression of TSTIP Proteins of Two Strains of NL4-3/BG505

[0187] Modification Design of 4-3/BG505 TSTIP

[0188] The base and amino acid sequences of gp160 of the two strains BG505/pNL4-3 on NCBI were used as templates for modification. Taking the modification of BG505 as an example, according to the three-dimensional cryoelectron microscope structure of BG505-SOSIP (PDB: 4tvp), part of the loop sequence (aa610-616, WNSSWSN) at the C-terminal of 27 of BG505 gp41 was removed, and the sequence of 10 amino acids including furin cleavage site at the C-terminal of gp120 (aa501-511, AKRRVVGREKR) was also removed. Subsequently, the truncated C-terminal of 27 of gp41 was linked to the N-terminal of gp120, and the truncated C-terminal of gp120 was linked to the N-terminal of the 8 domain of gp41. The amino acids of the complete gp140 after modification were arranged in sequence as follows: 6/7/27 (501-606)+part of the loop region between 27 and 8 (607-609)+gp120 (33-500)+part of the C-terminal sequence of the loop region between 27 and 8 (617-618)+8/9 (619-664).

[0189] In addition, 8His tag was added to the C-terminal of the above sequence to facilitate purification, and tPA signal peptide and kozak sequence were added in front of the sequence to promote secretion and expression. The protein designed above was named as BG505-TST (SEQ ID NO: 1). On the basis of the above structure, I559P and T332N mutations were introduced, and the protein designed was named as BG505-TSTIP (SEQ ID NO: 3). NL4-3 strain was modified in the same way to obtain NL4-3-TST (SEQ ID NO: 2) and NL4-3-TSTIP (SEQ ID NO: 4), respectively. The structure of each of the above proteins is shown in FIG. 1.

[0190] The designed amino acid sequences of BG505/pNL4-3-TSTIP were converted into base sequences suitable for mammalian cell expression, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis, and constructed between the ECORI and Xbal restriction sites of pcDNA3.1 vector. 1 l of the synthesized pcDNA3.1-BG505SOSIP/4-3TSTIP plasmid was taken to transform 50p1 of DH5a competent cells (purchased from Shenzhen Kangti Life Technology Co., Ltd.), which were plated on ampicillin-containing solid medium; after static culture at 37 C. for 10-12 hours, single colony was clearly visible, and was picked and placed into a test tube containing 3 ml of ampicillin-containing LB medium, and cultured under shaking at 220 rpm at 37 C. for 10 hours; 500 l of the bacterial solution was taken and mixed with 500111 of 50% glycerol, then cryopreserved at 20 C.

[0191] Extraction of PcDNA3.1 NL4-3/B G505 TSTIP Plasmid:

[0192] 10 l of the pcDA3.1 NL4-3/BG505 TSTIP bacterial solution cryopreserved at -20 C. was taken, transferred into a test tube containing 3 ml of ampicillin-containing LB medium, cultured for 10-12 h, then inoculated in a conical flask containing 500 ml of ampicillin-containing LB medium and cultured at 37 C. for 12 hours; the bacterial solution was collected and centrifuged at 7000 g for 10 minutes, the supernatant was discarded. The pcDNA3.1-BG505/NL4-3TSTIP plasmid was extracted by using Tiangen Endotoxin-Free Maxi Plasmid Kit.

[0193] Culture and Passage of 293F Cells 293F cells cryopreserved in 80 C. refrigerator were taken, thawed at 37 C., then centrifuged at 1300 rpm for 4 min; the supernatant was discarded in an ultra-clean bench, the cells were lightly flicked and resuspended in 293freestyle medium warmed at 37 C. in advance, then transferred into a conical flask containing 50 ml of warm culture medium, for suspension culture at 37 C., 5% CO2, 120 rpm; the cells were passaged when the cell density reached 2.0*10.sup.6, to gradually expand the culture system.

[0194] Transient Transfection

[0195] The 293F cells were transiently transfected with PEG2000. After the cell density reached 2.0*10.sup.6, the cells were harvested in a sterile 50 ml tube, centrifuged at 1300rpm for 4min; the cells were flicked, then resuspended in medium incubated at 37 C., transferred to a conical flask containing 450 ml of medium incubated at 37 C., and placed on a shaker at 37 C. for later use.

[0196] The extracted pcDNA3.1-BG505/NL4-3TSTIP plasmid and PEG2000 in a ratio of 1:2 was added into 50 ml of culture medium, mixed well and allowed to stand for 18min, then transferred into the above 450 ml of culture medium, for suspension-culture at 37 C., 5% CO2, 120rpm for 6 days to express BG505/NL4-3TSTIP protein. It should be aware the PEG operation should be carried out in the dark during transfection.

Example 2: Purification of NL4-3/BG505 TSTIP Protein

[0197] After 6 days of transient transfection, the cell culture medium was collected, centrifuged at 7000 g for 10 minutes with JA-14 rotor; the cell supernatant was collected, centrifuged at 20000g for 10 minutes; the supernatant was taken and filtered twice with a 0.22pm pore size membrane filter, and this sample was used for the next step of Ni-excel column purification.

[0198] Purification by Ni affinity chromatography using the AKTA system;

[0199] Instrument system: AKTA Pure type preparative liquid chromatograph;

[0200] Purification medium: Ni Sepharose excel affinity medium; buffer: buffers A and B, buffer A was 1PBS buffer, buffer B was 1PBS+250 mmol/L imidazole buffer;

[0201] System sample-loading flow rate: 8 mL/min; detection wavelength: UV @280 nm

[0202] System elution flow rate: 4 ml/min; detection wavelength: UV @280 nm

[0203] Elution conditions: 20 mM imidazole was used to elute impurity proteins, and the product eluted by 250 mM imidazole was collected. The eluate was dialyzed against 1PBS overnight with two dialysate changes. About 30 ml of low-concentration target protein was harvested, and concentrated in Vivaspin 20 ml, 100 KD ultrafiltration concentrator tube to 5 ml for later use.

Example 3: Identification of Biochemical Properties of NL4-3/BG505 TSTIP protein SDS-PAGE:

[0204] The concentrated sample in Example 2 and BG505/4-3SOSIP protein were diluted to 11.tg/111, two tubes of 50 l samples were then taken, added with 10 l of reducing 6 Loading Buffer and 10111 non-reducing 6Loading Buffer, respectively, to prepare reduced samples and non-reduced samples, the reduced samples were placed in a boiling water bath at 100 C. for 10 minutes. 10111 of the reduced and non-reduced samples were electrophoresed in 8% SDS-PAGE at a voltage of 80V for 120 min, and the electrophoresis bands were displayed after staining with Coomassie brilliant blue. The electrophoresis results are shown in FIGS. 2A to 2B. SDS-PAGE analysis showed that after one-step Ni-EXCEL purification, high-purity BG505/NL4-3TSTIP protein could be obtained, and the TSTIP protein showed a complete 140 KD band in the presence of DTT, while the BG505/NL4-3SOSIP protein presented a 120 KD band under reducing conditions, indicating that the gp120 and gp41 extracellular regions were stably and covalently linked in the protein designed by the inventors as expected.

[0205] Molecular Sieve Purification:

[0206] Instrument system: AKTA Pure type preparative liquid chromatograph;

[0207] Chromatographic column: superdex 200 16/600

[0208] Column volume: 120 ml

[0209] Buffer: PBS (20 mM phosphate buffer, pH7.5, 150 mM NaCl)

[0210] Detector wavelength: 280 nm

[0211] Flow rate: 1 ml/min

[0212] The sample was the purified concentrated protein of Example 2.

[0213] The purification procedure comprised: superdex200 16/600 was equilibrated with 1 column volume of PBS, the target protein purified in Example 2 was loaded using a 5 ml loading loop, and the molecular sieve purification of the purified sample was performed at inject mode, and the components of the sample would be eluted in sequence according to their molecular weights, from high to low, and the elution peaks at different elution volumes were collected, which are the target proteins in different multimer forms.

[0214] The molecular sieve purification results are shown in FIG. 3. It could be seen that the components of BG505/NL4-3TSTIP designed by the inventors were more simple than those of BG505/NL4-3, while the molecular sieve purification pattern of BG505/NL4-3TSTIP showed obvious peaks for monomers and multimers, indicating that the TSTIP protein and TST protein of the present invention had a higher trimer content.

[0215] SDS-PAGE of Molecular Sieve Purified Samples:

[0216] The samples of different elution volumes collected during the molecular sieve purification were concentrated in Vivaspin 20ml, 100 KD concentrator tubes to 1 g/l, and marked as 1, 2, 3, 4 according to the order of elution. 50 l of each of the samples was taken and added with 10 l of non-reducing 6 Loading Buffer, mixed well, 10 l of non-reduced sample was taken and electrophoresed at 80V voltage for 120 min in 8% SDS-PAGE, and the electrophoresis bands were displayed after staining with Coomassie brilliant blue. The electrophoresis results were shown in FIGS. 4A to 4C. It could be seen that the main component of the BG505/NL4-3TSTIP was gp140 trimer after superdex200-16/600 purification, while the BG505/NL4-3SOSIP contained a large amount of gp140 monomer.

[0217] Thermal Stability Analysis by Differential Scanning Calorimetry (DSC)

[0218] Instrument system: VP-Capillary produced by GE Healthcare

[0219] The sample was the purified concentrated protein of Example 2.

[0220] The sample-loading tank was washed once with an acidic or weakly alkaline washing solution, and washed 3 times with deionized water; 400 L of the sample and corresponding buffer solution thereof each was pipetted into EP tube, and centrifuged to remove sediment and air bubbles; 300 L of buffer solution was pipetted into the sample-loading tank and control tank, respectively, and rinsed 3 times, then 300 L of buffer solution was added to the sample-loading tank and control tank with avoiding air bubbles during the addition process; the DSC software and instrument were turned on, the number of scan cycles was set, and when the DP value in the scan cycle was stabilized between 0.2, it was allowed to perform circular scanning and the baseline equilibration was carried out for no less than 3 times; when the last circular scanning was completed, and the temperature dropped to between 30 C. and 10 C., the buffer in the sample tank was sucked out, and 300111 of protein sample to be tested was added quickly, and the scanning test was continued; the scan rate was set as: 90 C./h. After scanning, Origin7.0 was used to process the data. The results were shown in FIG. 5. The Tm of the protein BG505-TSTIP of the present invention was 74.51 C., the Tm of the control protein BG505-SOSIP was significantly lower than that of BG505-TSTIP, which was 68.18 C. The Tm of NL4-3-TSTIP was 63.41 C., and the Tm of NL4-3-SOSIP was 62.00 C.; although the Tm of the one based on NL4-3 strain did not increase significantly, it was improved to a certain extent compared with the reported optimal design NL4-3SOSIP. These results indicated that our design could improve the thermal stability of the protein to a certain extent, and its thermal stability could be greatly improved in some strains.

Example 4: Identification of BG505/NL4-3TSTIP Antigenicity and Immunogenicity

[0221] Enzyme-linked Immunosorbent Assay (ELISA)

[0222] Broad-spectrum neutralizing antibodies such as PGT121, PGT125, VRC01, 2G12, B12 and non-neutralizing antibodies such as 17b, 447-52D, F105, F240 were selected to carry out ELISA antigenicity analysis on BG505/NL4-3TSTIP and BG505/NL4-3SOSIP, and the specific process was as follows:

[0223] (1) BG505/NL4-3TSTIP and BG505/NL4-3SOSIP trimer proteins were diluted with 1xCB to 1 g/M1, coated at 100 L/well on a 96-well plate, and allowed to stand in a 37 C. incubator for 2 h;

[0224] (2) the plate was washed once and spin-dried, and blocked with ED (180 L/well) in a 37 C. incubator for 2 h;

[0225] (3) the plate was washed once and spin-dried. The 96-well u-bottom plate was taken, the antibody was diluted to 1 g/ml or 10g/ml, and added to the first well of the U-bottom plates, repeats of two wells were performed for each antibody, with 150 l at first well, and 3-fold dilution with 11 gradients. 100 l of the diluted antibody was transferred into the 96-well ELISA plates coated with BG505/NL4-3TSTIP and BG505/NL4-3SOSIP, and reacted at 37 C. for 1 hour.

[0226] (4) the plate was washed 5 times and spin-dried. The secondary antibody GAH-HRP (1:5000) was added into the 96-well plate, 100 L/well, and reacted at 37 C. for 45 minutes;

[0227] (5) the plate was washed 5 times, color development was performed at room temperature for min, then stopped, and detection was carried out at wavelength of 450 nm on a microplate reader; GraphPad Prism 5 (GraphPad, USA) software was used for data analysis. The results are shown in FIG. 6.

[0228] It could be seen that BG505/NL4-3TSTIP had strong reactivity with broad-spectrum neutralizing antibodies such as PGT121, PGT125, VRC01, 2G12, and B12, but weak reactivity with non-neutralizing antibodies such as 17b, F105, and F240. 17b, F105, and F240 were antibodies targeting non-neutralizing epitopes such as CDi, CD4bs, and gp41, respectively, which indicated that BG505/NL4-3TSTIP did not expose such non-neutralizing epitopes, but well presented key epitopes such as CD4bs, out Glycan, V3, etc. Overall, the reactivities of BG505/NL4-3TSTIP with various broad-spectrum neutralizing antibodies and non-neutralizing antibodies remained comparable to those of the control protein BG505/NL4-3SOSIP.

[0229] Immunological Evaluation of BG505/NL4-3TSTIP in Animals

[0230] White mice: female, 6 weeks old, purchased from Shanghai Slack Experimental Animal Co., Ltd. Four groups of immunized mice were set up, with 6 mice in each group, and BG505/NL4-3TSTIP and BG505/NL4-3SOSIP prepared in Example 2 were used as immune proteins. The proteins were diluted with normal saline, mixed with aluminum hydroxide adjuvant at a ratio of 1:1 by volume, so that the proteins were adsorbed on the adjuvant, and the mice were immunized intraperitoneally. The mouse immunization protocols were shown in Table 1.

TABLE-US-00002 TABLE 1 Mouse immunization protocol Group Immunogen Dose Period (weeks) A NL4-3TSTIP 10 g/animal 2 B NL4-3SOSIP 10 g/animal 2 C BG505/NL4-3TSTIP 10 g/animal 2 D BG505/NL4-3SOSIP 10 g/animal 2

[0231] The blood was collected from mouse eyeball before immunization, and immunization was carried out according to the immunization protocol in Table 1. The blood was collected from mouse eyeball before each immunization, and after the sixth injection of immunization, the blood was collected from mouse eyeball and the mice were treated with neck dislocation. After the blood sample was placed at 37 C. for 30 minutes, it was centrifuged at 13300 rpm for 10 minutes, and the serum was collected for HIV-1 pseudovirus neutralization and antibody titer determination.

[0232] The serum sample collected after the sixth injection was used to perform the virus neutralization experiment, and the results are shown in FIG. 7. For all four groups of mice, a strong neutralization response against NL4-3 was detected, but no neutralization response to the tier2 strain TRO11 of the same subtype was detected. The results showed that the BG505/NL4-3TSTIP of the present invention could induce a strong neutralizing antibody response against the corresponding strain, but it was difficult to generate a neutralizing response against other strains. In general, the protein of the present invention maintained a level of immunogenicity comparable to that of the control protein SOSIP. Those skilled in the art know that it is possible to induce virus cross-neutralizing antibodies by introducing amino acid mutations of cross-reactivity epitopes based on the protein of the present invention.

Example 5: Production and identification of B1.26-TSTIP Pseudoviral Particle

[0233] Construction of B1.26-TSTIP and NL4-3-TSTIP Pseudoviruses

[0234] The Bal26 TSTIP pseudovirus produced by the present inventors was obtained by co-transfecting 293FT cells (Invitrogen) with HIV-1 backbone plasmid pfNL43-dGPE-EGFP (Addgene) and envelope plasmid VRC8400-Bal26-TSTIP-gp160.

[0235] Acquisition of Envelope Gene and Backbone Gene

[0236] The backbone plasmid was pfNL43-dGPE-EGFP, purchased from addgene. The plasmid was obtained by modifying NL4-3 infectious clone, and contained gag, pol, tat, rev and other genes necessary for virus packaging, but its Env gene had been partially replaced by the EGFP gene, resulting in the silencing of the Env gene, so that the production of pseudovirus could be performed by co-transfecting only one expression plasmid expressing Env. The expression vector of the envelope gene used in the present invention was VRC8400 which was preserved in our laboratory. The inventor carried out the TSTIP design for the full length of the Env gene of Bal.26 based on the design of BG505 TSTIP protein, and cloned the full-length Bal.26 TSTIP gene (which encoded SEQ ID NO: 16) carrying the virus's own signal peptide between the EcoRV and BglII restriction sites of the VRC8400 vector, to construct a VRC8400-Bal.26-TSTIP-gp160 expression plasmid. The obtained pseudovirus was called BaL.26-SD-FS. In addition, a pseudovirus (BaL.26-WT) obtained based on the wild-type gp160 sequence of the B1.26 strain was used as a control.

[0237] Production of Pseudovirus:

[0238] The production of pseudovirus adopted the method of PEI transient transfection, the mixture of pfNL43-dGPE-EGFP plasmid and envelope plasmid of VRC8400-B1.26 TSTIP gp160 plasmid, and PEI were diluted in 90 l of normal saline respectively, and the two were fully mixed and allowed to stand for 18 minutes to form a plasmid-PEI complex, and co-transfected into 293FT cells at ratios of PEI:plasmid=2:1, pfNL43-dGPE-EGFP:VRC8400 Bal.26 TSTIP gp160=1:1, with the amount of plasmid of 20 m/plate. After 6 hours of transfection, fresh complete DMEM medium was replaced, and culturing was performed at 37 C., 5% CO.sub.2 for 48 hours to obtain a supernatant as a virus liquid.

[0239] Verification of Pseudovirus Packaging Capability

[0240] The above-mentioned transfected 293FT cells and cell transfection supernatant were harvested, lysed with cell lysis buffer for 1 hour, the cell lysate supernatant was collected by centrifugation, and subjected to western blot, and the specific steps were as follows:

[0241] (1) 20 l of cell lysate and virus liquid supernatant were taken and electrophoresed at 80V on 8% SDS polyacrylamide gel.

[0242] (2) after 2 hours, the gel was transferred to a nitrocellulose membrane with two layers of filter paper placed on each of its upside and downside, 1Trans-Blot Turbo Transfer Buffer (SDS) was used as transfer buffer for semi-dry transfer, with the transferring time of 30 min.

[0243] (3) the membrane was washed with deionized water, and the membrane was blocked with blocking solution 1 (purchased from Xiamen Wantai) for 2 h.

[0244] (4) the monoclonal antibody 3A7 screened in the laboratory was used as the primary antibody, the primary antibody was diluted in ED11 (purchased from Xiamen Wantai) at a concentration of 1 g/ml, and incubated for 1 hour at room temperature on a shaker.

[0245] (5) the membrane was washed with 1PBST washing solution for 3 times, 5 min for each time, and washed with deionized water once, the secondary antibody GAM-HRP was diluted in ED11 (purchased from Beijing Wantai) at a ratio of 1:5000, and incubated at room temperature for min on a shaker.

[0246] (6) the membrane was washed with 1PBST washing solution for 3 times, 5 min for each time, and washed with deionized water once, and then exposed for color development. The results of Western blot are shown in FIG. 8A, in which the Env protein was detected in both cell lysate and virus liquid.

[0247] At the same time, the virus liquid was diluted by 5-fold dilution for 6 gradients, and the p24 content in the virus liquid supernatant was detected by ELISA method, and the fitting curve was made according to the reaction of the p24 standard substance in the gradient dilution, and the fitting curve was Y=3618*X+151.3 (FIG. 8B), R.sup.2 was 0.996, which had a good linear relationship. The quantitative results of p24 were shown in Table 2. At the same time, the samples of the harvested pseudovirus liquid were observed by negative staining electron microscope, and the results were shown in FIG. 9. Based on the above results, the p24 content of the pseudovirus produced by the present invention had decreased to a certain extent, but within an acceptable range, the expression of Env protein could be detected in the supernatant, and obvious pseudoviral particles could be observed under negative staining electron microscope, indicating that the pseudovirus of the present invention could be normally packaged to form pseudoviral particles.

TABLE-US-00003 TABLE 2 Detection of p24 content Sample Dilution fold AOD630-450 p24 (ng/mL) BaL.26-SD-FS 25 0.402 40.08 BaL.26-WT 25 0.729 69.72 Negative control 1 0.008 0.18

[0248] Verification of Pseudovirus Infection Ability

[0249] The harvested virus liquid was diluted in a U-bottom 96-well plate, 100 l of virus liquid was added in the first well, 5-fold dilution was performed for 5 times in sequence, and each well contained 15 g/ml DEAE to promote the infectivity of pseudovirus to cells. TZM-b1 cells were cultured in a 96-well cell culture plate in advance at 37 C., 5% CO.sub.2 until the cell density reached 80%, the diluted pseudovirus liquid was transferred to TZM-b 1 cells, and after the cells were infected with the virus for 40-48 h, two methods (i.e., chemiluminescence and ELISPOT) were used to detect the infectivity of the produced pseudovirus to the target cells. The results were shown in FIG. 10, and both detection methods showed that the pseudovirus modified by TSTIP design almost completely lost its ability to infect, while the natural pseudovirus could normally infect TZM-b1 cells.

[0250] The verification of the production, packaging and infectivity of the NL4-3TSTIP and

[0251] NL4-3WT pseudoviral particles were carried out according to the verification methods for the production, packaging and infectivity of the above-mentioned B126 pseudovirus.

Example 6: Identification of Pseudoviral Particle Immunouenicity

[0252] Production and Purification of Pseudoviral Particle

[0253] According to the method for producing pseudovirus in Example 5, large scale production of pseudoviral particles (e.g., NL4-3-TSTIP and un-mutated NL4-3-WT pseudoviral particles) was performed by transfection of 293FT cells, and the virus supernatant was harvested and subjected to density gradient centrifugation and purification to obtain pseudoviral particles, and the specific process was as follows:

[0254] (1) 20% sucrose solution was prepared in advance and filtered with a 0.224tm syringe filter into a sterile tube;

[0255] (2) a set of internal and external tubes and the cap were cleaned and wiped with 75% alcohol, dried by tapping and irradiated with ultraviolet light for 30 minutes in a safety cabinet; the pre-concentrated virus was mixed and centrifuged at 3000 rpm for 7 minutes;

[0256] (3) 0.45 m syringe filter and 10 ml syringe were used to filter the virus supernatant into the ultracentrifuge internal tube (30 ml of virus liquid/tube); a 10 ml syringe was used to draw 5 ml/tube of 20% sucrose solution and connected with a 5 cm needle, and then it was inserted to the bottom of the tube set containing the virus liquid, to add sucrose solution slowly;

[0257] (4) the tube set was arranged in pairs 1-4, 2-5, 3-6 correspondingly, and labeled with sample signs. After addition of the samples, balance with electronic analytical balance was performed so that the error between the corresponding two tubes was less than 0.0005 g;

[0258] (5) the caps of tube sets were tightened after confirming there was no wrong, the tube sets were hung on the corresponding position on sw28 rotor; vacuuming was performed, and the parameters were set as 25000 rpm, 2.5 h, 4 C., the maximum up speed, and down speed with brakes;

[0259] (6) after centrifugation, the vacuum button was pressed to introduce air, the centrifuge tubes were gently taken out, the supernatant was discarded in a safety cabinet, sterile PBS was added to dissolve overnight, and the liquid was collected as a concentrated virus sample.

[0260] The obtained HIV-1 virus-like particles were resuspended in PBS, and the p24 and Env of the virus were quantified by the following methods.

[0261] Quantification of p254

[0262] 1. Antibody coating: 16G12 antibody was diluted with PB7.4, and coated at 100 ng/well (100 L) 16G12 on a 96-well plate, and the coating was carried out overnight at 4 C.

[0263] 2. Blocking: the 96-well plate was washed once with PB ST, spin-dried, added with 180 pL of ED solution, and blocked for 2 h at 37 C. or overnight at 4 C.

[0264] 3. Virus incubation: PBS was used as buffer solution to serially dilute the virus in a U bottom plate (p24 standard substance was added to the plate at 350, 700, 1400 and 2800 pg/mL), 100 L (volume) per well, then added with lysis buffer, 20 l (volume) per well. It was transferred to a 96-well plate and incubated at 37 C. for 1 h.

[0265] 4. Secondary antibody reaction: the 96-well plate was washed 5 times with PBST washing solution, spin-dried. 100 L of 2F2-HRP secondary antibody solution (1:10000 diluted in ED11 solution) was added to each well, and incubated in a 37 C. incubator for 45 min.

[0266] 5. Color development: the 96-well plate was washed 5 times with PBST washing solution, spin-dried, and 100 L of the A/B color development solutions mixed at equal volume was added to each well, and incubated at 37 C. for 10 min.

[0267] 6. Stopping: 50 L of sulfuric acid stopping solution was added to each well, the 96-well plate was placed in a microplate reader to read OD650-450 nm.

[0268] 7. The p24 standard substance was used as a reference to make a standard curve to calculate the p24 content in the virus.

[0269] Quantification of Env

[0270] 1. GNL coating: GNL at 500 ng/well (100 l) was coated on a 96-well plate, the buffer was PBS, and the coating was performed overnight at 4 C.

[0271] 2. Blocking: the plate was washed once with PBST, spin-dried, added with 180 L of ED solution, and blocked for 2 hours at 37 C. or overnight at 4 C.

[0272] 3. Virus coating: the virus liquid was diluted with ED as diluent solution (gp140 at a starting concentration of 1 g/ml was used as the standard), transferred into the 96-well plate, and incubated at 37 C. for 1 hour after sealing the plate.

[0273] 4. Primary antibody reaction: the plate was washed 5 times with PB ST washing solution, and spin-dried. 100 Ill of VRC01 (1 g/ml diluted in ED11 solution) was added to each well, and incubated at 37 C. for 1 hour after sealing the plate.

[0274] 4. Secondary antibody reaction: the plate was washed 5 times with PBST washing solution, and spin-dried. 100 L of GAH-HRP secondary antibody solution (1:5000 diluted in ED11 solution) was added to each well, and incubated at 37 C. for 45 min after sealing the plate.

[0275] 5. Color development: the 96-well plate was washed 5 times with PBST washing solution, and spin-dried. 100 L of the A/B color development solutions mixed at equal volume was added to each well, and incubated to react in a 37 C. incubator for 10 min.

[0276] 6. Stopping: 50 L of sulfuric acid stopping solution was added to each well, the 96-well plate was place in a microplate reader to read OD650-450 nm.

[0277] 7. The gp140 standard substance was used as a reference to make a standard curve to calculate the Env content in the virus.

[0278] Identification of Pseudovirus Immunogenicity

[0279] After quantification of p24 and Env of the NL4-3 pseudoviral particles, the mouse immunization experiment of the NL4-3 pseudoviral particles were performed with the immunization protocol shown in Table 3. The 6-week-old female mice, purchased from Shanghai Slack Experimental Animal Co., Ltd., were divided into five experimental groups, A/B/C/D/E, 5 mice in each group. Combined with aluminum adjuvant, a total of five injections of intraperitoneal immunization were performed, and the immunization period was 2 weeks/injection. The blood samples were collected from mouse eyeball before immunization at 0/2/4/6/8 weeks and at the week, and then the mice were treated with neck dislocation. The blood samples were placed at 37 C. for 30 minutes, then centrifuged at 13300 rpm for 10 minutes, and the serum samples were collected for determination of Env and P24 specific binding antibody titers and HIV-1 pseudovirus neutralizing antibody titers. The experimental results showed that the pseudovirus of the present invention could induce neutralizing antibody responses and exhibited good immunogenicity.

TABLE-US-00004 TABLE 3 Immunization protocol of NL4-3 pseudoviral particle Injection No. Group A Group B Group C Group D Group E 1 NL4-3TSTIP NL4-3 WT NL4-3TSTIP NL4-3SOSIP NL4-3TSTIP pseudovirus pseudovirus gp140(2 g) gp140(2 g) pseudovirus 2 g(Env) 2 g(Env) 2 g(Env) + NL4-3TSTIP gp140(2 g) 2 NL4-3TSTIP NL4-3 WT NL4-3TSTIP NL4-3SOSIP NL4-3TSTIP pseudovirus pseudovirus gp140(2 g) gp140(2 g) pseudovirus 2 g(Env) 2 g(Env) 2 g(Env) + NL4-3TSTIP gp140(2 g) 3 NL4-3TSTIP NL4-3 WT NL4-3TSTIP NL4-3SOSIP NL4-3TSTIP pseudovirus pseudovirus gp140(2 g) gp140(2 g) pseudovirus 2 g(Env) 2 g(Env) 2 g(Env) + NL4-3TSTIP gp140(2 g) 4 NL4-3TSTIP NL4-3 WT NL4-3TSTIP NL4-3SOSIP NL4-3TSTIP pseudovirus pseudovirus gp140(2 g) gp140(2 g) pseudovirus 2 g(Env) 2 g(Env) 2 g(Env) + NL4-3TSTIP gp140(2 g) 5 NL4-3TSTIP NL4-3 WT NL4-3TSTIP NL4-3SOSIP NL4-3TSTIP pseudovirus pseudovirus gp140(2 g) gp140(2 g) pseudovirus 2 g(Env) 2 g(Env) 2 g(Env) + NL4-3TSTIP gp140(2 g)

Example 7: Production and Characterization of NL4-3 Virus Particles

[0280] The full-length Env sequence of NL4-3 strain was designed with reference to the design of TSTIP protein, and the obtained full-length TSTIP gp160 gene (which encoded the amino acid sequence set forth in SEQ ID NO: 17) was codon-optimized to obtain base sequence suitable for expression in mammalian cells, and then sent to Sangon Biotech (Shanghai) Co., Ltd for gene synthesis, and cloned into the genomes of two strains to replace the wild-type Env gene. 1l of each of the synthesized plasmids was taken to transform Stb13 competent cell (purchased from Shanghai Weidi Biotechnology Co., Ltd.), then coated on an ampicillin-containing solid medium, and cultured at 30 C. for 12-14 hours; after a single colony was clearly visible, it was picked and placed in a test tube containing 3 ml of ampicillin-containing LB medium, cultured under shaking at 220 rpm at 30 C. for 12 hours; 500 l of the bacterial liquid was taken and mixed with 500 l of 50% glycerol, and then cryopreserved at 20 C. The bacterial liquid was inoculated into 500 ml of LB medium and cultured, for plasmid extraction. The extraction process of plasmid referred to the extraction process of pcDNA3.1 NL4-3/BG505 TSTIP plasmid in Example 1. The extracted plasmid was transiently transfected into 293FT adherent cells, with the transfection reagent of PEI, and by using the transfection method referring to the pseudovirus production process as mentioned in the production and identification of B1.26-TSTIP pseudoviral particle in Example 5. After 48 hours of transfection, the transfection supernatant was collected, the NL4-3 TSTIP virus particles were purified, and the collected virus particles were verified for their packaging ability and infectivity, and the specific methods referred to the methods described in Example 5. The experimental results showed that the virus obtained by the above method could be normally packaged to form virus particles, and the virus modified by TSTIP design almost completely lost its ability to infect.

Example 8: Identification of Immunogenicity of Virus Particles

[0281] According to the method of Example 7, a sufficient amount of virus particles was obtained, and detected for the p24 and Env content by using the p24 and Env quantification method in Example 6. NL4-3 TSTIP virus particle immunization experiment was carried out according to the immunization protocol shown in Table 4. Six-week-old female white mice were purchased from Shanghai Slack Experimental Animal Co., Ltd., and two experimental groups A/B were set up, five mice in each group. Taking Env content as a reference, the group A was immunized with virus particles containing 2 g-Env, and the group B was immunized with 2 g of purified gp140 protein. Combined with aluminum adjuvant, intraperitoneal immunization was carried out, the immunization period was 2 weeks/injection, and there was a total of 5 injections for the immunization. The blood samples of mice were collected from eyeball before the immunization at weeks and at the 10 th week, and then the mice were treated with neck dislocation. After the blood samples were placed at 37 C. for 30 minutes, they were centrifuged at 13300 rpm for 10 minutes, and the serum samples were collected for the determination of Env and P24-specific binding antibody titers and HIV-1 pseudovirus neutralizing antibody titers. The experimental results showed that the virus of the present invention could induce neutralizing antibody responses and exhibited good immunogenicity.

TABLE-US-00005 TABLE 4 Immunization protocol of NL4-3 virus particle Injection No. Group A Group B 1 NL4-3TSTIP pseudovirus 2 NL4-3TSTIP gp140(2 g(Env) g) 2 NL4-3TSTIP pseudovirus 2 NL4-3TSTIP gp140(2 g(Env) g) 3 NL4-3TSTIP pseudovirus 2 NL4-3TSTIP gp140(2 g(Env) g) 4 NL4-3TSTIP pseudovirus 2 NL4-3TSTIP gp140(2 g(Env) g) 5 NL4-3TSTIP pseudovirus 2 NL4-3TSTIP gp140(2 g(Env) g)

Example 9: Design and Characterization of Various TSTIP-based Proteins

[0282] Based on the amino acid sequence of BG505 TSTIP (SEQ ID NO: 3), WNSSWSN and AKRRVVGREKR, which needed to be deleted due to the design of TSTIP, were introduced at the linkage region of 27 and gp120 and the linkage region of gp120 and 8 of BG505 TSTIP respectively to obtain SEQ ID NO: 5 (BGTSTIP-Full).

[0283] Based on the amino acid sequence of NL4-3TSTIP (SEQ ID NO: 4), WNSSWSN and AKRRVVGREKR, which needed to be deleted due to the design of TSTIP, were introduced at the linkage region of 27 and gp120 and the linkage region of gp120 and 8 of NL4-3TSTIP respectively to obtain SEQ ID NO: 6 (NL4-3TSTIP Full).

[0284] GGGGS (SEQ ID NO: 28) was introduced at the linkage region of 27 and gp120 and the linkage region of gp120 and 8 of BG505 TSTIP, respectively, to obtain SEQ ID NO: 7 (BG505-TSTIP G1).

[0285] GGGGS was introduced at the linkage region of 27 and gp120 and the linkage region of gp120 and 8 of NL4-3TSTIP, respectively, to obtain SEQ ID NO: 8 (NL4-3-TSTIP G1).

[0286] GGGGSGGGGS (SEQ ID NO: 29) was introduced at the linkage region of 27 and gp120 and the linkage region of gp120 and 8 of BG505 TSTIP, respectively, to obtain SEQ ID NO: 9 (BG505-TSTIP G2).

[0287] GGGGSGGGGS was introduced at the linkage region of 27 and gp120 and the linkage region of gp120 and 8 of NL4-3TSTIP, respectively, to obtain SEQ ID NO: 10 (NL4-3-TSTIP G2).

[0288] According to the steps of Examples 1, 2, 3, and 4, a series of studies were carried out on the six proteins obtained above. The results of SDS-polyacrylamide gel electrophoresis of the six proteins were shown in FIG. 11A, in which the purity of BGTSTIP-Full, NL4-3TSTIP Full and 8NL4-3-TSTIP G1 proteins decreased in some extent after one-step Ni column purification; the Coomassie brilliant blue staining of BGTSTIP-G1 and NL4-3 TSTIP G2 under reducing conditions showed >180 KD bands, which might be the dimer forms thereof, while BG505-TSTIP G2 showed similar purity and molecular weight to BG505 TSTIP.

[0289] The four high-purity proteins were subjected to analytical ultracentrifugation, and the results were shown in FIG. 11B, in which this result was consistent with the results of SDS polyacrylamide gel electrophoresis in the previous step (14A), that was, the molecular weights of BGTSTIP G1 and NL4-3TSTIP G2 were greater than the molecular weight of monomer gp140, while the purity of BG505 TSTIP was significantly lower than that of BGTSTIP. This showed that the linkage region of 27 and gp120, and the linkage region of gp120 and 8 had obvious effects on the forms of Env protein. Moreover, different modifications of the same strain could result in differences of expression level, and the mutation of Ile.fwdarw.Pro at position 559 could effectively increase the protein production. Specifically, the expression levels were summarized in Table 3.

[0290] The ELISA experiment results of the six proteins with various reported human monoclonal antibodies were shown in FIG. 12. Even some proteins had a relatively lower purity, the purified six proteins could all be well recognized by various antibodies, which proved their activity.

[0291] According to the above immunization protocol, we immunized BABL/C mice with the six purified proteins. The results of animal immunization experiments showed that all the six proteins could induce neutralizing antibody responses in mice, as shown in FIG. 13. In addition, pseudoviral particles and virus particles were further constructed based on these proteins, and mouse immunization experiments were carried out to detect their immunogenicity.

TABLE-US-00006 TABLE 3 Summary of protein expression levels BG505 Env BGTSTIP BGSOSIP BGTSTIP-G1 BGTSTIP-G2 BGTSTIP-full Expression 40 20 9 27 6 level (mg/L) NL4-3 Env 4-3TSTIP 4-3SOSIP 4-3TSTIP-G1 4-3TSTIP-G2 4-3TSTIP-full Expression 37 19 3 25 5 level (mg/L)

Example 10: Application of TSTIP Modification in Strains of Multiple Subtypes

[0292] In order to further confirm the effect of the TSTIP design on different strains, the inventors applied a design method similar to BG505/NL4-3-TSTIP to the Env proteins of various strains. Table 4 showed 12 global-representative strains. Since the expression and identification of proteins of subtype A and subtype B strains had been completed, one strain of each subtype except subtype A and subtype B was selected from the 12 global pseudovirus strains, to carry out the TSTIP modification of Env protein in the gp140 proteins of the selected 5 strains, including 25710 (C), X1632 (G), CH119 (BC), CNE8 (AE), and 246F3 (AC). According to a series of experimental steps and methods such as cloning, expression and purification, and characterization as described above, the TSTIP-gp140 proteins of the five strains were studied, and the series of results were shown in FIGS. 14A to 14D.

[0293] The results of SDS polyacrylamide gel electrophoresis in FIG. 14A showed that the TSTIP proteins of the five strains all had a relatively higher purity after one-step Ni column purification, and a molecular weight of about 140 KD under the reducing SDS PAGE, indicating that the subunits in the proteins of the present invention were linked together by stable covalent interaction as expected.

[0294] FIG. 14B showed the superdex200 16/600 molecular sieve chromatography results of the five TSTIP proteins, which showed that the elution peaks were relatively single, indicating that the components of the five proteins of the present invention were relatively uniform in natural state, with trimers as majority.

[0295] FIG. 14C showed the enzyme-linked immunoassay (ELISA) results of the five proteins with various reported antibodies, which showed that the proteins of different strains had different recognition and binding abilities to the antibody of the same type. The results showed that the TSTIP proteins of the five strains produced by the inventors all had good activity.

[0296] FIG. 14D showed the neutralization experiment of serum samples after immunizing BALB/C mice with the five proteins, and the results indicated that the immunization with TSTIP proteins of different strains could stimulate mice to produce a neutralizing response.

TABLE-US-00007 TABLE 4 Twelve global pseudoviruses Isolate strain Subtype Region TRO11 B Italy 25710 C India 398F1 A Tanzania CNE8 CRF01_AE China X2278 B Spain BJOX002000 CRF07_BC China X1632 G Spain CE1176 C Malawi 246F3 AC Tanzania CH119 CRF07_BC China CE0217 C Malawi CNE55 CRF01_AE China

TABLE-US-00008 TABLE 5 Summary of TSTIP protein expression levels of five subtypes TSTIP 246F3 25710 CH119 CNE8 X1632 Expression 14.7 24 60 27 36 level (mg/L)

Example 11: Design and Activity Identification of other TSTIP-based Proteins

[0297] 11.1 Protein Design

[0298] 1. BG-B1(1/1)

[0299] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of GGGGS after it, and WNSSWSN was added back before 8 with further insertion of GGGGS before it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:30.

[0300] 2. BG-B1(1/2)

[0301] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of GGGGS after it, and

[0302] WNSSWSN was added back before 8 with further insertion of (GGGGS) 2 before it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:31.

[0303] 3. BG-B1(2/2)

[0304] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of (GGGGS) 2 after it, and WNSSWSN was added back before 8 with further insertion of (GGGGS) 2 before it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:32.

[0305] 4. BG-B1(2/1)

[0306] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of (GGGGS) 2 after it, and WNSSWSN was added back before 8 with further insertion of GGGGS before it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:33.

[0307] BG-B1(2/3)

[0308] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of (GGGGS) 2 after it, and WNSSWSN was added back before 8 with further insertion of (GGGGS) 3 before it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:34.

[0309] 6. BG-B2(1-1)

[0310] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of GGGGS after it, and WNSSWSN was added back before 8 with further insertion of GGGGS before it; in addition, a cys mutation was introduced between gp120 and gp41 subunits by referring to the design of

[0311] SOSIP in order to expect the formation of a disulfide bond, which specifically comprised that Ala at position 501 was mutated to cys, and Thr at position 605 was mutated to cys. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:35.

[0312] 7. BG-B2(1-2)

[0313] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of GGGGS after it, and WNSSWSN was added back before 8 with further insertion of (GGGGS) 2 before it; in addition, a cys mutation was introduced between gp120 and gp41 subunits by referring to the design of SOSIP in order to expect the formation of a disulfide bond, which specifically comprised that Ala at position 501 was mutated to cys, and Thr at position 605 was mutated to cys. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:36.

[0314] 8. BG-B2(1-3)

[0315] The amino acid sequence AKRRVVG (not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, AKRRVVG was added back after (326 with further insertion of GGGGS after it, and WNSSWSN was added back before 8 with further insertion of (GGGGS) 3 before it; in addition, a cys mutation was introduced between gp120 and gp41 subunits by referring to the design of SOSIP in order to expect the formation of a disulfide bond, which specifically comprised that Ala at position 501 was mutated to cys, and Thr at position 605 was mutated to cys. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:37.

[0316] 9. BG-C1(1/1)

[0317] The amino acid sequence AKRRVVG (or its homologous sequence, not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, WNSSWSN was added back after 27 with further insertion of GGGGS after it, and AKRRVVG was added back before 8 with further insertion of GGGGS after it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:38.

[0318] 10. BG-C1(1/2)

[0319] The amino acid sequence AKRRVVG (or its homologous sequence, not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, WNSSWSN was added back after 27 with further insertion of GGGGS after it, and AKRRVVG was added back before 8 with further insertion of (GGGGS) 2 after it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO:39.

[0320] 11. BG-C1(1/3)

[0321] The amino acid sequence AKRRVVG (or its homologous sequence, not including the furin cleavage site) deleted after (326 and the sequence WNSSWSN (or homologous sequence) deleted after 27 were added back, that was, WNSSWSN was added back after 27 with further insertion of GGGGS after it, and AKRRVVG was added back before 8 with further insertion of (GGGGS) 3 after it. The modified sequence based on the BG505 strain was set forth in SEQ ID NO: 40.

[0322] 11.2 SDS-PAGE:

[0323] These proteins were prepared and purified using the methods described in the above Examples 1-2, the concentrated samples were diluted to 1 g/l, then two tubes of 50 l samples were taken, and 10 l of reducing loading buffer and 10 l of non-reducing loading buffer were added, respectively, to prepare reduced samples and non-reduced samples, and the reduced samples were placed in a boiling water bath at 100 C. for 10 minutes. The reduced samples and non-reduced samples in an amount of 10 l were taken and electrophoresed at 80V for 120 min in 8% SDS PAGE, and the electrophoresis bands were displayed after Coomassie brilliant blue staining.

[0324] The results of electrophoresis were shown in the panel (I) of FIGS. 15A to 15K. The SDS-PAGE analysis showed that after one-step Ni-EXCEL purification, the above-mentioned designs could obtain high-purity proteins; under the condition of adding reducing loading buffer, except for BG-C1(1/1), the main bands showed by other optimized proteins were all 140 KD, while bands of multimer were showed under the condition of non-reducing loading buffer. This indicated that in the optimized designed proteins, similar to the original TSTIP protein, the gp120 and gp41 extracellular regions were still connected via a stable covalent linkage.

[0325] 11.3 Molecular Sieve Purification:

[0326] Instrument system: AKTA Pure type preparative liquid chromatograph;

[0327] Chromatographic column: superdex 200 10/300

[0328] Column volume: 24 ml

[0329] Buffer: PBS (20 mM phosphate buffer, pH7.5, 150 mM NaCl)

[0330] Detector wavelength: 280 nm

[0331] Flow rate: 0.5 ml/min

[0332] The samples were purified and concentrated proteins.

[0333] The purification procedure comprised: the superdex200 10/300 was equilibrated with 1 column volume of PBS, a target protein to be purified was loaded with 5O01 sample loop, and molecular sieve purification of the sample to be purified was performed at inject mode. The components of the sample would be eluted according to molecular weights thereof in order, from high to low, and a diagram of peaks after purification was saved. The molecular sieve purification results were shown in the panel (II) of FIGS. 15A to 15K. Each of the proteins showed an obvious single elution peak on the molecular sieve spectrum. Among them, BG-B1(1/1), BG-B1(1/2), BG-B1(2-1), BG-B1(2-2), BG-B1(2-3), BG-B2(1-1), BG-B2(1-2), BG-B2(1-3), BG-C1(1/2), BG-B1(1/3) all had main elution peaks at an elution volume of about 9.5 ml, which were peaks of trimer, while BG-C1 (1/1) had an elution volume of 14 ml showing a peak of dimer.

[0334] 11.4 Homogeneity Analysis and Molecular Weight Prediction of Proteins After Analytical Ultracentrifugation (AUC)

[0335] The above-mentioned purified proteins were subjected to analytical ultracentrifugation, and the AUC results were shown in the panel (III) of FIGS. 15A to 15K. The results showed that BG-B1(1/1), BG-B1(1/2), BG-B 1(2-1), BG-B1(2-2), BG-B1(2-3), BG-B2(1-1), BG-B2(1-2), BG-B2(1-3), BG-C1(1/2), BG-B1(1/3) were mainly trimers, while BG-C1(1/1) was mainly dimers, and this result was consistent with result of molecular sieve.

[0336] 11.5 Enzyme-linked Immunosorbent Assay (ELISA)

[0337] Using the enzyme-linked immunosorbent assay described in Example 4, the antigenicity of these above-mentioned purified proteins was identified. The results were shown in the panel (IV) of FIGS. 15A to 15K. The results showed that these proteins had antigenicity, and the modification of linker at the linkage region could adjust their antigenic activity.

[0338] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that: according to all the teachings that have been disclosed, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the claims appended hereto and any equivalents thereof.