WO2022100703A1 - 经改造的人免疫缺陷病毒膜蛋白及其应用 - Google Patents

经改造的人免疫缺陷病毒膜蛋白及其应用 Download PDF

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WO2022100703A1
WO2022100703A1 PCT/CN2021/130414 CN2021130414W WO2022100703A1 WO 2022100703 A1 WO2022100703 A1 WO 2022100703A1 CN 2021130414 W CN2021130414 W CN 2021130414W WO 2022100703 A1 WO2022100703 A1 WO 2022100703A1
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amino acid
sequence
seq
protein
recombinant protein
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PCT/CN2021/130414
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French (fr)
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顾颖
邓婷婷
张慧
黄芳
陈格格
林燕玲
李少伟
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Priority to EP21891223.6A priority Critical patent/EP4245767A1/en
Priority to US18/252,836 priority patent/US20240000919A1/en
Publication of WO2022100703A1 publication Critical patent/WO2022100703A1/zh

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Definitions

  • the present invention relates to the field of virology.
  • the present invention relates to a newly designed HIV-1 Env trimer protein and HIV-1 pseudoviruses and true viruses expressing the Env trimer protein, and their use in the prevention and/or treatment of HIV Infectious uses.
  • Human immunodeficiency virus type 1 HIV-1 (Human immunodeficiency virus-1) is the main pathogen that induces AIDS. It belongs to the genus of lentivirus of the family Retroviridae.
  • the virus particle consists of a core/envelope and a diploid single strand of genetic material.
  • the single-stranded genome is about 9.8kb, which encodes three structural proteins Env, Gag, Pol and six accessory proteins: vpr, vif, vpu, nef, tat, rev. Accessory proteins coordinate with each other to form a regulatory network for HIV-1 viral replication.
  • the Pol gene encodes a polymerase precursor protein, which is cleaved to form protease (PR), reverse transcriptase (RT) and integrase (IN), which are key enzymes for virus maturation, replication and infection.
  • the Gag protein encoded by the Gag gene is the main structural protein of the virus and is responsible for the assembly process of the virus, accounting for 50% of the entire virus mass; the N-terminus to the C-terminus of the Gag protein is MA, CA, NC and p6.
  • MA is a matrix protein that gathers near the plasma membrane of infected cells to recruit another structural protein, Env, to initiate the virus assembly process;
  • CA is a capsid protein, and the capsid protein of mature HIV-1 virus is cone-shaped.
  • NC is a nucleocapsid protein, which can capture the genome in the process of virus packaging and make it into a complete virus particle; p6 has binding sites for other proteins, including accessory proteins, which are related to HIV- 1 replication, transcription and other processes are closely related.
  • PR enzyme cleavage sites between different components of Gag protein which gradually become mature particles through its action (Sundquist, W.I. and H.G. Krausslich, HIV-1 assembly, budding, and maturation. Cold Spring Harb Perspect Med, 2012.).
  • Env is the only antigenic substance on the surface of HIV-1. The number of Env trimers on the surface of each virus is very small, about 7-14, but it can effectively induce the infection of host cells, which shows its importance.
  • Env is a heterotrimeric protein, and its precursor is gp160 protein with a molecular weight of 160KD, which is cleaved into gp120 and gp41 by furin enzyme during the process of virus maturation, and gp120 and gp41 form a heterotrimer through non-covalent interaction; gp120 exists extracellularly.
  • gp120 recognizes the receptor molecule CD4 on the host cell and promotes virus infection.
  • There are key epitopes such as CD4bs, V1V2, V3, CD4i on gp120, and the CD4bs epitope recognizes the receptor molecule.
  • gp41 contains extracellular region, membrane proximal region (MPER), transmembrane region (TM) and intracellular region (CT), on which there are fusion membrane peptide (FP), membrane proximal region (MPER), gp120 and gp41 interface Iso-epitope, after gp120 recognizes the CD4 molecule, it will induce a conformational change of gp120, exposing the coreceptor binding site to bind to the coreceptor on the cell, further leading to a conformational change, so that the FP epitope of gp41 is instantly exposed from the viral membrane , and inserted into host cells to mediate viral infection (Guttman, Garcia et al. CD4-induced activation in a soluble HIV-1 Env trimer, structure. 2014.).
  • Env is also the main component of a number of HIV-1 vaccine clinical studies.
  • Early HIV vaccine studies mostly used gp120 monomer as an antigen, which retained some of the known Nab epitopes, but proved difficult to induce Tier 2 neutralizing antibodies , Phase III clinical trials have proved that it does not have clinical protective effects. The researchers believe that this may be due to the existence of many epitopes on the gp120 monomer that do not exist on the native Env, and gradually shifted the focus to the native Env trimer, but because the transmembrane region of gp41 is extremely hydrophobic and incompatible with the cell. As the inner region is less antigenic, the researchers used the method of removing its transmembrane region and intracellular region to increase the soluble expression of Env.
  • gp140 This type of HIV-1 antigen molecule was named gp140 (Sanders, R.W.and J.P.Moore.Native-like Env trimers as a platform for HIV-1 vaccine design. Immunol Rev, 2017.).
  • BG505 SOSIP, NFL2P, UFO and other designs are all based on gp140.
  • BG505-SOSIP As an example, its trimer content is significantly increased, and its thermal stability is about 14 °C higher than that of monomer gp120. But it is very weak (Sanders, Derking et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies, 2013.). Immune mice with BG505-SOSIP trimer, although it can induce stronger IgG, Tfh and GC responses, but it cannot induce the production of Tier2 neutralizing antibodies.
  • Inactivated or attenuated virus is the main active ingredient of many vaccines, but so far there is no effective vaccine and cure for AIDS.
  • Inactivated or attenuated virus contains all the antigenic components of the virus, and can induce the body to produce humoral immunity and Cellular immunity induces a strong cytotoxic T lymphocyte response (CTL), and the live attenuated SIV vaccine has been proven to successfully protect rhesus monkeys from SIV virus infection (Protection by Live, Attenuated Simian Immunodefificiency Virus against Heterologous Challenge. Journal of Virology , 1999.).
  • CTL cytotoxic T lymphocyte response
  • HIV-1 virus vaccine due to the pathogenicity and incurability of HIV-1, the attenuated or inactivated HIV-1 vaccine has the potential to infect the human body, which limits the application of HIV-1 inactivated or attenuated virus in the development of AIDS vaccines.
  • a modified HIV-1 virus particle that has completely lost its pathogenicity and cannot regain its ability to infect through reverse mutation.
  • the preparation of HIV-1 virus vaccine without potential infection risk is a very novel and safe vaccine design idea, which is expected to be It has brought breakthrough progress in the field of AIDS vaccine research and development.
  • the present invention provides a recombinant protein comprising gp120 and an extracellular domain of gp41 (gp41ectodomain, gp41ECTO), wherein the gp120 is located between ⁇ 27 and ⁇ 8 of the gp41ECTO.
  • the recombinant protein of the invention is an engineered gp140 protein.
  • gp120 is located between ⁇ 27 and ⁇ 8 of said gp41ECTO
  • the expression "gp120 is located between ⁇ 27 and ⁇ 8 of said gp41ECTO” means that the position of gp120 in the recombinant protein of the present invention is between ⁇ 27 and ⁇ 8 of gp41ECTO, and is not intended to limit it by The specific way to put in between ⁇ 27 and ⁇ 8.
  • gp120 can be inserted directly between adjacent amino acids between ⁇ 27 and ⁇ 8 of gp41ECTO, or gp120 can replace one or more consecutive amino acids between ⁇ 27 and ⁇ 8 of gp41ECTO.
  • gp120 can be native or modified and gp41ECTO can be native or modified.
  • gp41ECTO described in the technical scheme of the present invention is natural, which means that the gp41ECTO does not contain other artificial modifications except that gp120 is included between ⁇ 27 and ⁇ 8.
  • modification preferably refers to the deletion, addition or substitution of one or more amino acid residues.
  • the recombinant protein comprises from N-terminal to C-terminal: ⁇ 6, ⁇ 7, ⁇ 27 of gp41ECTO; gp120; ⁇ 8, ⁇ 9 of gp41ECTO.
  • one or more of the connecting regions between ⁇ 27 and ⁇ 8 of the gp41ECTO eg, 1-12, 5-12, 5-10; such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
  • the linking region corresponds to amino acid positions 607-618 of the gp160 sequence of isolate HXB2.
  • the gp41ECTO is in one or more (eg, 1-7, eg, 5-7; eg, 1) in the region corresponding to amino acid positions 610-616 of the gp160 sequence of isolate HXB2 , 2, 3, 4, 5, 6, or 7) consecutive amino acids were replaced with gp120.
  • the gp41ECTO is replaced with gp120 in 7 consecutive amino acids in a region corresponding to amino acid positions 610-616 of the gp160 sequence of isolate HXB2.
  • the junction region between ⁇ 27 and ⁇ 8 of the gp41ECTO is truncated by the sequence shown in SEQ ID NO: 26 (WNSSWSN) or the amino acid sequence at the corresponding position.
  • amino acid sequence at the corresponding position refers to the amino acid sequence at the equivalent position to WNSSWSN when gp160 sequences of different strains are optimally aligned, ie when the sequences are aligned for the highest percent identity.
  • the gp120 is located between amino acid positions 606 and 619 corresponding to the gp160 sequence of isolate HXB2.
  • the gp120 is inserted between adjacent amino acids in the junction region between ⁇ 27 and ⁇ 8 of gp41ECTO.
  • the linking region corresponds to amino acid positions 607-618 of the gp160 sequence of isolate HXB2.
  • the gp120 is located between amino acid positions 609 and 610 corresponding to the gp160 sequence of isolate HXB2.
  • the gp120 is located between amino acid positions 616 and 617 corresponding to the gp160 sequence of isolate HXB2.
  • the gp120 inserted in gp41ECTO can be a native gp120.
  • the native gp120 protein corresponds to amino acid positions 31-511 of the gp160 sequence of isolate HXB2.
  • the gp120 inserted in gp41ECTO may be a modified gp120, which may contain one or more amino acid mutations (eg, substitutions, deletions, or insertions) compared to native gp120.
  • the gp120 is a modified gp120 that contains a mutation in the furin recognition site compared to native gp120 to prevent the furin site from being cleaved.
  • Methods for removing furin dependence by cleavage site modification are well known to those skilled in the art, and may include, for example, amino acid substitutions, insertions or deletions in the furin recognition site sequence, or the furin recognition site Sequences or parts thereof are replaced with additional sequences (eg linker sequences).
  • the furin site corresponds to amino acid positions 508-511 of the gp160 sequence of isolate HXB2.
  • the furin recognition site is set forth in SEQ ID NO:41 (REKR).
  • the furin recognition site in the modified gp120 is deleted compared to the native gp120.
  • the gp120 is a modified gp120 that is C-terminally truncated by 1-11 (eg, 4-11; eg, 1, 2, 3, 4) compared to native gp120 5, 6, 7, 8, 9, 10, or 11) amino acids.
  • the modified gp120 comprises one or more (eg, 1-11, 4-11; eg, 1-11, 4-11; eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) deletions of consecutive amino acids.
  • the modified gp120 is deleted in a region corresponding to amino acid positions 501-511 of the gp160 sequence of isolate HXB2.
  • the C-terminus of the modified gp120 is truncated to the amino acid sequence of the sequence shown in SEQ ID NO: 27 (AKRRVVGREKR) or its corresponding position.
  • amino acid sequence at the corresponding position refers to the amino acid sequence at the equivalent position to AKRRVVGREKR when gp160 sequences of different strains are optimally aligned, ie when the sequences are aligned for the highest percent identity.
  • the modified gp120 is deleted in a region corresponding to amino acid positions 508-511 of the gp160 sequence of isolate HXB2.
  • the C-terminus of the modified gp120 is truncated to the amino acid sequence of the sequence shown in SEQ ID NO: 41 (REKR) or its corresponding position.
  • amino acid sequence at the corresponding position refers to the amino acid sequence at the equivalent position to REKR when gp160 sequences of different strains are optimally aligned, ie when the sequences are aligned for the highest percent identity.
  • the gp120 is a modified gp120 that is N-terminally truncated by 1-5 (eg, 1-4; eg, 1, 2, 3, 4) compared to native gp120 single, or 5) amino acids.
  • the modified gp120 is deleted in a region corresponding to amino acid positions 31-34 of the gp160 sequence of isolate HXB2. In certain embodiments, the modified gp120 is replaced with an exogenous nucleic acid sequence in a region corresponding to amino acid positions 31-34 of the gp160 sequence of isolate HXB2. In certain embodiments, the exogenous nucleic acid sequence consists of 4 amino acid residues.
  • the modified gp120 is deleted at the amino acid residue corresponding to amino acid position 499 of the gp160 sequence of isolate HXB2.
  • the modified gp120 is located between amino acid positions 606 and 610 corresponding to the gp160 sequence of isolate HXB2, that is, the modified gp120 replaces the gp160 in gp41ECTO corresponding to isolate HXB2 The region of amino acid positions 607-609 of the sequence.
  • the gp120 and gp41ECTO comprise a disulfide bond.
  • the recombinant protein has a disulfide bond between amino acid positions 501 and 605 corresponding to the gp160 sequence of isolate HXB2.
  • the recombinant protein is substituted with Cys at residues corresponding to amino acid positions 501 and 605 of the gp160 sequence of isolate HXB2.
  • gp120 inserted in gp41ECTO may also comprise one or more amino acid mutations to increase exposure of broadly neutralizing antibody recognition epitopes and/or stabilize the HIV envelope protein trimer structure.
  • the gp120 comprises one or more of the following mutations:
  • the gp120 comprises the substitution T332N to increase the exposure of the epitope recognized by the broad-spectrum neutralizing antibody
  • the gp120 contains substitutions E64K and H66R to stabilize the trimer conformation, making it less prone to morphing into an open conformation, thereby not easily exposing the CD4i non-neutralizing epitope;
  • the gp120 contains the substitution A316W to enhance the hydrophobic interaction between the gp120 subunits, hinder the movement of the V3 region, and avoid the exposure of the non-neutralizing epitopes in the V3 region;
  • N-linked glycosylation site near the CD4bs epitope of the gp120 is replaced to prevent glycosylation; preferably, the PNGS is selected from N276, N301, N360, N463;
  • the gp120 contains an internal disulfide bond; preferably, the gp120 contains an internal disulfide bond between I201C and A433C;
  • (6) further comprising a disulfide bond between the gp120 and gp41ECTO; for example, the recombinant protein has 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.
  • gp120 is included between ⁇ 27 and ⁇ 8 of gp41ECTO.
  • other parts of the gp41ECTO can still be the corresponding sequences in natural gp41, and can also contain artificial modifications.
  • the gp41ECTO may comprise a stabilizing mutation. Mutations for stabilizing the HIV envelope protein trimer structure are known to those skilled in the art, see eg WO 03/022869. In certain embodiments, the stabilizing mutation is I559P, and the numbering of the positions is according to the numbering in gp160 of HIV-1 isolate HXB2.
  • gp120 inserted in gp41ECTO can be directly connected to gp41ECTO, or can be connected to it through a peptide linker.
  • the N-terminus and/or C-terminus of said gp120 is optionally linked to said gp41ECTO via a peptide linker.
  • the peptide linker comprises or consists of the sequence represented by (GmS)n, wherein m is selected from an integer of 1-4 and n is selected from an integer of 1-3.
  • the peptide linker comprises or consists of the sequence shown in (GmS)n, wherein m is 4 and n is 1, 2 or 3, preferably 1 or 2.
  • the gp41ECTO and gp120 can be from the same or different HIV-1 strains. In certain embodiments, the gp41ECTO and gp120 are from the same HIV-1 strain.
  • the gp41ECTO and gp120 can be from any subtype of HIV-1, such as group M, N, O or P, or subtypes A, B, C, D, F, G, H, J or K etc.
  • the HIV-1 strain is selected from subtypes A, B, C, G, BC, AE, DC.
  • the HIV-1 strain is selected from the group consisting of BG505, NL4-3, 246F3, 25710, CH119, CNE8, X1632, Bal.26.
  • gp160 of BG505 has the sequence set forth in SEQ ID NO:18.
  • the gp160 of NL4-3 has the sequence set forth in SEQ ID NO:19.
  • the gp160 of 25710 has the sequence set forth in SEQ ID NO:20.
  • the gp160 of X1632 has the sequence set forth in SEQ ID NO:21.
  • gp160 of CH119 has the sequence set forth in SEQ ID NO:22.
  • the gp160 of CNE8 has the sequence set forth in SEQ ID NO:23.
  • the gp160 of 246F3 has the sequence set forth in SEQ ID NO:24.
  • gp160 of Bal. 26 has the sequence set forth in SEQ ID NO:25.
  • the recombinant proteins of the present invention comprise an amino acid sequence selected from the group consisting of:
  • the recombinant protein of the invention optionally comprises at its N-terminus or C-terminus one or more sequences selected from the group consisting of a signal peptide, a translation initiation sequence (eg, a Kozak consensus sequence), a tag sequence .
  • the signal peptide is tPA.
  • the translation initiation sequence is a Kozak consensus sequence (Kozak M., Nucleic Acids Research, 1984, 12, 857-872), which may comprise at least a portion of the sequence CCRGCCAUGG, wherein R may be A or G. Position -3 (ie, 3 nucleotides upstream of the ATG codon) and position +4 have the greatest effect on translation (Kozak M., Nucleic Acids Research, 1987, 15, 8125-8148).
  • the consensus sequence may also be RXXAUGG, XXAUGG or RXXAUG.
  • the tag sequence is a purification tag, such as a polyhistidine tag, a myc tag, or an HA tag.
  • the recombinant protein optionally comprises a signal peptide and/or a translation initiation sequence (eg, a Kozak consensus sequence) at its N-terminus.
  • the recombinant protein optionally includes a tag sequence at its C-terminus.
  • the present invention provides a fusion protein comprising the recombinant protein of the first aspect and the transmembrane region and intracellular region sequences of gp41 linked to its C-terminus.
  • fusion proteins of the invention are engineered gp160 proteins.
  • the transmembrane and intracellular region sequences of the gp41 are from the same HIV-1 strain as the gp41ECTO in the recombinant protein.
  • fusion proteins of the present invention comprise an amino acid sequence selected from the group consisting of:
  • amino acid substitutions, deletions or additions eg 1, 2 compared to the sequence from which it is derived 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions
  • fusion proteins of the present invention optionally comprise at its N-terminus or C-terminus one or more sequences selected from the group consisting of a signal peptide, a translation initiation sequence (eg, a Kozak consensus sequence), a tag sequence .
  • the signal peptide is a signal peptide contained in native gp160.
  • the signal peptide, the transmembrane and intracellular domain sequences of gp41, and gp41ECTO and gp120 in the recombinant protein are from the same HIV-1 strain.
  • the tag sequence is a purification tag, such as a polyhistidine tag, a myc tag, or an HA tag.
  • the recombinant protein optionally comprises a signal peptide and/or a translation initiation sequence (eg, a Kozak consensus sequence) at its N-terminus.
  • the recombinant protein optionally includes a tag sequence at its C-terminus.
  • the present invention provides a multimer comprising a plurality of monomers, wherein each monomer is independently selected from the recombinant proteins described in the first aspect, or independently selected from the recombinant proteins described in the second aspect. fusion protein.
  • the monomers are identical to each other.
  • the multimer is a trimer or dimer.
  • the recombinant protein or fusion protein or multimers thereof of the present invention can be prepared by various methods known in the art, for example, by genetic engineering methods (recombinant technology), or by chemical synthesis methods (eg, Fmoc solid-phase method) )produce.
  • the recombinant protein or fusion protein or multimer thereof of the present invention is not limited by the manner in which it is produced.
  • the present invention provides an isolated nucleic acid molecule comprising nucleotides encoding the recombinant protein of the first aspect, the fusion protein of the second aspect, or the multimer of the third aspect sequence.
  • the present invention provides a vector comprising the isolated nucleic acid molecule as described above.
  • the vector is, for example, a plasmid, cosmid, phage, and the like.
  • the present invention provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, eg, mouse cells, human cells, etc.).
  • the host cell is a mammalian cell, such as a human cell.
  • the present invention provides a method for preparing the recombinant protein of the first aspect, the fusion protein of the second aspect or the multimer of the third aspect, comprising: culturing the first aspect under suitable conditions The host cell of aspect six, and recovering the recombinant protein, fusion protein or multimer from the cell culture.
  • the recombinant protein or fusion protein exists as a multimer (eg, a trimer or a dimer).
  • the protein of the first or second aspect of the invention or the multimer of the third aspect may be displayed on particles such as liposomes, virus-like particles (VLPs), nanoparticles, virions or exosomes to enhance Antigen presentation efficacy in vivo.
  • particles such as liposomes, virus-like particles (VLPs), nanoparticles, virions or exosomes to enhance Antigen presentation efficacy in vivo.
  • the present invention provides a particle displaying the recombinant protein of the first aspect, the fusion protein of the second aspect or the multimer of the third aspect on its surface.
  • the particles are liposomes or nanoparticles.
  • the recombinant proteins, fusion proteins or multimers of the invention are fused to and/or displayed on liposomes.
  • Liposomes are spherical vesicles with at least one lipid bilayer.
  • multimeric proteins (eg, trimeric or dimeric proteins) of the invention can be incorporated into Divalent chelating atoms (eg Ni 2+ or Co 2+ ) in the head group of the derivatized lipids in the liposomes are not covalently coupled to these liposomes.
  • the multimeric proteins (eg, trimeric or dimeric proteins) of the invention are covalently coupled to the surface of the liposome, eg, through a maleimide functional group incorporated on the surface of the liposome .
  • a multimeric protein (eg, a trimeric or dimeric protein) of the invention can be coupled thereto, for example, via a C-terminal cysteine added to the multimeric protein.
  • recombinant proteins, fusion proteins or multimers of the invention are fused to self-assembling particles or displayed on nanoparticles.
  • Antigen nanoparticles are assemblies of polypeptides that present multiple copies of an antigen (eg, the HIV Env protein of the present invention), which result in multiple binding sites (affinity) and can provide improved antigen stability and immunogenicity.
  • an antigen eg, the HIV Env protein of the present invention
  • the preparation of self-assembling protein nanoparticles and their use in vaccines is well known to those skilled in the art, see e.g. Zhao L et al (2014) Vaccine 32:327-337, López-Sagaseta J et al (2016) Computational and Struct Biotechnol J 14:58-68.
  • self-assembling nanoparticles can be based on ferritin, bacterioferritin, or DPS.
  • DPS nanoparticles displaying proteins on the surface are eg described in WO 2011/082087. Illustration of trimeric HIV-1 antigen on such particles has been described, for example, in. He L et al. (2016) Na t Commun. 2016 Jun 28;7:1 2041.
  • Other self-assembling protein nanoparticles and their preparation are disclosed, for example, in WO 2014/124301 and US 2016/0122392, which are incorporated herein by reference.
  • the present invention provides a pseudoviral particle comprising the recombinant protein of the first aspect, the fusion protein of the second aspect or the multimer of the third aspect on its surface.
  • the pseudovirion of the present invention has a typical pseudovirion shape, but has completely lost its ability to infect. Compared with traditional protein vaccines, this kind of pseudovirion has both the envelope and capsid protein of HIV-1 virus. The components are highly similar to the natural virus in shape, but have completely lost the ability to infect, have good immunity, and can be used as a virus vaccine.
  • the pseudovirion is produced by a lentiviral or retroviral packaging system.
  • the pseudovirion is obtained by co-expressing (i) a vector comprising the nucleic acid molecule of the fourth aspect and (ii) a packaging vector (eg, a backbone plasmid) in a host cell.
  • a packaging vector eg, a backbone plasmid
  • the packaging vector is capable of expressing gag, pol, tat, rev genes.
  • the packaging vector is a vector comprising the HIV-1 genome deleted for the env gene.
  • the packaging vector is a plasmid.
  • the backbone plasmid includes, eg, pSPAX, pNL4-3.Luc.R-E-, pSG3 ⁇ env, pfNL43-dGPE-EGFP.
  • the vector described in (i) is a eukaryotic expression vector, including but not limited to VRC8400, PTT5, pCDN3.1, and the like.
  • the present invention provides a method for preparing the pseudoviral particle of the present invention, comprising: (1) co-transfecting the expression vector and the packaging vector comprising the nucleic acid molecule described in the fourth aspect into a host cell; ( 2) expressing the protein encoded by the expression vector and the packaging vector in a host cell, and the protein can spontaneously assemble into HIV pseudovirus; and (3) collecting HIV pseudovirus.
  • the host cells are eukaryotic cells, eg, mammalian cells, eg, primate cells, eg, human cells.
  • the present invention provides a packaging system for producing the above pseudoviral particles, comprising: (i) an expression vector comprising the nucleic acid molecule of the fourth aspect, (ii) a packaging vector.
  • the present invention provides an engineered HIV virus expressing the fusion protein of the second aspect or a multimer (eg trimer or dimer) thereof as its envelope protein.
  • the HIV virus is HIV-1 virus.
  • the modified HIV virus of the present invention will lose the ability to infect cells and animals, and as the immunogen of the vaccine, it can simulate the immune response process of HIV-1 infection of the human body to the greatest extent, and may generate protective immunity while having good safety. .
  • the modified HIV true virus of the present invention can also simulate the natural mutation of HIV-1 virus during cell culture, and this mutation cannot be reverted into infectious virus particles, because the virus replication process cannot be completed with the coding version.
  • the gene fragments of the invented Env protein are rearranged. Therefore, this kind of live virus vaccine will have the immunity of natural virus to the body, but it is impossible to have infectivity and pathogenic ability, so it is also a feasible vaccine strategy.
  • the genome of the engineered HIV virus comprises the following modifications: the wild-type env gene is replaced by a nucleotide sequence encoding the fusion protein of the second aspect.
  • the present invention provides an isolated nucleic acid molecule or vector comprising a nucleotide sequence encoding the genome of the engineered HIV virus described above.
  • the present invention provides a composition comprising the recombinant protein of the first aspect, the fusion protein of the second aspect, or the multimer of the third aspect, and the fourth aspect.
  • the composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the composition is an immunogenic composition or vaccine.
  • the recombinant protein of the first aspect, the fusion protein of the second aspect, or the multimer of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the fifth aspect The vector described in the sixth aspect, the host cell described in the sixth aspect, the particle described in the eighth aspect, the pseudovirion described in the ninth aspect, the modified HIV virus described in the tenth aspect, or the eleventh aspect. isolated nucleic acid molecules or vectors as immunogens.
  • the composition is a protein vaccine comprising the recombinant protein of the first aspect, the fusion protein of the second aspect or the multimer of the third aspect, or the recombinant protein of the eighth aspect
  • the described particles were used as immunogens.
  • the composition is a viral vaccine comprising the pseudovirion of the ninth aspect, or the engineered HIV virus of the tenth aspect as an immunogen.
  • the composition is a nucleic acid vaccine comprising the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the isolated nucleic acid molecule or vector of the eleventh aspect as an immunogen.
  • nucleic acid vaccine refers to a DNA or RNA (eg, plasmid, eg, expression plasmid) based vaccine, optionally further comprising an adjuvant.
  • the nucleic acid vaccine comprises DNA or RNA.
  • the DNA or RNA may be naked or may be encapsulated within a delivery or/and protective envelope.
  • the coat may be the coat of adenovirus, adeno-associated virus, lentivirus, retrovirus, etc., or may be other materials synthesized by chemical methods that can perform similar functions.
  • the pharmaceutically acceptable carrier and/or excipient comprises an adjuvant.
  • the adjuvant for co-administration or inclusion in the composition according to the invention should preferably be a potentially safe, well tolerated and effective adjuvant in humans.
  • adjuvants are well known to those skilled in the art, and non-limiting examples include QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TER amide, PSC97B, Adjumer , PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, aluminium salts such as aluminium phosphate (eg AdjuPhos) or aluminium hydroxide and MF59.
  • aluminium salts such as aluminium phosphate (eg AdjuPhos) or aluminium hydroxide and MF59.
  • compositions of the present invention may further comprise an antiretroviral agent.
  • the antiretroviral agent comprises: a nucleoside reverse transcriptase inhibitor such as abacavir, AZT, didanosine, emtricitabine , lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, etc.; non-nucleoside reverse transcriptase inhibitors, eg delavirdine, efavirenz, nevirapine, protease inhibitors eg amprenavir, atazanavir, indinavir , lopinavir, nelfinavir, osamprenavir, ritonavir, saquinavir, tipranavir, etc., as well as fusion
  • the immunogenic composition or vaccine of the invention and the antiretroviral agent are present as separate components or as a single formulation. In certain embodiments, the immunogenic composition or vaccine of the invention and the antiretroviral agent are present as separate components or as a single formulation. In certain embodiments, the immunogenic composition or vaccine of the invention and the antiretroviral agent may be administered simultaneously, separately or sequentially.
  • compositions of the invention can be formulated into any dosage form known in the medical art, eg, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules , elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the compositions of the present invention should be sterile and stable under the conditions of manufacture and storage.
  • a preferred dosage form is an injection.
  • Such injectable preparations can be sterile injectable solutions.
  • sterile injectable solutions can be prepared as sterile lyophilized powders (eg, by vacuum drying or freeze-drying) for ease of storage and use.
  • sterile lyophilized powders can be dispersed in a suitable vehicle, eg, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg, 0.9% (w/v) NaCl), Dextrose solutions (eg, 5% dextrose), surfactant-containing solutions (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered solutions), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution eg, 0.9% (w/v) NaCl
  • Dextrose solutions eg, 5% dextrose
  • surfactant-containing solutions eg, 0.01% polysorbate 20
  • pH buffered solutions eg, phosphate buffere
  • compositions of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal , intracytoplasmic reticulum, inguinal, intravesical, topical (eg, powder, ointment, or drops), or nasal route.
  • parenteral administration eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection.
  • the route and/or mode of administration will vary depending on the intended purpose.
  • the immunogenic compositions of the present invention should be administered in an amount sufficient to induce an immune response against HIV-1.
  • Appropriate amounts of immunogens can be determined based on the particular disease or disorder to be treated or prevented, the severity, the age of the subject, and other personal attributes of the particular subject (e.g., the general state of the subject's health and the robustness of the subject's immune system). Determination of effective doses is also guided by animal model studies, followed by human clinical trials, and by administration regimens that significantly reduce the occurrence or severity of the symptom or condition of the target disease in a subject.
  • compositions eg, immunogenic compositions
  • the compositions can readily be used in a variety of therapeutic or prophylactic applications to treat HIV-1 infection or elicit an immune response to HIV-1 in a subject.
  • the composition can be administered to a subject to induce an immune response to HIV-1, eg, to induce the production of broadly neutralizing antibodies against HIV-1.
  • the immunogenic compositions of the present invention can be administered to provide prophylactic protection against viral infection.
  • the present invention provides methods for inducing an immune response against HIV in a subject or for preventing and/or treating HIV infection in a subject, comprising administering to a subject in need thereof
  • the subject administers an immunologically effective amount of the recombinant protein described in the first aspect, the fusion protein described in the second aspect, or the multimer described in the third aspect, the isolated nucleic acid molecule described in the fourth aspect, and the fifth aspect.
  • the method may further comprise administering to the subject other combinations of agents for the treatment or prevention of HIV infection.
  • the method comprises administering an antiretroviral agent, such as a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, a fusion protein inhibitor, and the like.
  • Prophylactic administration of an immunogenic composition for the prevention or amelioration of any subsequent infection to attenuate the expected severity, duration or extent of infection and/or associated disease symptoms following exposure or suspected exposure to the virus or after actual initiation of infection .
  • the subject to be treated is a subject suffering from or at risk of developing HIV infection, eg, as a result of exposure or potential exposure to HIV.
  • a subject can be monitored for HIV-1 infection, or symptoms associated with HIV-1 infection.
  • the recombinant proteins, fusion proteins, multimers, isolated nucleic acid molecules, vectors, host cells, particles, pseudovirions, engineered HIV viruses, or compositions (eg, immunogenic compositions) of the invention Provided at or after the onset of symptoms of the disease or infection, eg, after the onset of symptoms of HIV-1 infection or after diagnosis of HIV-1 infection.
  • the present invention also relates to the recombinant protein of the first aspect, the fusion protein of the second aspect or the multimer of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the fifth aspect
  • the vector of aspect, the host cell of the sixth aspect, the particle of the eighth aspect, the pseudovirion of the ninth aspect, the engineered HIV virus of the tenth aspect, or the eleventh aspect Use of the isolated nucleic acid molecule or vector in the preparation of a medicament for inducing an immune response against HIV in a subject and/or for preventing and/or treating HIV infection in a subject.
  • the agent is a vaccine.
  • the subject is a human.
  • the present invention also relates to the recombinant protein of the first aspect, the fusion protein of the second aspect, the multimer of the third aspect, or the particle of the eighth aspect for the preparation of protein vaccines Use of the protein vaccine for inducing an immune response against HIV in a subject and/or for preventing and/or treating HIV infection in a subject.
  • the present invention also relates to the use of the pseudovirion of the ninth aspect, or the engineered HIV virus of the tenth aspect, for the preparation of a viral vaccine for use in a subject Inducing an immune response against HIV and/or for preventing and/or treating HIV infection in a subject.
  • the present invention also relates to the use of the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the isolated nucleic acid molecule or the vector of the eleventh aspect for preparing a nucleic acid vaccine,
  • the nucleic acid vaccine is used for inducing an immune response against HIV in a subject and/or for preventing and/or treating HIV infection in a subject.
  • Env refers to the envelope protein (envelope) on the surface of HIV-1 virus, Env is synthesized on the surface of HIV-1 in the form of full-length gp160 (full-length molecular weight 160KD), and the translated protein After the virus matures, the precursor is cleaved by protease into gp120 (molecular weight of 120KD) and gp41. gp120 is located on the surface of the virus and is a molecule that recognizes CD4 receptors to induce infection.
  • gp41 contains an extracellular region, a transmembrane region and an intracellular region, with a molecular weight of 41KD, gp120 and gp41 form heterodimers through non-covalent interactions between subunits, and heterodimers form a typical trimer structure through non-covalent interactions, the trimeric protein is Env .
  • gp140 refers to a protein consisting of gp120 and gp41 extracellular segment with the intracellular and transmembrane regions of gp41 deleted, and the trimer formed by this monomeric protein may be referred to as the gp140 triad aggregates.
  • gp140 trimers include trimeric proteins whose conformation is further stabilized by introducing disulfide bonds, point mutations, etc. into the monomer.
  • BG505 SOSIP refers to, on the basis of the full-length Env of the BG505 strain, the intracellular segment and the transmembrane region sequence are removed, and the cys mutation is introduced at the 501st amino acid and the 605th amino acid, so as to be in gp120 and A disulfide bond is formed between the subunits of the extracellular segment of gp41, and the pre-fusion conformation of gp140 trimer is further stabilized by the Ile mutation at amino acid 559.
  • This modification method is named SOSIP, and this modified protein is BG505 SOSIP. Deletion of the intracellular and transmembrane domains of gp160 will facilitate soluble expression of the membrane protein.
  • Furin refers to a major protein converting enzyme in the exocrine pathway that catalyzes the cleavage of Arg-X-Y-Arg carboxy-terminal peptide bonds in proteins (X is any amino acid, Y is Arg or Lys), resulting in the mature protein.
  • Furin recognizes the REKR sequence between the gp120 and gp41 subunits of immature HIV-1 Env and cleaves full-length gp160 into gp120 and gp41, which undergo conformational rearrangement and maturation.
  • the HXB2 numbering system is a reference numbering system for HIV protein and nucleic acid sequences, using the HIV-1 HXB2 strain sequence as a reference to the sequences of all other HIV strains.
  • One of ordinary skill in the art is familiar with the HXB2 numbering system.
  • the amino acid numbering described herein, such as 559, 501, 605, etc., are all given according to the HXB2 numbering system, which can be determined by using the amino acid numbering of the strain HXB2 as a template and through sequence homology alignment. Those skilled in the art understand that It does not refer to the actual amino acid number of the protein.
  • a region corresponding to a specific amino acid position of the gp160 sequence of the isolate HXB2 means that when the gp160 sequence of the strain to be compared is optimally aligned with the gp160 sequence of the isolate HXB2, that is, when the sequence Positions in the strains to be compared that are equivalent to specific amino acid positions in isolate HXB2 are performed when alignments are made to obtain the highest percent identity.
  • subtypes As used herein, "subtypes,” “strains,” etc. of HIV are well known to those skilled in the art. It is now known that HIV-1 has at least 13 subtypes, including A, B, C, D, E, F, G, H, I, J, K, O, N subtypes, which are classified as M, O, N Three groups, A-K belong to the M group. There are many strains in different subtypes, one strain corresponds to one Env sequence. As used in the invention, BG505 is a strain in subtype A, and NL4-3 and Bal.26 are strains in subtype B.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • packaging vector refers to a vector capable of expressing proteins other than envelope proteins that are necessary for the formation of HIV virus-like particles, such as gag, pol, tat and vpu proteins of HIV.
  • Packaging vectors can usually be constructed by modifying or deleting envelope protein-encoding genes and regulatory genes in the complete HIV genome.
  • packaging vectors for assembling HIV virus-like particles/pseudoviruses and methods of constructing such packaging vectors are known in the art.
  • the term "backbone plasmid” refers to a plasmid encoding structural proteins such as gag, pol, etc., used in pseudovirus production.
  • the packaging plasmids, envelope plasmids, transfer plasmids, and regulatory plasmids need to be co-transfected into packaging cells to generate pseudovirus particles that are convenient for detection.
  • the transfer plasmids generally contain reporter genes that can indicate the infection of pseudoviruses.
  • the reporter gene can be integrated into the packaging plasmid, the regulation gene can regulate the transcription and translation of pseudovirus structural genes, and it can also be integrated with the packaging plasmid.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus, etc.
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the term "pseudovirus” refers to a virus-like particle formed from viral capsid or envelope proteins, which typically does not encapsulate nucleic acid or encapsulates genetically deleted or modified viral nucleic acid. Generally speaking, because the pseudovirus does not contain nucleic acid or the contained viral nucleic acid genome is incomplete, the pseudovirus only has the ability of a single round of infection, but does not have the replication ability to produce progeny viruses, and has high biological safety. sex.
  • Tier2 strain is well known to those skilled in the art, HIV-1 strains can be neutralized by some specific antibodies, and HIV-1 strains are neutralized by some specific antibodies. It is divided into three levels: Tier1, Tier2 and Tier3. Tier1 is the most easily neutralized strain, and Tier2 is the least neutralized strain.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by an adenine, or both A position in each of the polypeptides is occupied by a lysine)
  • the molecules are identical at that position.
  • the "percent identity” between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6).
  • comparisons are made when two sequences are aligned for maximum identity.
  • Such alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.).
  • Align program DNAstar, Inc.
  • Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0) can also be used, using the PAM120 weight residue table , a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences.
  • the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences .
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient , which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, ionic strength enhancers, Agents for maintaining osmotic pressure, agents for delaying absorption, diluents, adjuvants, preservatives, stabilizers, etc.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate salts and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like.
  • Adjuvants include, but are not limited to, aluminum adjuvants (eg, aluminum hydroxide), Freund's adjuvants (eg, complete Freund's adjuvant), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning generally understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug (such as the inhibitory activity against ubiquitination of PSD-95), including but not limited to sodium glutamate, gelatin, SPGA, Sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation Products (such as lactalbumin hydrolyzate), etc.
  • Env As the main antigenic protein of HIV-1 virus surface recognition receptor molecule, Env is also the key molecule for HIV-1 vaccine development.
  • the Env on the surface of the natural virus is a non-covalently linked heterotrimer.
  • the conformation of Env gradually changes from the closed prefusion conformation to the open conformation.
  • the Env trimer protein of the present invention can be efficiently expressed in mammalian cells, the trimer content is increased, the components are more uniform, and the thermal stability is significantly improved.
  • the Env trimer protein of the present invention can effectively expose key epitopes such as CD4bs/V3, but is not conducive to the exposure of non-neutralizing epitopes, and has the potential to be used as a protein vaccine.
  • the HIV pseudovirus and true virus obtained based on the Env trimer protein are highly similar to the natural virus in shape and thus have immunity to the body from the natural virus, but have lost the ability to infect and cause disease, and thus exhibit the ability to act as a virus.
  • the potential of vaccines has important clinical value.
  • Figures 1A-1B show the monomeric protein spatial structure (1A) and secondary structure distribution topologies ( 1B).
  • Figures 2A-2B show the results of reduced SDS polyacrylamide gel electrophoresis of the engineered gp140 trimers after one-step Ni Sepharose excel purification.
  • Figure 2A shows the BG505 strain
  • Figure 2B shows the NL4-3 strain.
  • 1 is TSTIP protein
  • 2 is TST protein
  • 3 is SOSIP protein.
  • Both TSTIP and TST showed a single band under reducing conditions with a molecular weight of about 140KD
  • SOSIP showed two bands under reducing conditions, which were gp120 and gp41 extracellular segment, respectively. It shows that the protein of the present invention adopts a stable valence link between subunits as expected.
  • Figure 3 shows the purification results of the molecular sieve superdex200-16/600 modified gp140 trimer, A is the BG505 strain, and B is the NL4-3 strain.
  • the molecular sieve chromatographic results of TSTIP protein and TST protein showed a single elution peak, while the chromatographic results of SOSIP protein showed three elution peaks, indicating that the components of the protein TSTIP and TST of the present invention were more homogeneous than those of SOSIP. .
  • Figures 4A-4C show the results of non-reducing polyacrylamide gel electrophoresis after the modified gp140 trimer was purified by molecular sieves.
  • A is BG505 TSTIP
  • B is NL4-3 TSTIP
  • C is BG505 SOISP.
  • the results showed that after purification by molecular sieve, the main component of the protein TSTIP of the present invention was trimer, and the content of trimer was higher than that of SOSIP.
  • FIG. 5 shows the results of differential scanning calorimetry (differential scanning calorimetry, DSC) Tm determination of the modified gp140 trimer.
  • the results showed that the Tm of the protein BG505 TSTIP of the present invention was 74.5°C, and the Tm of the NL4-3 TSTIP was 63.41°C, which were 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. It indicated that the thermal stability of the engineered protein was higher than that of the corresponding control protein SOSIP.
  • Figure 6 shows the results of enzyme-linked immunosorbent assay (ELISA) of the engineered gp140 trimer with various reported human monoclonal antibodies.
  • the antibodies used included broadly neutralizing antibodies VRC01, B12, PGT121, PGT125, 2G12, non-broadly neutralizing antibodies F105, F240, 17b, and the receptor molecule CD4 of HIV-1. It is shown that the binding ability of the proteins BGTSTIP and NL4-3 TSTIP of the present invention and various kinds of reported broad-spectrum neutralizing antibodies and non-broad-spectrum neutralizing antibodies is basically equivalent to that of the control proteins BG505 SOSIP and NL4-3 SOSIP, and can be well recognized and combined. CD4 receptor molecule. And like SOSIP, the recognition of CD4 molecule and the engineered protein induces a conformational change of the inventive protein, which in turn exposes the 17b binding site.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 7 shows the virus neutralization experiment of immune sera against B subtype NL4-3 and B subtype 2626 strain viruses after immunizing BALB/C mice with the modified gp140 trimer; wherein, M1 to M6 refer to different mice respectively .
  • the results showed that the engineered protein TSTIP and the control protein could induce strong auto-neutralizing responses after immunizing mice, but could not induce the same isoform Tier2 neutralizing responses.
  • the engineered protein TSTIP and the control protein SOSIP showed comparable immunogenicity.
  • Figure 8A shows the results of a western blot of pseudoviruses based on engineered gp140 trimers.
  • Figure 8B shows the quantification of p24 based on pseudoviruses engineered to the gp140 trimer. The results show that both the transfected cells and the collected virus supernatant express the full-length TSTIP protein, and the content of p24 in the pseudovirus supernatant of the present invention is lower than that of the wild-type pseudovirus, but a certain amount of p24 can still be detected. and Env, indicating that the pseudovirus of the present invention can be normally packaged into complete pseudovirus particles, but the yield may be reduced to a certain extent.
  • Figure 9 shows negative staining electron microscopy results of pseudovirions based on engineered gp140 trimers. Pseudovirions clearly similar to wild-type pseudoviruses can be seen under a negative-stain electron microscope.
  • Figure 10 shows the detection results of the infectivity of the pseudovirus based on the modified gp140 trimer detected by the Elispot method with LacZ as the reporter gene. It is shown that the wild-type pseudovirus maintains normal infectivity, while the pseudovirus of the present invention has lost the infectivity, which is in line with the inventor's expectation.
  • Figure 11A shows the results of reduced SDS polyacrylamide gel electrophoresis of the engineered gp140 trimer BG505/NL4-3 TSTIP G1/G2/Full. showed that the molecular weights of BG505 TSTIP-G2 and NL4-3 TSTIP-G1 were larger than expected, the two inventive proteins may still be dimers under reducing conditions, and BGTSTIP-Full, NL4-3-G1 and NL4-3-Full were not purified to a purer protein of interest. In contrast, the inventive protein of TSTIP has a stronger scope of use.
  • Figure 11B shows the AUC results of the modified gp140 trimer BG505/NL4-3 TSTIP G1/G2/Full, which are consistent with the SDS-PAGE results.
  • Figure 12 shows the ELISA results of the modified gp140 trimer BG505/NL4-3 TSTIP G1/G2/Full and various reported antibodies. It is shown that all six proteins can react with various antibodies and have certain activities, but the reactivity of some proteins is weaker than that of the invented protein TSTIP.
  • Figure 13 shows the virus neutralization experiment of the B subtype NL4-3 strain by immune sera after immunizing BALB/C mice with various engineered gp140 trimers. The results showed that all six proteins could induce neutralizing antibody responses in mice.
  • Figure 14A shows the SDS acrylamide gel electrophoresis results of the modified gp140 trimers of various strains. The results show that the modified proteins of each strain are well expressed and can be purified to obtain higher purity. Under SDS-PAGE, the inventive protein showed a single band, which proved that the subunits were stable covalently linked.
  • Figure 14B shows the purification results of five strains of TSTIP protein molecular sieve superdex200 16/600, showing that the peaks of the five inventive protein molecular sieves are relatively single, indicating that the protein homogeneity is good. as the subject.
  • Figure 14C shows the results of the enzyme-linked immunosorbent assay of five strains and various antibodies, showing that the five inventive proteins have certain reactivity with various reported antibodies, which proves that their activity is good.
  • Figure 14D shows the results of the neutralization experiment between the sera and pseudoviruses of the five strains after TSTIP protein immunization.
  • the strains detected by the CH119 TSTIP and X1632 TSTIP immune sera were the corresponding CH119 and X1632 strains; the 25710 and 246F3 TSTIP sera detected the The strain was BJOX200; the strain detected by CNE8 TSTIP immune serum was TRO11.
  • the results showed that weak virus neutralization was detected in the fourth dose of immune sera of the five strains of TSTIP protein, indicating that the TSTIP protein immunization of different strains could stimulate the neutralization response of mice.
  • FIGS. 15A-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.
  • Figure 15A BG-B1 (1/1);
  • Figure 15B BG-B1 (1/2);
  • Figure 15C BG-B1 (2/2);
  • Figure 15D BG-B1 (2/1);
  • Figure 15E BG-B1 (2/3);
  • Figure 15F BG-B2 (1-1);
  • Figure 15J BG-C1 (1/2);
  • Figure 15K BG-C1 (1/3).
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995, was performed as described; restriction enzymes were used according to the conditions recommended by the product manufacturer.
  • restriction enzymes were used according to the conditions recommended by the product manufacturer.
  • Example 1 Expression of TSTIP protein of two strains of NL4-3/BG505
  • the base and amino acid sequences of the two strains of BG505/pNL4-3-gp160 on NCBI were used as transformation templates.
  • the transformation of BG505 as an example, according to the cryo-electron microscope three-dimensional structure of BG505-SOSIP (PDB: 4tvp), the partial loop sequence (aa610-616, WNSSWSN) of the C-terminus of ⁇ 27 of BG505 gp41 was removed, and the C-terminus of gp120 included furin enzyme Sequence of 10 amino acids including the excision site (aa501-511, AKRRVVGREKR).
  • the truncated C-terminus of ⁇ 27 of gp41 was then ligated to the N-terminus of gp120, and the truncated C-terminus of gp120 was ligated to the N-terminus of the ⁇ 8 domain of gp41.
  • the amino acid sequence of the modified complete gp140 is as follows: ⁇ 6/ ⁇ 7/ ⁇ 27 (501-606) + part of the loop region between ⁇ 27 and ⁇ 8 (607-609) + gp120 (33-500) + between ⁇ 27 and ⁇ 8 C-terminal sequence of loop region part (617-618)+ ⁇ 8/ ⁇ 9 (619-664).
  • BG505-TST SEQ ID NO: 1
  • BG505-TSTIP SEQ ID NO: 3
  • NL4-3 was transformed in the same way to obtain NL4-3-TST (SEQ ID NO: 2) and NL4-3-TSTIP (SEQ ID NO: 4), respectively.
  • Figure 1 The structural schematic diagram of each of the above proteins is shown in Figure 1.
  • the designed amino acid sequence of BG505/pNL4-3-TSTIP was converted into bases suitable for mammalian cell expression and sent to Shanghai Sangon for gene synthesis, and constructed between the ECORI and Xbal restriction sites of pcDNA3.1 vector. Take 1 ul of the synthesized pcDNA3.1-BG505SOSIP/4-3TSTIP plasmid to transform 50 ul of DH5 ⁇ competent cells (purchased from Shenzhen Kangti Life Technology Co., Ltd.), spread it on ampicillin-resistant solid medium, and culture at 37°C for 10- 12 hours until a single colony is clearly visible, pick a single colony into a test tube containing 3 ml of ampicillin-resistant LB medium, shake at 220 rpm for 10 hours at 37 °C, and mix 500 ul of bacterial liquid with 500 ul of 50% glycerol and freeze it in -20°C.
  • Frozen 293F cells were taken from -80°C refrigerator, thawed at 37°C and centrifuged at 1300 rpm for 4 min. The supernatant was discarded in the ultra-clean bench, and the cells were flicked and resuspended in 293freestyle medium incubated at 37°C in advance. In the flask of the incubation medium, suspension culture was carried out at 37°C, 5% CO2, 120 rpm, and when the cell density reached 2.0*10 6 , the cells were passaged, and the culture system was gradually expanded.
  • 293F cells were transiently transfected with PEG2000. After the cells reached a density of 2.0*106, the cells were collected in a sterile 50ml tube, centrifuged at 1300rpm for 4min, and the cells were flicked and resuspended in the 37°C incubation medium, and transferred to a 450ml 37°C incubation medium. In a base Erlenmeyer flask, placed on a shaker at 37°C for later use.
  • the cell culture medium was collected, and the cell supernatant was collected after centrifugation at 7000g with a JA-14 rotor for 10min. Column purification.
  • Ni Sepharose excel affinity medium buffer: divided into A and B buffers, A is 1 ⁇ PBS buffer, and B pump is 1 ⁇ PBS+250mmol/L imidazole buffer;
  • Example 3 Identification of biochemical properties of NL4-3/BG505 TSTIP protein
  • Buffer PBS (20 mM phosphate buffer, pH 7.5, 150 mM NaCl)
  • the sample is the purified protein concentrate of Example 2
  • the purification procedure is: 1 column volume of PBS to balance superdex200 16/600, 5ml loading loop to load the target protein purified in Example 2, molecular sieve purification of the purified sample in inject mode, the sample will be based on the molecular weight of different components
  • the size, from high to low, is eluted in sequence, and the elution peaks under different elution volumes are collected, that is, the target protein in the form of different aggregates.
  • the samples collected in the molecular sieve purification process under different elution volumes were concentrated to 1ug/ul in 20ml of Vivaspin and a 100KD concentrating tube, and labeled 1, 2, 3, and 4 in the order of elution.
  • Take 50ul of each part of the sample add 10ul of non-reducing 6 Loading Buffer, mix well, take 10ul of non-reducing samples and electrophorese them in 8% SDS-PAGE at 80V for 120min. After staining with Coomassie brilliant blue, electrophoresis bands are displayed.
  • the electrophoresis results are shown in Figure 4A-4C.
  • the main component of purified BG505/NL4-3TSTIP was gp140 trimer, while BG505/NL4-3SOSIP contained a large amount of gp140 monomer.
  • the sample is the purified protein concentrate of Example 2
  • the value When the value is stable between ⁇ 0.2, it can be scanned cyclically, and the baseline balance is not less than 3 times; when the last cycle of scanning is over, and the temperature drops to between 30°C and 10°C, the buffer in the sample tank is sucked out. Quickly add 300ul of the protein sample to be tested, and continue to scan the test; the scan rate is set to: 90°C/h. After the scan is completed, use Origin7.0 to process the data. The results are shown in Figure 5.
  • the Tm of the invention protein BG505-TSTIP 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 At 62.00 °C, although the Tm of the NL4-3 strain did not increase significantly, it was improved to a certain extent compared with the reported optimized design NL4-3SOSIP. These results indicate that our design can improve the thermal stability of the protein to a certain extent, and its thermal stability may be greatly improved in some strains.
  • Select broad-spectrum neutralizing antibodies such as PGT121, PGT125, VRC01, 2G12, B12, and non-neutralizing antibodies such as 17b, 447-52D, F105, F240, etc. to carry out ELISA antigenic analysis on BG505/NL4-3TSTIP and BG505/NL4-3SOSIP.
  • the specific process is as follows:
  • BG505/NL4-3TSTIP has strong reactivity with broad-spectrum neutralizing antibodies such as PGT121, PGT125, VRC01, 2G12, and B12, but weaker reactivity with non-neutralizing antibodies such as 17b, F105, and F240.
  • 17b, F105, and F240 are antibodies targeting non-neutralizing epitopes such as CDi, CD4bs, and gp41, respectively, which indicates that BG505/NL4-3TSTIP does not expose such non-neutralizing epitopes, but well presents such non-neutralizing epitopes as CD4bs, out glycan, V3 and other key epitopes.
  • the reactivity of BG505/NL4-3TSTIP with various broad-spectrum neutralizing antibodies and non-neutralizing antibodies remained comparable to the control protein BG505/NL4-3SOSIP.
  • mice female, 6 weeks old, purchased from Shanghai Slack Laboratory Animal Co., Ltd. Four groups of immunized mice were set, with 6 mice in each group, and BG505/NL4-3TSTIP and BG505/NL4-3SOSIP prepared in Example 2 were used as immune proteins.
  • the protein was diluted with physiological saline, mixed with aluminum hydroxide adjuvant in a volume ratio of 1:1, the protein was adsorbed on the adjuvant, and mice were immunized intraperitoneally.
  • the immunization protocol for mice is set up in Table 1.
  • Eyeball blood of mice was collected before immunization, and immunized according to the immunization scheme in Table 1. Eyeball blood of mice was collected before each immunization. After immunization to the sixth needle, eyeball blood was collected, and then the mice were subjected to neck dissection treatment. The blood samples were placed at 37° C. for 30 min, and then centrifuged at 13,300 rpm for 10 min, and serum was collected for HIV-1 pseudovirus neutralization and antibody titer determination.
  • the Bal26 TSTIP pseudovirus produced by the present inventors was obtained by co-transfecting 293FT (Invitrogen) cells with HIV-1 backbone plasmid pfNL43-dGPE-EGFP (Addgene) and envelope plasmid VRC8400-Bal26-TSTIP-gp160.
  • the backbone plasmid is pfNL43-dGPE-EGFP, purchased from addgene. This plasmid is transformed from NL4-3 infectious clone, and contains genes such as gag, pol, tat, and rev necessary for virus packaging, but its Env gene has been partially replaced by EGFP gene, resulting in the silencing of the Env gene. Pseudovirus production is performed by transfecting an expression plasmid expressing Env.
  • the expression vector of the envelope gene used in the present invention is VRC8400, and the VRC8400 vector is preserved in this laboratory.
  • the inventor designed TSTIP for the full length of the Env gene of Bal.26 according to the design idea of the BG505 TSTIP protein, and cloned the full-length Bal.26 TSTIP gene (which encodes SEQ ID NO: 16) carrying the virus's own signal peptide into VRC8400 Between the EcoRV and BglII restriction sites of the vector, the VRC8400-Bal.26-TSTIP-gp160 expression plasmid was constructed. The obtained pseudovirus was named BaL.26-SD-FS. In addition, a pseudovirus was obtained as a control (BaL.26-WT) based on the wild-type gp160 sequence of the Bal.26 strain.
  • pseudovirus adopts the method of PEI transient transfection.
  • the above transfected 293FT cells and cell transfection supernatant were harvested, lysed with cell lysate for 1 h, centrifuged to collect the cell lysate supernatant, and performed western blot together with the above cell lysate.
  • the specific steps are as follows:
  • the primary antibody was diluted in ED11 (purchased from Xiamen Wantai) at a concentration of 1ug/ml, and incubated at room temperature on a shaker for 1 h.
  • the virus solution was diluted 5 times in 6 gradients, and the p24 content in the supernatant virus solution was detected by ELISA method, and a fitting curve was prepared from the reaction of the gradient diluted p24 standard.
  • the quantitative results of p24 are shown in Table 2. Meanwhile, negative staining electron microscopy was performed on the harvested pseudovirus liquid samples, as shown in Fig. 9.
  • the content of p24 of the pseudovirus produced by the present invention has decreased to a certain extent, but within an acceptable range, the expression of Env protein can be detected in the supernatant, and an obvious pseudovirus can be observed under negative staining. particles, indicating that the pseudovirus of the present invention can be normally packaged to form pseudovirus particles.
  • the harvested virus solution was diluted in a U-bottom 96-well plate, and 100ul virus solution in the first well was sequentially diluted 5 times for 5 times. Each well contained 15ug/ml DEAE to promote the ability of pseudovirus to infect cells.
  • TZM-b1 cells were cultured in 96-well cell culture plates in advance, and when the cell density reached 80% at 37°C and 5% CO2, the diluted pseudovirus solution was transferred to TZM-b1 cells. After -48h, the infectivity of the produced pseudoviruses to target cells was detected by chemiluminescence and ELISPOT methods. The results are shown in Figure 10. Both detection methods showed that the pseudovirus transformed with TSTIP almost completely lost its infectivity, while the natural pseudovirus could infect TZM-b1 cells normally.
  • the verification of the production, packaging and infectivity of the NL4-3TSTIP and NL4-3WT pseudovirus particles was carried out according to the above-mentioned methods for verification of the production, packaging and infectivity of the Bal26 pseudovirus.
  • HIV-1 virus-like particles were resuspended in PBS, and viral p24 and Env were quantified by the following method.
  • Antibody coating 16G12 antibody was diluted with PB7.4, and 100ng/well (100 ⁇ L) of 16G12 was coated on a 96-well plate, and coated overnight at 4°C.
  • Blocking Wash the 96-well plate once with PBST, spin dry, add 180 ⁇ L of ED solution, and block at 37°C for 2 hours or 4° overnight.
  • Virus incubation Use PBS as buffer to dilute the virus in U bottom plate (p24 standard is added to the plate according to 350, 700, 1400 and 2800pg/mL), 100 ⁇ L volume per well, add lysate, 20ul volume per well. Transfer to a 96-well plate and incubate at 37°C for 1 h.
  • Secondary antibody reaction wash the plate 5 times with PBST washing solution, and spin dry the 96-well plate. Add 100 ⁇ L of 2F2-HRP secondary antibody solution (1:10000 dilution in ED11 solution) to each well. Incubate for 45 min in a 37°C incubator.
  • Color development wash the plate 5 times with PBST wash solution, spin dry the 96-well plate and add 100 ⁇ L equal volume of mixed A/B color development solution to each well. Incubate at 37°C for 10 min.
  • Termination Add 50 ⁇ L of sulfuric acid stop solution to each well, and put the 96-well plate into a microplate reader to read OD650-450nm.
  • GNL coating Coat GNL on a 96-well plate with 500ng/well (100ul), and the buffer is PBS. Coat overnight at 4°C.
  • Blocking Wash the plate once with PBST, spin dry, add 180 ⁇ L of ED solution, and block at 37°C for 2 hours or 4° overnight.
  • Virus coating Dilute the virus solution with ED as a diluent (use gp140 with 1 ug/ml in the first well as the standard), transfer it to a 96-well plate, seal the plate, and incubate at 37°C for 1 hour.
  • Secondary antibody reaction Wash 5 times with PBST washing solution and spin dry. Add 100 ⁇ L of GAH-HRP secondary antibody solution (1:5000 dilution in ED11 solution) to each well. After sealing, incubate at 37°C for 45min.
  • Color development wash 5 times with PBST washing solution, spin dry the 96-well plate and add 100 ⁇ L equal volume of mixed A/B color developing solution to each well. Incubate the reaction in a 37°C incubator for 10 min.
  • Termination Add 50 ⁇ L of sulfuric acid stop solution to each well, and put the 96-well plate into a microplate reader to read OD650-450nm.
  • an experimental protocol was designed to carry out the mouse immunization experiment of the NL4-3 pseudovirion.
  • the immunization protocol was set as shown in Table 3.
  • Shanghai Slack Laboratory Animal Co., Ltd. purchased 6-week-old magnetic mice, and set up five groups of experiments A/B/C/D/E, with 5 mice in each group.
  • a total of five injections of intraperitoneal immunization were performed, and the immunization cycle was 2 weeks/injection.
  • Eyeball blood was collected from mice at 0/2/4/6/8 weeks and 10 weeks before immunization, and then the mice were subjected to neck dissection.
  • Example 7 Production and identification of NL4-3 eukaryotic particles
  • the Env full-length sequence of the NL4-3 strain was designed with reference to the design of the TSTIP protein, and the obtained full-length TSTIP gp160 gene (which encodes the amino acid sequence shown in SEQ ID NO: 17) is codon-optimized to be suitable for mammalian cell expression
  • the bases were sent to Shanghai Sangon for gene synthesis, and were cloned and replaced at the position of the wild-type Env gene in the genomes of the two strains. Take 1 ul of the synthetic plasmid to transform Stbl3 competent (purchased from Shanghai Weidi Biotechnology Co., Ltd.), spread it on ampicillin-resistant solid medium, and stand at 30 ° C for 12-14 hours until a single colony is clearly visible.
  • a single colony was taken into a test tube containing 3 ml of ampicillin-resistant LB medium, cultured with shaking at 220 rpm at 30°C for 12 hours, and 500ul of bacterial solution was mixed with 500ul of 50% glycerol and frozen at -20°C. And inoculate the bacterial liquid to 500ml LB medium for cultivation, to carry out plasmid extraction, and the plasmid extraction process refers to the pcDNA3.1 NL4-3/BG505 TSTIP plasmid extraction process in Example 1.
  • the extracted plasmid was transiently transfected into 293FT adherent cells, and the transfection reagent was PEI.
  • transfection method refer to the production process of pseudovirus in Example 5 Bal.26-TSTIP pseudovirus particle production and identification.
  • the transfection supernatant was collected after 48 hours of transfection, and the purification of NL4-3 TSTIP true virus particles was carried out, and the collected true virus particles were subjected to packaging ability verification and infection ability verification, and the specific method was with reference to the method described in Example 5.
  • the experimental results show that the true virus obtained by the above method can be normally packaged to form true virus particles, and the true virus transformed by TSTIP almost completely loses the ability to infect.
  • Example 7 A sufficient amount of euviral particles was obtained according to the method of Example 7, and the p24 and Env content information of the purified euviral particles was obtained by the p24 and Env quantification method in Example 6.
  • the experimental protocol was designed to carry out the NL4-3 TSTIP eukaryotic particle immunization experiment, and the immunization protocol is shown in Table 4. 6-week-old magnetic mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd., and two groups of A/B experiments were set up, with five mice in each group. Taking the Env content as a reference, group A was immunized with true virus particles containing 2ug-Env, and group B was immunized with 2ug purified gp140 protein.
  • the immunization cycle was 2 weeks/injection, and a total of 5 injections were immunized.
  • Eyeball blood was collected from mice at 0/2/4/6/8 weeks and 10 weeks before immunization, and then the mice were subjected to neck dissection.
  • the blood samples were placed at 37° C. for 30 min, and then centrifuged at 13,300 rpm for 10 min.
  • the serum was collected for the determination of Env and P24-specific binding titers and the determination of HIV-1 pseudovirus neutralizing antibody titers.
  • the experimental results show that the eukaryotic virus of the present invention can induce neutralizing antibody responses and exhibit good immunogenicity.
  • NL4-3TSTIP Based on the amino acid sequence of NL4-3TSTIP (SEQ ID NO: 4), WNSSWSN and AKRRVVGREKR, which need to be deleted due to the design of TSTIP, were introduced at the junction of ⁇ 27 and gp120 of NL4-3TSTIP and the junction of gp120 and ⁇ 8 to obtain SEQ ID NO: 6 (NL4-3TSTIP Full).
  • GGGGS (SEQ ID NO:28) was introduced at the junction of ⁇ 27 and gp120 of BG505 TSTIP and the junction of gp120 and ⁇ 8, respectively, to obtain SEQ ID NO:7 (BG505-TSTIP G1).
  • GGGGS was introduced at the junction of ⁇ 27 to gp120 and the junction of gp120 to ⁇ 8 of NL4-3TSTIP, resulting in SEQ ID NO: 8 (NL4-3-TSTIP G1).
  • GGGGSGGGGS (SEQ ID NO: 29) was introduced at the junction between ⁇ 27 and gp120 of BG505 TSTIP and the junction between gp120 and ⁇ 8 to obtain SEQ ID NO: 9 (BG505-TSTIP G2).
  • GGGGSGGGGS was introduced at the junction of ⁇ 27 to gp120 and the junction of gp120 to ⁇ 8 of NL4-3TSTIP to obtain SEQ ID NO: 10 (NL4-3-TSTIP G2).
  • the inventors analyzed four high-purity proteins, and the results are shown in Figure 11B. This result is consistent with the previous SDS polyacrylamide gel electrophoresis results (14A).
  • the molecular weights of BGTSTIP G1 and NL4-3TSTIP G2 are greater than The molecular weight of monomer gp140, and the purity of BG505 TSTIP was significantly lower than that of BGTSTIP. This indicates that ⁇ 27, the junction of gp120, and the junction of gp120, ⁇ 8 have obvious effects on the expression of Env protein.
  • different transformations on the same strain also have certain differences in expression levels.
  • the mutation of Ile ⁇ Pro at site 559 can effectively improve protein production. Specifically, the expression levels are summarized in Table 3.
  • mice We immunized BABL/C mice with the six purified proteins according to the above immunization scheme.
  • the results of animal immunization experiments showed that all six proteins could induce neutralizing antibody responses in mice, as shown in Figure 13.
  • pseudovirus particles and true virus particles were further constructed based on these proteins, and mouse immunization experiments were performed to detect their immunogenicity.
  • the inventors applied the design method similar to BG505/NL4-3-TSTIP to the Env proteins of various strains.
  • Table 4 shows 12 global representative strains. Since the expression and identification of the A subtype and B subtype strains have been completed, we have selected 1 strain of other subtype strains except for A and B subtypes in the 12 global pseudovirus disks, and carried out Env The transformation of protein TSTIP, the selected strains include 25710 (C), X1632 (G), CH119 (BC), CNE8 (AE), 246F3 (AC) five strains of gp140 protein. According to a series of experimental steps and methods such as cloning, expression and purification, and property identification as described above, the TSTIP-gp140 proteins of five strains were studied, and the series results are shown in Figures 14A-14D.
  • Figure 14B is the chromatographic result of the molecular sieve superdex200 16/600 of the five TSTIP proteins, and the elution peaks are relatively single, indicating that the five inventive proteins have relatively uniform components in the natural state, and the content of trimers is mostly.
  • Figure 14C is the enzyme-linked immunoassay results (ELISA) of five proteins and a variety of reported antibodies. There are differences in the recognition and binding abilities of different strains to the same antibodies. The results show that the five strains produced by the present inventors TSTIP proteins have good activity.
  • FIG 14D shows the neutralization experiment of sera after immunizing BALB/C mice with five proteins. The results show that the immunization of TSTIP proteins of different strains can stimulate the neutralization response of mice.
  • Example 11 Other protein design and activity identification based on TSTIP
  • AKRRVVG Complete the amino acid sequence AKRRVVG (or its homologous sequence, not including furin cleavage site) after the original deleted ⁇ 26 and the sequence WNSSWSN (or homologous sequence) after ⁇ 27, add WNSSWSN to the back of ⁇ 27, and after GGGGS was introduced, AKRRVVG was supplemented before ⁇ 8, and GGGGS was added after it.
  • the modified sequence based on the BG505 strain is shown in SEQ ID NO:38.
  • AKRRVVG Complete the amino acid sequence AKRRVVG (or its homologous sequence, not including furin cleavage site) after the original deleted ⁇ 26 and the sequence WNSSWSN (or homologous sequence) after ⁇ 27, add WNSSWSN to the back of ⁇ 27, and after GGGGS was introduced, AKRRVVG was supplemented before ⁇ 8, and (GGGGS) 3 was added after.
  • the modified sequence based on the BG505 strain is shown in SEQ ID NO:40.
  • Buffer PBS (20 mM phosphate buffer, pH 7.5, 150 mM NaCl)
  • Samples are purified and concentrated proteins.
  • the purification procedure is: 1 column volume of PBS to balance superdex200 10/300, 500ul loading loop to purify the target protein, and molecular sieve purification of the purified sample in inject mode, the sample will be based on the molecular weight of different components, from high to low , are eluted in sequence, and the peaks after purification are saved.
  • Molecular sieve purification results are shown in panel (II) in Figures 15A-15K. Each protein showed a distinct single elution peak on the molecular sieve pattern.
  • the Tito volume of the main elution peak was all In the vicinity of 9.5 ml, there is a trimer peak, while the elution volume of BG-C1 (1/1) is 14 ml, which is a dimer peak.
  • the inventors performed analytical ultra-ionization analysis on the above purified protein, and the AUC results are shown in panel (III) in Figures 15A-15K.
  • the results show that 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) are mainly trimers, BG- C1(1/1) is mainly a dimer, which is consistent with the molecular sieve results.
  • the antigenicity of the purified protein was identified using the enzyme-linked immunosorbent assay method in Example 4 above.
  • the results are shown in panel (IV) in Figures 15A-15K. The results show that these proteins are antigenic, and that modifying the way the linker at the junction can tune antigenic activity.

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Abstract

提供一种经过崭新设计的HIV-1 Env三聚体蛋白和表达所述Env三聚体蛋白的HIV-1假病毒和真病毒，及它们用于预防和/或治疗HIV感染的用途。

Description

经改造的人免疫缺陷病毒膜蛋白及其应用 技术领域
本发明涉及病毒学领域。具体而言，本发明涉及一种经过崭新设计的HIV-1 Env三聚体蛋白和表达所述Env三聚体蛋白的HIV-1假病毒和真病毒，及它们用于预防和/或治疗HIV感染的用途。
背景技术
人免疫缺陷病毒1型HIV-1(Human immunodeficiency virus-1)，是诱发艾滋病的主要病原体，属逆转录病毒科慢转录病毒属，病毒颗粒由核心/包膜和遗传物质二倍体单股正链RNA组成，单链基因组约为9.8kb，其编码三个结构蛋白Env、Gag、Pol和六个辅助蛋白：vpr、vif、vpu、nef、tat、rev。辅助蛋白相互协调，形成了HIV-1病毒复制的调控网络。Pol基因编码聚合酶前体蛋白，经切割后形成蛋白酶(PR)、反转录酶(RT)和整合酶(IN)，是病毒成熟，复制和感染的关键酶。Gag基因编码的Gag蛋白是病毒的主要结构蛋白，并负责病毒的组装过程，占整个病毒质量的50％；Gag蛋白的N端到C端依次为MA、CA、NC和p6。MA为基质蛋白，其聚集到感染细胞的质膜附近招募另一种结构蛋白Env，启动病毒组装过程；CA为衣壳蛋白，成熟HIV-1病毒的衣壳蛋白为锥形，通过蛋白间相互作用驱动病毒成熟；NC为核衣壳蛋白，在病毒包装的过程中能捕获基因组，使其成为完整病毒颗粒；p6上存在包括辅助蛋白在内的其他蛋白的结合位点，这些蛋白与HIV-1的复制、转录等过程息息相关。Gag蛋白的不同组分间存在PR酶切位点，经其作用逐渐成为成熟颗粒(Sundquist,W.I.and H.G.Krausslich,HIV-1 assembly,budding,and maturation.Cold Spring Harb Perspect Med,2012.)。Env是HIV-1表面唯一的抗原物质，每一个病毒表面Env三聚体的数量非常少，约为7-14个，但却能有效引发宿主细胞的感染，可见其重要性。Env为异源三聚体蛋白，前体为分子量160KD的gp160蛋白，在病毒成熟的过程中被furin酶酶切为gp120和gp41，gp120和gp41通过非共价相互作用形成异源三聚体；gp120存在于胞外，病毒感染时，gp120识别宿主细胞上的受体分子CD4,促发病毒感染，gp120上存在CD4bs、V1V2、V3、CD4i等关键表位，识别受体分子的为CD4bs表位，gp41包含胞外区、近膜区(MPER)、跨膜区(TM)及胞内区(CT)，其上存在融膜肽(FP)、近膜区(MPER)、gp120与gp41交界面等表位，gp120识别CD4分子后，会诱导gp120构象改变，暴露出辅助受体结合位点与细胞上的 辅助受体结合，进一步导致构象改变，使gp41的FP表位从病毒膜上瞬间暴露，并插入到宿主细胞中，以介导病毒感染(Guttman,Garcia et al.CD4-induced activation in a soluble HIV-1 Env trimer,structure.2014.)。
Env也是目前多项HIV-1疫苗临床研究的主要成分，早期的艾滋病疫苗研究多以gp120单体作为抗原，其保留了部分已知的Nab表位，但却被证明难以诱导Tier2类中和抗体，III期临床试验证明其不具备临床上的保护效果。研究者认为这可能是gp120单体上存在许多天然Env上不存在的表位，遂逐渐将焦点转移至类天然Env三聚体，但因gp41的跨膜区具有极强的疏水性且和胞内区一样抗原性较低，研究者采用除去其跨膜区及胞内区的方法增加Env的可溶性表达，这类HIV-1抗原分子被命名为gp140(Sanders,R.W.and J.P.Moore.Native-like Env trimers as a platform for HIV-1 vaccine design.Immunol Rev,2017.)。BG505 SOSIP、NFL2P、UFO等设计皆是在gp140基础上进行的，通过在gp120、gp41胞外区间引入二硫键，在gp41上引入稳定突变I559P或将gp120和gp41之间的furin酶切位点用linker取代等方式增强亚基间的相互作用和三聚体的稳定性，这些设计能够有效地暴露Nab表位而不暴露非中和表位，被认为较好地模拟了病毒上的天然三聚体构象。该类抗原及其通过纳米技术制备的病毒样颗粒抗原相继被运用于动物实验，被证实仅能诱导出针对同型病毒的中和抗体,广谱性有限。因此，HIV-1免疫原设计仍需另辟蹊径。
经改造后的gp140的各项理化性质皆得到优化。以BG505-SOSIP为例，其三聚体含量明显提升，热稳定性较单体gp120提高约14℃，与多种bNab的结合能力得到保留，而与CD4i等一些非中和表位抗体的结合却非常弱(Sanders,Derking et al.A next-generation cleaved,soluble HIV-1 Env trimer,BG505 SOSIP.664 gp140,expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies,2013.)。以BG505-SOSIP三聚体免疫小鼠，虽能诱导更强的IgG、Tfh及GC应答，但却无法诱导产生Tier2中和抗体，表位图谱分析表明诱导产生的抗体多针对三聚体base区(Hu,Crampton et al.2015,Murine Antibody Responses to Cleaved Soluble HIV-1 Envelope Trimers Are Highly Restricted in Specificity,2015.)，这一区域少糖基掩盖，但不存在于天然病毒表面。这一结果提示我们，进行Env三聚体免疫原设计，尽可能减少非中和抗体的暴露，稳定三聚体构象，将进一步推进HIV-1疫苗研究，但Env三聚体作为疫苗候选分子仍具有一定的局限性，如难以诱导Tier2中和应答，难以诱导产生多毒株交叉中和效应。
灭活或减毒病毒是许多疫苗的主要有效成分，但截至目前艾滋病尚无有效疫苗及根 治措施，灭活或减毒病毒含有病毒上所有的抗原组分，并能同时诱导机体产生体液免疫和细胞免疫，诱发强的细胞毒性T淋巴细胞应答(CTL)，且SIV减毒活疫苗被证实能成功地保护恒河猴感染SIV病毒(Protection by Live,Attenuated Simian Immunodefificiency Virus against Heterologous Challenge.Journal of Virology,1999.)。但由于HIV-1的致病性和不可治愈性，减毒或灭活HIV-1疫苗有潜在感染人体的危险，限制了HIV-1灭活或减毒病毒在艾滋病疫苗研发中的应用，获得一种经改造完全丧失了致病性且不能通过回复突变重获感染能力的HIV-1病毒颗粒,制备无潜在感染风险的HIV-1病毒疫苗是一种十分新颖且安全的疫苗设计思路，有望对艾滋病疫苗研发领域带来突破性的进展。
发明内容
经改造的gp140/gp160蛋白及三聚体
在第一方面，本发明提供了一种重组蛋白，其包含gp120和gp41胞外结构域(gp41ectodomain,gp41ECTO)，其中，所述gp120位于所述gp41ECTO的β27和α8之间。在某些实施方案中，本发明的重组蛋白是经改造的gp140蛋白。
在本文中，表述“gp120位于所述gp41ECTO的β27和α8之间”是指，gp120在本发明的重组蛋白中的位置是介于gp41ECTO的β27和α8之间，而并不旨在限定其被放入β27和α8之间的具体方式。例如，gp120可以直接插入在gp41ECTO的β27和α8之间的相邻氨基酸之间，或者gp120可以替换gp41ECTO的β27和α8之间的一个或多个连续氨基酸。此外，gp120可以是天然的或经修饰的，gp41ECTO可以是天然的或经修饰的。容易理解，在本发明的技术方案中描述gp41ECTO是天然的，是指除了在β27和α8之间包含gp120之外，该gp41ECTO不包含其他人为修饰。在本文中，术语“修饰”优选地是指一个或多个氨基酸残基的缺失、添加或替换。
在某些实施方案中，所述重组蛋白从N端至C端方向包含：gp41ECTO的α6、α7、β27；gp120；gp41ECTO的α8、α9。
在某些实施方案中，所述gp41ECTO的β27和α8之间的连接区域中的一个或多个(例如，1-12个，5-12个，5-10个；例如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个，11个，或12个)连续氨基酸被替换为gp120。在某些实施方案中，所述连接区域对应于分离株HXB2的gp160序列的氨基酸位置607-618。
在某些实施方案中，所述gp41ECTO在对应于分离株HXB2的gp160序列的氨基酸位置610-616的区域中的一个或多个(例如，1-7个，例如5-7个；例如1个，2个，3个， 4个，5个，6个，或7个)连续氨基酸被替换为gp120。
在某些示例性实施方案中，所述gp41ECTO在对应于分离株HXB2的gp160序列的氨基酸位置610-616的区域的7个连续氨基酸被替换为gp120。在某些实施方案中，所述gp41ECTO的β27和α8之间的连接区域被截去SEQ ID NO:26所示的序列(WNSSWSN)或其对应位置的氨基酸序列。表述“对应位置的氨基酸序列”是指，当不同毒株的gp160序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，与WNSSWSN处于等同位置的氨基酸序列。在某些实施方案中，所述gp120位于对应于分离株HXB2的gp160序列的氨基酸位置606和619之间。
在某些实施方案中，所述gp120插入在gp41ECTO的β27和α8之间的连接区域中的相邻氨基酸之间。在某些实施方案中，所述连接区域对应于分离株HXB2的gp160序列的氨基酸位置607-618。在某些实施方案中，所述gp120位于对应于分离株HXB2的gp160序列的氨基酸位置609和610之间。在某些实施方案中，所述gp120位于对应于分离株HXB2的gp160序列的氨基酸位置616和617之间。
gp120的改变
在一些实施方案中，插入在gp41ECTO中的gp120可以是天然gp120。在某些实施方案中，所述天然gp120蛋白对应于分离株HXB2的gp160序列的氨基酸位置31-511。
在另一些实施方案中，插入在gp41ECTO中的gp120可以是经修饰的gp120，其相比于天然gp120，可以包含一个或多个氨基酸的突变(例如置换、缺失或插入)。
在某些实施方案中，所述gp120是经修饰的gp120，其与天然gp120相比，弗林蛋白酶识别位点包含突变以防止弗林蛋白酶位点被切割。通过切割位点修饰以去除弗林蛋白酶依赖性的方法是本领域技术人员熟知的，例如可以包括对弗林蛋白酶识别位点序列进行氨基酸的置换、插入或缺失，或者将弗林蛋白酶识别位点序列或其部分替换为另外的序列(例如接头序列)。
在某些实施方案中，所述弗林蛋白酶位点对应于分离株HXB2的gp160序列的氨基酸位置508-511。在某些示例性实施方案中，所述弗林蛋白酶识别位点如SEQ ID NO:41(REKR)所示。
在某些实施方案中，与天然gp120相比，所述经修饰的gp120中的弗林蛋白酶识别位点被删除。
在某些实施方案中，所述gp120是经修饰的gp120，其与天然gp120相比，C端截短了1-11个(例如4-11个；例如1个，2个，3个，4个，5个，6个，7个，8个，9个，10 个，或11个)氨基酸。
在某些实施方案中，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置501-511的区域中包含一个或多个(例如，1-11个，4-11个；例如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个，或11个)连续氨基酸的缺失。
在某些示例性实施方案中，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置501-511的区域被删除。在某些实施方案中，所述经修饰的gp120的C末端被截去SEQ ID NO:27所示的序列(AKRRVVGREKR)或其对应位置的氨基酸序列。表述“对应位置的氨基酸序列”是指，当不同毒株的gp160序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，与AKRRVVGREKR处于等同位置的氨基酸序列。
在某些示例性实施方案中，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置508-511的区域被删除。在某些实施方案中，所述经修饰的gp120的C末端被截去SEQ ID NO:41所示的序列(REKR)或其对应位置的氨基酸序列。表述“对应位置的氨基酸序列”是指，当不同毒株的gp160序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，与REKR处于等同位置的氨基酸序列。
在某些实施方案中，所述gp120是经修饰的gp120，其与天然gp120相比，N端截短了1-5个(例如1-4个；例如1个，2个，3个，4个，或5个)氨基酸。
在某些实施方案中，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置31-34的区域被删除。在某些实施方案中，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置31-34的区域被替换为外源核酸序列。在某些实施方案中，所述外源核酸序列由4个氨基酸残基组成。
在某些实施方案中，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置499的氨基酸残基被删除。
在某些实施方案中，所述经修饰的gp120位于对应于分离株HXB2的gp160序列的氨基酸位置606和610之间，也就是说所述经修饰的gp120替换gp41ECTO中对应于分离株HXB2的gp160序列的氨基酸位置607-609的区域。
在某些实施方案中，所述gp120和gp41ECTO之间包含二硫键。在某些实施方案中，所述重组蛋白在对应于分离株HXB2的gp160序列的氨基酸位置501和605之间具有二硫键。在某些实施方案中，所述重组蛋白在对应于分离株HXB2的gp160序列的氨基酸位置501和605上的残基被置换为Cys。
在某些实施方案中，插入在gp41ECTO中的gp120还可以包含一个或多个氨基酸突变，以增加广谱中和抗体识别表位的暴露和/或稳定HIV包膜蛋白三聚体结构。
在某些实施方案中，所述gp120包含下列的一种或多种突变：
(1)所述gp120包含置换T332N，以增加广谱中和抗体识别表位的暴露；
(2)所述gp120包含置换E64K和H66R，以稳定三聚体构象，使其不易变构为结开放构象，从而不容易暴露CD4i非中和表位；
(3)所述gp120包含置换A316W，以增强gp120亚基之间的疏水相互作用，阻碍V3区的移动，避免V3区非中和表位的暴露；
(4)所述gp120的CD4bs表位附近的N-连接糖基化位点(PNGS)被置换以防止糖基化；优选地，所述PNGS选自N276、N301、N360、N463；
(5)所述gp120包含内部二硫键；优选地，所述gp120包含I201C和A433C之间的内部二硫键；
(6)所述gp120和gp41ECTO之间还包含二硫键；例如，所述重组蛋白在E49C和L555C之间具有二硫键；
以上位置的编号是根据HIV-1分离株HXB2的gp160中的编号。
gp41ECTO的改变
在本发明的重组蛋白中，gp41ECTO的β27和α8之间包含gp120。然而所述gp41ECTO的其他部分依然可以是天然gp41中的相应序列，也可以包含人为修饰。
在某些实施方案中，所述gp41ECTO可以包含稳定化突变。用于稳定HIV包膜蛋白三聚体结构的突变是本领域技术人员已知的，参见例如WO 03/022869。在某些实施方案中，所述稳定化突变是I559P，所述位置的编号根据HIV-1分离株HXB2的gp160中的编号。
在本发明的重组蛋白中，插入在gp41ECTO中的gp120可以直接与gp41ECTO连接，也可以通过肽接头与其连接。
在某些实施方案中，所述gp120的N端和/或C端任选地通过肽接头与所述gp41ECTO连接。在某些实施方案中，所述肽接头包含(GmS)n所示的序列或由其组成，其中m选自1-4的整数，n选自1-3的整数。在某些示例性实施方案中，所述肽接头包含 (GmS)n所示的序列或由其组成，其中m为4，n为1、2或3，优选1或2。
在本发明的重组蛋白中，所述gp41ECTO和gp120可以来自相同或不同的HIV-1毒株。在某些实施方案中，所述gp41ECTO和gp120来自相同的HIV-1毒株。
在本发明的重组蛋白中，所述gp41ECTO和gp120可以来自HIV-1的任何亚型，例如组M、N、O或P，或亚型A、B、C、D、F、G、H、J或K等。在某些实施方案中，所述HIV-1毒株选自亚型A、B、C、G、BC、AE、DC。在某些示例性实施方案中，所述HIV-1毒株选自BG505、NL4-3、246F3、25710、CH119、CNE8、X1632、Bal.26。
在某些实施方案中，BG505的gp160具有如SEQ ID NO:18所示的序列。在某些实施方案中，NL4-3的gp160具有如SEQ ID NO:19所示的序列。在某些实施方案中，25710的gp160具有如SEQ ID NO:20所示的序列。在某些实施方案中，X1632的gp160具有如SEQ ID NO:21所示的序列。在某些实施方案中，CH119的gp160具有如SEQ ID NO:22所示的序列。在某些实施方案中，CNE8的gp160具有如SEQ ID NO:23所示的序列。在某些实施方案中，246F3的gp160具有如SEQ ID NO:24所示的序列。在某些实施方案中，Bal.26的gp160具有如SEQ ID NO:25所示的序列。
在某些示例性实施方案中，本发明的重组蛋白包含选自下列的氨基酸序列：
(1)由SEQ ID NO:1所示序列的第40位至第651位氨基酸残基构成的氨基酸序列；
(2)由SEQ ID NO:2所示序列的第40位至第652位氨基酸残基构成的氨基酸序列；
(3)由SEQ ID NO:3所示序列的第40位至第651位氨基酸残基构成的氨基酸序列；
(4)由SEQ ID NO:4所示序列的第40位至第652位氨基酸残基构成的氨基酸序列；
(5)由SEQ ID NO:5所示序列的第40位至第665位氨基酸残基构成的氨基酸序列；
(6)由SEQ ID NO:6所示序列的第40位至第678位氨基酸残基构成的氨基酸序列；
(7)由SEQ ID NO:7所示序列的第40位至第661位氨基酸残基构成的氨基酸序列；
(8)由SEQ ID NO:8所示序列的第40位至第666位氨基酸残基构成的氨基酸序列；
(9)由SEQ ID NO:9所示序列的第40位至第671位氨基酸残基构成的氨基酸序列；
(10)由SEQ ID NO:10所示序列的第40位至第676位氨基酸残基构成的氨基酸序列；
(11)由SEQ ID NO:11所示序列的第36位至第607位氨基酸残基构成的氨基酸序列；
(12)由SEQ ID NO:12所示序列的第36位至第646位氨基酸残基构成的氨基酸序列；
(13)由SEQ ID NO:13所示序列的第36位至第648位氨基酸残基构成的氨基酸序列；
(14)由SEQ ID NO:14所示序列的第36位至第638位氨基酸残基构成的氨基酸序列；
(15)由SEQ ID NO:15所示序列的第36位至第639位氨基酸残基构成的氨基酸序列；
(16)由SEQ ID NO:16所示序列的第36位至第836位氨基酸残基构成的氨基酸序列；
(17)由SEQ ID NO:30所示序列的第36位至第673位氨基酸残基构成的氨基酸序列；
(18)由SEQ ID NO:31所示序列的第36位至第678位氨基酸残基构成的氨基酸序列；
(19)由SEQ ID NO:32所示序列的第36位至第683位氨基酸残基构成的氨基酸序列；
(20)由SEQ ID NO:33所示序列的第36位至第678位氨基酸残基构成的氨基酸序列；
(21)由SEQ ID NO:34所示序列的第36位至第688位氨基酸残基构成的氨基酸序列；
(22)由SEQ ID NO:35所示序列的第36位至第670位氨基酸残基构成的氨基酸序列；
(23)由SEQ ID NO:36所示序列的第36位至第676位氨基酸残基构成的氨基酸序列；
(24)由SEQ ID NO:37所示序列的第36位至第680位氨基酸残基构成的氨基酸序列；
(25)由SEQ ID NO:38所示序列的第36位至第673位氨基酸残基构成的氨基酸序列；
(26)由SEQ ID NO:39所示序列的第36位至第678位氨基酸残基构成的氨基酸序列；
(27)由SEQ ID NO:40所示序列的第36位至第683位氨基酸残基构成的氨基酸序列；或
(28)(1)-(27)任一项所述序列的变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)或者具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且所述变体保留了其所源自的序列的特性。
在某些实施方案中，本发明的重组蛋白在其N端或C端任选地包含一种或多种选自下列的序列：信号肽、翻译起始序列(例如Kozak共有序列)、标签序列。
在某些实施方案中，所述信号肽是tPA。
在某些实施方案中，所述翻译起始序列是Kozak共有序列(Kozak M.，NucleicAcids Research，1984，12，857-872)，此共有序列可包含至少部分的序列CCRGCCAUGG，其中R可以是A或G。位置-3(即ATG密码子上游3个核苷酸处)和位置+4对翻译影响最大(Kozak M.，NucleicAcids Research，1987，15，8125-8148)。由此，该共有序列也可以是RXXAUGG、XXAUGG或RXXAUG。
在某些实施方案中，所述标签序列是纯化标签，例如多组氨酸标签、myc标签或HA标签。
在某些实施方案中，所述重组蛋白在其N端任选地包含信号肽和/或翻译起始序列(例如Kozak共有序列)。
在某些实施方案中，所述重组蛋白在其C端任选地包含标签序列。
在第二方面，本发明提供了融合蛋白，其包含第一方面所述的重组蛋白以及与其C端连接的gp41的跨膜区和胞内区序列。在某些实施方案中，本发明的融合蛋白是经改造的gp160蛋白。
在某些实施方案中，所述gp41的跨膜区和胞内区序列与所述重组蛋白中的gp41ECTO来自相同的HIV-1毒株。
在某些示例性实施方案中，本发明的融合蛋白包含选自下列的氨基酸序列：
(1)由SEQ ID NO:16所示序列的第33位至第836位氨基酸残基构成的氨基酸序列；
(2)由SEQ ID NO:17所示序列的第34位至第837位氨基酸残基构成的氨基酸序列；
(3)(1)-(2)任一项所述序列的变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)或者具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且所述变体保留了其所源自的序列的特性。
在某些实施方案中，本发明的融合蛋白在其N端或C端任选地包含一种或多种选自下列的序列：信号肽、翻译起始序列(例如Kozak共有序列)、标签序列。
在某些实施方案中，所述信号肽是天然gp160所包含的信号肽。在某些实施方案中，所述信号肽、所述gp41的跨膜区和胞内区序列、以及重组蛋白中的gp41ECTO和gp120来自相同的HIV-1毒株。
在某些实施方案中，所述标签序列是纯化标签，例如多组氨酸标签、myc标签或HA标签。
在某些实施方案中，所述重组蛋白在其N端任选地包含信号肽和/或翻译起始序列(例如Kozak共有序列)。
在某些实施方案中，所述重组蛋白在其C端任选地包含标签序列。
在第三方面，本发明提供了包含多个单体的多聚体，其中，各单体各自独立地选自第一方面所述的重组蛋白，或者各自独立地选自第二方面所述的融合蛋白。
在某些实施方案中，所述各单体彼此相同。
在某些实施方案中，所述多聚体是三聚体或二聚体。
包膜蛋白及三聚体的制备
本发明的重组蛋白或融合蛋白或其多聚体可以通过本领域已知的各种方法来制备，例如，通过基因工程方法(重组技术)产生，也可以通过化学合成方法(例如Fmoc固相方法)产生。本发明的重组蛋白或融合蛋白或其多聚体不受其产生方式的限定。
在第四方面，本发明提供了一种分离的核酸分子，其包含编码第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体的核苷酸序列。
在第五方面，本发明提供了一种载体，其包含如上所述的分离的核酸分子。在某些实施方案中，所述载体是例如质粒，粘粒，噬菌体等。
在第六方面，本发明提供了一种宿主细胞，其包含如上所述的分离的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。优选地，所述宿主细胞是哺乳动物细胞，例如人类细胞。
在第七方面，本发明提供了制备第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体的方法，其包括，在合适的条件下培养第六方面的宿主细胞，和从细胞培养物中回收所述重组蛋白、融合蛋白或多聚体。在某些实施方案中，所述重组蛋白或融合蛋白以多聚体(例如三聚体或二聚体)形式存在。
展示平台
本发明第一或第二方面所述的蛋白或第三方面所述的多聚体可以展示在粒子如脂质体、病毒样粒子(VLP)、纳米粒子、病毒体或外来体上，以增强体内抗原呈递功效。
因此，在第八方面，本发明提供了一种粒子，在其表面上展示第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体。
在某些实施方案中，所述粒子是脂质体或纳米粒子。
在某些实施方案中，本发明的重组蛋白、融合蛋白或多聚体融合到和/或展示在脂质体上。脂质体是具有至少一个脂质双层的球形囊泡。在某些实施方案中，本发明的多聚体蛋白(例如三聚体或二聚体蛋白)可以例如通过静电相互作用(例如通过向多聚体的C端添加His-标签)和并入到脂质体中衍生化脂质的头部基团中二价螯合原子(如Ni 2+或Co 2+) 而非共价偶联到这些脂质体上。在某些实施方案中，本发明的多聚体蛋白(例如三聚体或二聚体蛋白)共价偶联到脂质体表面，例如通过整合在脂质体表面的马来酰亚胺官能团。在某些实施方案中，本发明的多聚体蛋白(例如三聚体或二聚体蛋白)可以与其偶联，例如通过多聚体蛋白中添加的C端半胱氨酸。用于制备与脂质体偶联的HIV Env三聚体和表征它们的方法是已知的，其详细描述于例如Bale S等人.J Virol.2017；91(16):e00443-17，该文献通过引用并入本文。
在某些实施方案中，本发明的重组蛋白、融合蛋白或多聚体与自组装粒子融合或展示在纳米粒子上。抗原纳米粒子是呈递多个拷贝的抗原(例如本发明的HIV Env蛋白)的多肽的组装，其导致多结合位点(亲合力)并且可以提供改进的抗原稳定性和免疫原性。制备自组装蛋白纳米粒子和其用于疫苗中的用途是本领域技术人员熟知的，参见例如Zhao L等人(2014)Vaccine 32:327-337，López-Sagaseta J等人(2016)Computational and Struct Biotechnol J 14:58-68。作为非限制性实例，自组装纳米粒子可以基于铁蛋白、细菌铁蛋白或DPS。在表面上展示蛋白的DPS纳米粒子例如描述于WO 2011/082087中。这种粒子上的三聚体HIV-1抗原的说明已经例如在.He L等人(2016)Na t Commun.2016Jun 28；7:1 2041中描述过。其他自组装蛋白纳米粒子及其制备例如在WO 2014/124301和US 2016/0122392中披露，它们通过引用结合在此。
假病毒颗粒
在第九方面，本发明提供了一种假病毒颗粒，在其表面包含第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体。
本发明的假病毒颗粒呈现典型的假病毒颗粒形态，但已完全丧失感染能力，这类假病毒颗粒相较于传统的蛋白疫苗而言，既具备HIV-1病毒的包膜，衣壳蛋白等组分，与天然病毒形态高度相似，又完全丧失了感染能力，具有较好的免疫力，可以作为一种病毒疫苗。
在某些实施方案中，所述假病毒颗粒通过慢病毒包装系统或逆转录病毒包装系统产生。
在某些实施方案中，所述假病毒颗粒通过在宿主细胞中共表达(i)包含第四方面所述的核酸分子的载体和(ii)包装载体(例如骨架质粒)获得。
在某些实施方案中，所述包装载体能够表达gag、pol、tat、rev基因。在某些实施方案中，所述包装载体为包含缺失了env基因的HIV-1基因组的载体。在某些实施方案中， 所述包装载体为质粒。在某些实施方案中，所述骨架质粒包括如pSPAX、pNL4-3.Luc.R-E-、pSG3Δenv、pfNL43-dGPE-EGFP。
在某些实施方案中，(i)中所述的载体为真核表达载体，包括但不限于VRC8400、PTT5、pCDN3.1等。
在另一个方面，本发明提供了一种制备本发明的假病毒颗粒的方法，其包括：(1)将包含第四方面所述的核酸分子的表达载体和包装载体共转染宿主细胞；(2)在宿主细胞中表达所述表达载体和包装载体所编码的蛋白，所述蛋白能够自发组装成HIV假病毒；和(3)收集HIV假病毒。
在某些优选的实施方案中，所述宿主细胞为真核细胞，例如哺乳动物细胞，例如灵长类动物细胞，例如人类细胞。
在另一个方面，本发明提供了一种用于产生上述假病毒颗粒的包装系统，其包括：(i)包含第四方面所述的核酸分子的表达载体、(ii)包装载体。
经改造的HIV病毒
在第十方面，本发明提供了一种经改造的HIV病毒，其表达第二方面所述的融合蛋白或其多聚体(例如三聚体或二聚体)作为其包膜蛋白。在某些实施方案中，所述HIV病毒是HIV-1病毒。
本发明的经改造的HIV病毒将失去感染细胞和动物的能力，作为疫苗的免疫原可以最大程度地模拟HIV-1感染人体的免疫反应过程，可能产生保护性免疫，同时又具有良好的安全性。本发明的经改造的HIV真病毒在进行细胞培养时，也可模拟HIV-1病毒的天然变异，这种变异无法回复突变成具有感染性的病毒粒子，因为病毒复制过程无法完成与编码本发明的Env蛋白的基因片段重排，因此，该类活病毒疫苗将具有天然病毒对机体的免疫力，但又不可能有感染力和致病能力，因此也是一种可行的疫苗策略。
在某些实施方案中，所述经改造的HIV病毒的基因组包含以下改造：野生型env基因被替换为编码第二方面所述的融合蛋白的核苷酸序列。
在第十一方面，本发明提供了一种分离的核酸分子或载体，其包含编码上述经改造的HIV病毒的基因组的核苷酸序列。
组合物
在第十二方面，本发明提供了一种组合物，其包含第一方面所述的重组蛋白、第二 方面所述的融合蛋白或第三方面所述的多聚体、第四方面所述的分离的核酸分子、第五方面所述的载体、第六方面所述的宿主细胞、第八方面所述的粒子、第九方面所述的假病毒颗粒、第十方面所述的经改造的HIV病毒、或第十一方面所述的分离的核酸分子或载体。
在某些实施方案中，所述组合物还包含药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述组合物是免疫原性组合物或疫苗。在此类实施方案中，第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体、第四方面所述的分离的核酸分子、第五方面所述的载体、第六方面所述的宿主细胞、第八方面所述的粒子、第九方面所述的假病毒颗粒、第十方面所述的经改造的HIV病毒、或第十一方面所述的分离的核酸分子或载体作为免疫原。
在某些实施方案中，所述组合物是蛋白疫苗，其包含第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体、或第八方面所述的粒子作为免疫原。
在某些实施方案中，所述组合物是病毒疫苗，其包含第九方面所述的假病毒颗粒、或第十方面所述的经改造的HIV病毒作为免疫原。
在某些实施方案中，所述组合物是核酸疫苗，其包含第四方面所述的分离的核酸分子、第五方面所述的载体、或第十一方面所述的分离的核酸分子或载体作为免疫原。在本文中，术语“核酸疫苗”是指，基于DNA或RNA(例如质粒，如表达质粒)的疫苗，其任选地还包含佐剂。
在某些实施方案中，所述核酸疫苗包含DNA或RNA。在某些实施方案中，所述DNA或RNA可以是裸露的或可以包裹于具有传递或/和保护功能的外壳内。在某些实施方案中，所述外壳可以是腺病毒、腺相关病毒、慢病毒、逆转录病毒等的外壳，也可以是采用化学方法合成的能行使相似功能的其他材料。
在某些实施方案中，所述药学上可接受的载体和/或赋形剂包含佐剂。用于共同施用或包含在根据本发明的组合物中佐剂优选地应该是在人体中潜在安全、良好耐受和有效的佐剂。这样的佐剂是本领域技术人员熟知的，并且非限制性实例包括QS-21、Detox-PC、MPL-SE、MoGM-CSF、TiterMax-G、CRL-1005、GERBU、TER酰胺、PSC97B、Adjumer、PG-026、GSK-I、GcMAF、B-alethine、MPC-026、Adjuvax、CpG ODN、Betafectin、铝盐如磷酸铝(例如AdjuPhos)或氢氧化铝以及MF59。
本发明的免疫原性组合物或疫苗还可以与本领域已知的其他药剂组合用于治疗或预防HIV感染。因此，在某些实施方案中，本发明的组合物还可以包含抗逆转录病毒药剂。 在某些实施方案中，所述抗逆转录病毒药剂包含：核苷逆转录酶抑制剂，例如阿巴卡韦(abacavir)、AZT、去羟肌苷(didanosine)、恩曲他滨(emtricitabine)、拉米夫定(lamivudine)、司他夫定(stavudine)、替诺福韦(tenofovir)、扎西他滨(zalcitabine)、齐多夫定(zidovudine)等；非核苷逆转录酶抑制剂，例如地拉韦啶(delavirdine)、依发韦仑(efavirenz)、奈韦拉平(nevirapine)，蛋白酶抑制剂，例如安普那韦(amprenavir)、阿扎那韦(atazanavir)、茚地那韦(indinavir)、洛匹那韦(lopinavir)、奈非那韦(nelfinavir)、osamprenavir、利托那韦(ritonavir)、沙奎那韦(saquinavir)、替拉那韦(tipranavir)等，以及融合蛋白抑制剂如恩夫韦地(enfuvirtide)等。
在某些实施方案中，本发明的免疫原性组合物或疫苗和所述抗逆转录病毒药剂作为单独组分存在或作为单一配方存在。在某些实施方案中，本发明的免疫原性组合物或疫苗和所述抗逆转录病毒药剂作为单独组分存在或作为单一配方存在。在某些实施方案中，本发明的免疫原性组合物或疫苗和所述抗逆转录病毒药剂可以同时、分开或相继施用。
本发明的组合物(例如免疫原性组合物)可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本发明的组合物(例如免疫原性组合物)可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。
本发明的免疫原性组合物应以足以诱导针对HIV-1的免疫应答的量施用。可根据待治疗或预防的特定疾病或病症、严重程度、对象的年龄以及特定对象的其他个人属性(例 如，对象健康的一般状态和对象免疫系统的稳健性)来确定免疫原的合适的量。有效剂量的确定还通过动物模型研究引导，随后进行人体临床试验，并且通过显著降低对象中目标疾病症状或病症的发生或严重性的施用方案来指导。
应用
本发明的组合物(例如免疫原性组合物)可以容易地用于在受试者中治疗HIV-1感染或引发对HIV-1的免疫应答的多种治疗或预防应用中。例如，可以将组合物施用于受试者以诱导对HIV-1的免疫应答，例如诱导产生针对HIV-1的广泛中和抗体。对于有发生HIV感染风险的受试者，可以施用本发明的免疫原性组合物以提供针对病毒感染的预防性保护。
因此，在另一方面，本发明提供了用于在受试者中诱导针对HIV的免疫应答或用于在受试者中预防和/或治疗HIV感染的方法，其包括向有此需要的受试者施用免疫学有效量的第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体、第四方面所述的分离的核酸分子、第五方面所述的载体、第六方面所述的宿主细胞、第八方面所述的粒子、第九方面所述的假病毒颗粒、第十方面所述的经改造的HIV病毒、或第十一方面所述的分离的核酸分子或载体。
在某些实施方案中，所述方法还可以包括向所述受试者施用其他药剂组合用于治疗或预防HIV感染。在某些实施方案中，所述方法包括施用抗逆转录病毒药剂，例如核苷逆转录酶抑制剂，非核苷逆转录酶抑制剂，蛋白酶抑制剂，或融合蛋白抑制剂等。
对于预防性应用，本发明的重组蛋白、融合蛋白、多聚体、分离的核酸分子、载体、宿主细胞、粒子、假病毒颗粒、经改造的HIV病毒、或组合物(例如免疫原性组合物)在任何症状之前，例如在感染之前提供。免疫原性组合物的预防性施用用于预防或改善任何后续感染，以在暴露或怀疑暴露于病毒之后或在实际开始感染之后减弱感染和/或相关疾病症状的预期严重性、持续时间或程度。因此，在一些实施方案中，待治疗的对象是患有HIV感染或有发生HIV感染风险的对象，例如由于暴露于或可能暴露于HIV。在施用治疗有效量的所公开的治疗组合物后，可以监测对象的HIV-1感染，或与HIV-1感染相关的症状。
对于治疗应用，本发明的重组蛋白、融合蛋白、多聚体、分离的核酸分子、载体、宿主细胞、粒子、假病毒颗粒、经改造的HIV病毒、或组合物(例如免疫原性组合物)在疾病或感染的症状发作时或之后，例如在HIV-1感染的症状发生之后或在诊断出HIV- 1感染之后提供。
在另一方面，本发明还涉及第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体、第四方面所述的分离的核酸分子、第五方面所述的载体、第六方面所述的宿主细胞、第八方面所述的粒子、第九方面所述的假病毒颗粒、第十方面所述的经改造的HIV病毒、或第十一方面所述的分离的核酸分子或载体，在制备用于在受试者中诱导针对HIV的免疫应答和/或用于在受试者中预防和/或治疗HIV感染的药剂中的应用。优选地，所述药剂为疫苗。优选地，所述受试者是人。
在另一方面，本发明还涉及第一方面所述的重组蛋白、第二方面所述的融合蛋白或第三方面所述的多聚体、或第八方面所述的粒子用于制备蛋白疫苗的用途，所述蛋白疫苗用于在受试者中诱导针对HIV的免疫应答和/或用于在受试者中预防和/或治疗HIV感染。
在另一方面，本发明还涉及第九方面所述的假病毒颗粒、或第十方面所述的经改造的HIV病毒用于制备病毒疫苗的用途，所述病毒疫苗用于在受试者中诱导针对HIV的免疫应答和/或用于在受试者中预防和/或治疗HIV感染。
在另一方面，本发明还涉及第四方面所述的分离的核酸分子、第五方面所述的载体、或第十一方面所述的分离的核酸分子或载体用于制备核酸疫苗的用途，所述核酸疫苗用于在受试者中诱导针对HIV的免疫应答和/或用于在受试者中预防和/或治疗HIV感染。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、核酸化学、免疫学等实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“Env”是指HIV-1病毒表面的包膜蛋白(envelope)，Env在HIV-1表面以全长gp160(全长分子量160KD)的形式合成，翻译成的蛋白前体在病毒成熟后被蛋白酶裂解为gp120(分子量为120KD)和gp41，gp120位于病毒表面，是识别CD4受体诱发感染的分子，gp41含有胞外区跨膜区和胞内区，分子量约为41KD，gp120和gp41通过亚基之间的非共价相互作用形成异源二聚体，异源二聚体通过非共价相互作用形成典型的三聚体结构，该三聚体蛋白即为Env。
如本文中所使用的，术语“gp140”是指删除了gp41胞内段和跨膜区的由gp120和 gp41胞外段组成的蛋白，由该单体蛋白形成的三聚体可以称为gp140三聚体。gp140三聚体包括通过在单体中引入二硫键、点突变等方式使构象进一步稳定的三聚体蛋白。本文中所述的“BG505 SOSIP”是指，在BG505毒株全长Env的基础上，去除了胞内段和跨膜区序列，并在501位氨基酸和605位氨基酸引入cys突变以在gp120和gp41胞外段亚基之间形成二硫键，再进一步通过将559位氨基酸的Ile突变位pro稳定gp140三聚体融合前构象，这一改造方法被命名为SOSIP，这一改造蛋白即为BG505 SOSIP。删除gp160的胞内区和跨膜区将有助于膜蛋白的可溶性表达。
如本文中所使用的，术语“弗林蛋白酶”(furin)是指一种外泌途径中的主要的蛋白质转化酶，催化切割蛋白中的Arg-X-Y-Arg羧基端肽键(X为任一氨基酸，Y为Arg或者Lys)，产生成熟蛋白。弗林蛋白酶识别未成熟HIV-1 Env gp120和gp41亚基之间的REKR序列将全长gp160切割为gp120和gp41，使其发生构象重排并成熟。
如本文中所使用的，HXB2编号系统是HIV蛋白和核酸序列的参考编号系统，使用HIV-1 HXB2毒株序列作为所有其他HIV毒株序列的参考。本领域普通技术人员熟悉HXB2编号系统。本文所描述的氨基酸编号如559、501、605等均是根据HXB2编号系统给出，可以通过以毒株HXB2的氨基酸编号为模板，并通过序列同源比对后确定，本领域技术人员理解其并不是指蛋白的实际氨基酸编号。在本文中，表述“对应于分离株HXB2的gp160序列的特定氨基酸位置的区域”是指，当将待比较毒株的gp160序列与分离株HXB2的gp160序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，待比较毒株中与分离株HXB2中的特定氨基酸位置等同的位置。
如本文中所使用的，HIV的“亚型”、“毒株”等为本领域技术人员所熟知。现在已知HIV-1至少有13个亚型，包括A、B、C、D、E、F、G、H、I、J、K、O、N亚型，被归为M、O、N三组，A-K属于M组。不同亚型中存在很多种毒株，一种毒株对应一种Env序列。如发明中所使用的BG505即为A亚型中的一种毒株，NL4-3、Bal.26为B亚型中的毒株。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物 病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
在本发明中，术语“包装载体”是指这样的载体，其能够表达除包膜蛋白以外的、形成HIV病毒样颗粒所必需的其他蛋白，例如HIV的gag、pol、tat和vpu蛋白等。通常可以通过将HIV完整基因组中的包膜蛋白编码基因和调控基因进行修饰或删除从而构建包装载体。此外，用于组装HIV病毒样颗粒/假病毒的包装载体以及构建此类包装载体的方法是本领域已知的。
在本发明中，术语“骨架质粒”是指用于假病毒生产中编码产生gag、pol等结构蛋白的质粒。当使用慢病毒系统生产假病毒时，需包装质粒、包膜质粒、转移质粒、调节质粒共同转染包装细胞，才能产生便于检测的假病毒颗粒，转移质粒一般含有报告基因可以指示假病毒感染入胞过程，报告基因可以整合入包装质粒，调节基因调节假病毒结构基因的转录翻译等过程，也可与包装质粒整合。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“假病毒”是指，由病毒衣壳蛋白或包膜蛋白所形成的病毒样颗粒，通常其不包裹核酸或包裹经基因删除或修饰后的病毒核酸。通常而言，由于假病毒内不包含核酸或所包含的病毒核酸基因组不完整，因此，假病毒只具有单轮感染的能力，而不具有产生子代病毒的复制能力，具有较高的生物安全性。
如本文中所使用的，术语“Tier2毒株”为本领域技术人员所熟知，HIV-1毒株能被一些特异性抗体中和，根据被中和的难易程度，HIV-1毒株被分为Tier1、Tier2和Tier3三个等级，Tier1为最易被中和的毒株，Tier2为最不易被中和的毒株。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的 函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指，在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，离子强度增强剂，维持渗透压的试剂，延迟吸收的试剂，稀释剂，佐剂，防腐剂，稳定剂等。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性(例如对PSD-95泛素化的抑制活性)，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉， 蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
发明的有益效果
Env作为HIV-1病毒表面识别受体分子的主要抗原蛋白，也是HIV-1疫苗研发的关键分子。天然病毒表面的Env为非共价连接的异源三聚体，当病毒结合宿主细胞上的CD4分子之后，Env的构象逐渐从闭合的融合前构象转变为开放构象。研究表明Env在融合前构象下有更多bNab表位暴露。基于天然Env三聚体的不稳定性，进行HIV-1Env免疫原设计以稳定其构象尤为重要。
本发明的Env三聚体蛋白可以在哺乳动物细胞中进行高效的表达，三聚体含量提高，组分更为均一，热稳定性明显提升。本发明的Env三聚体蛋白可有效暴露诸如CD4bs/V3等关键表位，而不利于非中和表位的暴露，具备作为蛋白疫苗的潜力。此外，基于该Env三聚体蛋白获得的HIV假病毒及真病毒，与天然病毒形态高度相似因此具有天然病毒对机体的免疫力，但又丧失了感染能力和致病能力，因此展现出作为病毒疫苗的潜力，具有重要临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1A-1B显示了改造之前gp140三聚体(BG505 SOSIP，PDB No.4TVP)和推测的改造后三聚体(BG505 TSTIP)的单体蛋白空间结构(1A)和二级结构分布拓扑图(1B)。
图2A-2B显示了改造gp140三聚体在经过一步Ni Sepharose excel纯化后的还原SDS聚丙烯酰胺凝胶电泳结果。图2A为BG505毒株，图2B为NL4-3毒株。1为TSTIP蛋白，2为TST蛋白，3为SOSIP蛋白。TSTIP及TST在还原条件下均呈现单条带，分子量约为140KD，SOSIP在还原条件下呈现两条带，分别为gp120和gp41胞外段。说明本发明蛋白如预期在亚基间采用的是稳定的供价连接。
图3显示的是改造gp140三聚体的分子筛superdex200-16/600的纯化结果，A为BG505毒株，B为NL4-3毒株。TSTIP蛋白和TST蛋白的分子筛层析结果显示为单个洗脱峰，而SOSIP蛋白的层析结果则呈现三个洗脱峰，则说明本发明蛋白TSTIP和TST的组分相较于与SOSIP更加均一。
图4A-4C显示的是改造gp140三聚体经分子筛纯化后的非还原聚丙烯酰胺凝胶电泳结果。A为BG505 TSTIP，B为NL4-3 TSTIP，C为BG505 SOISP。结果显示经过分子筛纯化后，本发明蛋白TSTIP主要成分为三聚体，且三聚体含量高于SOSIP。
图5显示的是改造gp140三聚体的差式扫描量热法(differential scanning calorimetry，DSC)Tm测定结果。结果显示本发明蛋白BG505 TSTIP的Tm为74.5℃，NL4-3 TSTIP的Tm为63.41℃，高于对照蛋白BG505 SOSIP的Tm：68.2℃和对照蛋白NL4-3 SOSIP的Tm：62℃。表明改造蛋白的热稳定性高于对应的对照蛋白SOSIP。
图6显示的是改造gp140三聚体与多种已报道人单克隆抗体的酶连免疫吸附实验(ELISA)结果。所使用的抗体包括广谱中和抗体VRC01、B12、PGT121、PGT125、2G12，非广谱中和抗体F105、F240、17b，以及HIV-1的受体分子CD4。显示本发明蛋白BGTSTIP、NL4-3 TSTIP与各类已报道广谱中和抗体，非广谱中和抗体的结合能力于对照蛋白BG505 SOSIP、NL4-3 SOSIP基本相当,能很好的识别和结合CD4受体分子。且与SOSIP一样，CD4分子与改造蛋白的识别慧诱发发明蛋白构象改变，进而暴露17b结合位点。
图7显示的是改造gp140三聚体免疫BALB/C小鼠后免疫血清对B亚型NL4-3，B亚型2626毒株病毒的病毒中和实验；其中，M1～M6分别指不同小鼠。结果显示改造蛋白TSTIP和对照蛋白免疫小鼠后能诱导的较强的自体中和应答，但不能诱导同亚型Tier2中和应答。改造蛋白TSTIP和对照蛋白SOSIP显示出相当的免疫原性。
图8A显示的是基于改造gp140三聚体的假病毒的western blot结果。图8B显示的是基于改造gp140三聚体的假病毒的p24的定量结果。结果显示转染细胞和收取的病毒上清均有全长TSTIP蛋白的表达，本发明的假病毒上清中的p24的含量相较野生型假病毒有所下降，但仍能检测到一定量p24和Env，说明本发明假病毒能够正常包装成完整的假病毒颗粒，但产量可能会有一定程度降低。
图9显示的是基于改造gp140三聚体的假病毒颗粒的负染电镜结果。显示在负染电镜下可以看到明显的类似于野生型假病毒的假病毒颗粒。
图10显示的是以LacZ为报告基因的Elispot法检测的基于改造gp140三聚体的假 病毒的感染能力检测结果。显示野生型假病毒保持正常的感染能力，而本发明假病毒已经丧失了感染能力，这符合发明人得预期。
图11A显示的是改造gp140三聚体BG505/NL4-3 TSTIP G1/G2/Full的还原SDS的聚丙烯酰胺凝胶电泳结果。显示BG505 TSTIP-G2和NL4-3 TSTIP-G1的分子量比预期大，两种发明蛋白在还原条件下可能仍为二聚体，BGTSTIP-Full、NL4-3-G1和NL4-3-Full未纯化到较纯的目的蛋白。相较之下TSTIP的发明蛋白具有更强的使用范围。
图11B显示的是改造gp140三聚体BG505/NL4-3 TSTIP G1/G2/Full的AUC结果，与SDS-PAGE结果保持一致。
图12显示的是改造gp140三聚体BG505/NL4-3 TSTIP G1/G2/Full与多种已报道抗体的酶联免疫吸附实验结果。显示六种蛋白均与能与多种抗体反应，具有一定的活性，但部分蛋白的反应性弱于发明蛋白TSTIP.
图13显示的是多种改造gp140三聚体免疫BALB/C小鼠后免疫血清对B亚型NL4-3毒株的病毒中和实验。结果显示六种蛋白均可以诱导小鼠产生中和抗体应答。
图14A显示的是多种毒株的改造gp140三聚体的的SDS丙烯酰胺凝胶电泳结果，结果显示各毒株改造蛋白均得到较好的表达，并能纯化获得较高的纯度，在还原SDS-PAGE下，发明蛋白呈现单条带，证明其亚基间为稳定的共价连接。
图14B为五种毒株TSTIP蛋白分子筛superdex200 16/600纯化结果，显示五种发明蛋白分子筛的峰较为单一，说明蛋白均一性较好，本发明思路运用于不同毒株时蛋白也以三聚体为主体。
图14C为五种毒株与多种抗体的酶联免疫吸附实验结果，显示五种发明蛋白，与多种已报道抗体均有一定反应性，证明其活性良好。
图14D为五种毒株的TSTIP蛋白免疫后血清与假病毒的中和实验结果，其中CH119 TSTIP和X1632 TSTIP免疫血清检测的毒株为对应的CH119和X1632毒株；25710和246F3 TSTIP血清检测的毒株为BJOX200；CNE8 TSTIP免疫血清检测的毒株为TRO11。结果显示，五种毒株的TSTIP蛋白第四针免疫血清均检测到较弱的病毒中和，说明不同毒株的TSTIP蛋白免疫能够刺激小鼠产生中和应答。
图15A-15K显示了实施例11中各TSTIP蛋白构建体的电泳(I)、分子筛纯化(II)、分析型超速离心(III)、ELISA(IV)的实验结果图。图15A：BG-B1(1/1)；图15B：BG-B1(1/2)；图15C：BG-B1(2/2)；图15D：BG-B1(2/1)；图15E：BG-B1(2/3)；图15F： BG-B2(1-1)；图15G：BG-B2(1-2)；图15H：BG-B2(1-3)；图15I：BG-C1(1/1)；图15J：BG-C1(1/2)；图15K：BG-C1(1/3)。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2021130414-appb-000001
Figure PCTCN2021130414-appb-000002
Figure PCTCN2021130414-appb-000003
Figure PCTCN2021130414-appb-000004
Figure PCTCN2021130414-appb-000005
Figure PCTCN2021130414-appb-000006
Figure PCTCN2021130414-appb-000007
Figure PCTCN2021130414-appb-000008
Figure PCTCN2021130414-appb-000009
Figure PCTCN2021130414-appb-000010
Figure PCTCN2021130414-appb-000011
Figure PCTCN2021130414-appb-000012
Figure PCTCN2021130414-appb-000013
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：NL4-3/BG505两毒株TSTIP蛋白的表达
4-3/BG505 TSTIP改造设计
以NCBI上BG505/pNL4-3-gp160两毒株的碱基及氨基酸序列为改造模板。以BG505的改造为例，依据BG505-SOSIP的冷冻电镜三维结构(PDB:4tvp)，去除BG505 gp41的β27的C末端的部分loop序列(aa610-616，WNSSWSN)，以及gp120的C端包括furin酶切位点在内的10个氨基酸的序列(aa501-511，AKRRVVGREKR)。随后将gp41的β27的截短C末端与gp120的N端相连，将gp120的截短C端与gp41的α8结构域的N端相连。改造后的完整gp140的氨基酸排列顺序如下：α6/α7/β27(501-606)+β27和α8之间的部分loop区(607-609)+gp120(33-500)+β27和α8之间的loop区部分C端序列(617-618)+α8/α9(619-664)。
另外，在上述序列的C末端加上8His标签便于纯化，在序列前加入tPA信号肽及kozak序列促进分泌表达，经如上设计的蛋白被命名为BG505-TST(SEQ ID NO:1)。在上述结构基础上，引入I559P、T332N突变，经如上设计的蛋白被命名为BG505-TSTIP(SEQ ID NO:3)。以同样的方法改造NL4-3，分别获得NL4-3-TST(SEQ ID NO:2)和NL4-3-TSTIP(SEQ ID NO:4)。上述各蛋白的结构示意图如图1所示。
将设计好的BG505/pNL4-3-TSTIP氨基酸序列转换为适合哺乳动物细胞表达的碱基后送上海生工进行基因合成，并构建到pcDNA3.1载体的ECORI和Xbal酶切位点之间。取1ul合成的pcDNA3.1-BG505SOSIP/4-3TSTIP质粒转化50ulDH5α感受态细 胞(购自深圳康体生命科技有限公司)，涂布于氨苄青霉素抗性的固体培养基，37℃静置培养10-12小时至单菌落清晰可见，挑取单菌落至含有3ml氨苄青霉素抗性LB培养基的试管中，37℃220转/分振荡培养10小时，取500ul菌液与500ul50％甘油混合后冻存于-20℃。
PcDNA3.1NL4-3/BG505 TSTIP质粒提取：
于-20℃取出冻存的pcDA3.1 NL4-3/BG505 TSTIP菌液，取10ul至含有3ml氨苄抗性LB培养基的试管培养10-12h后接种于含有500ml氨苄抗性LB培养基的锥形瓶中，37℃培养12h后，收取菌液7000g离心10min，弃上清。采用天根无内毒素大提试剂盒，提取pcDNA3.1-BG505/NL4-3TSTIP质粒
293F细胞培养与传代
-80℃冰箱取冻存的293F细胞，37℃化冻后1300rpm离心4min，于超净台内弃上清，轻弹细胞并用提前37℃温育的293freestyle培养基重悬后，转移至含50ml温育培养基的三角瓶中，37℃5％CO2120转/min悬浮培养，待细胞密度达2.0*10 6时进行传代，逐渐扩大培养体系。
瞬时转染
采用PEG2000瞬时转染293F细胞，待细胞至2.0*106密度后，无菌50ml管收取细胞，1300rpm离心4min，轻弹细胞后37℃温育培养基重悬，并转移至含有450ml37℃温育培养基的三角瓶中，置于37℃摇床备用。
将提取的pcDNA3.1-BG505/NL4-3TSTIP质粒与PEG2000按1：2比例置于50ml培养基中充分混匀后静置18min，转移至上述450ml培养基中，37℃5％CO2120转/min悬浮培养6天，进行BG505/NL4-3TSTIP蛋白的表达。转染过程中注意PEG的避光操作。
实施例2：NL4-3/BG505 TSTIP蛋白纯化
瞬时转染6天后，收取细胞培养液，JA-14转头7000g离心10min后取细胞上清，20000g离心10min后取上清0.22um孔径滤膜过滤两次，以该样品进行下一步Ni-excel柱纯化。
利用AKTA系统进行Ni亲和层析纯化；
仪器系统：AKTA Pure型制备型液相色谱仪；
纯化介质：Ni Sepharose excel亲和介质；缓冲液：分为A、B缓冲液，A液为 1×PBS缓冲液，B泵为1×PBS+250mmol/L咪唑缓冲液；
系统上样流速：8mL/min；检测波长:UV@280nm
系统洗脱流速：4ml/min；检测波长:UV@280nm
洗脱条件：用20mM咪唑洗脱杂蛋白，收集250mM咪唑洗脱产物。将洗脱液透析于1×PBS过夜，期间更换两次透析液。收获约30ml低浓度目的蛋白，Vivaspin20ml，100KD超滤浓缩管浓缩至5ml备用。
实施例3：NL4-3/BG505 TSTIP蛋白生化性质鉴定
SDS-PAGE:
将实施例2中的浓缩样品以及BG505/4-3SOSIP蛋白稀释至1ug/ul后取两管50ul样品，分别加入10ul还原6Loading Buffer和10ul非还原6Loading Buffer，制备还原样品和非还原样品，还原样品于100℃沸水水浴10min。取10ul还原和非还原样品于8％SDS-PAGE中以80V电压电泳120min，经考马斯亮蓝染色后显示电泳条带，电泳结果见图2A-2B。经SDS-PAGE分析显示经一步Ni-EXCEL纯化，可得高纯度BG505/NL4-3TSTIP蛋白，且TSTIP蛋白在DTT存在的条件下为完整140KD条带，而BG505/NL4-3SOSIP蛋白在还原条件下呈现120KD条带，表明本发明人设计的蛋白如预期采用了稳定的共价连接方式将gp120和gp41胞外段相连。
分子筛纯化：
仪器系统：AKTA Pure型制备型液相色谱仪；
层析柱：superdex 200 16/600
柱体积：120ml
缓冲液：PBS(20mM磷酸缓冲液，pH7.5，150mMNacl)
检测器波长：280nm
流速：1ml/min
样品为实施例2纯化浓缩蛋白
纯化程序为：1倍柱体积的PBS平衡superdex200 16/600，5ml上样环上样实施例2中纯化获得的目的蛋白，inject模式下进行纯化样品的分子筛纯化，样品会按不同组分的分子量大小，由高到低，依次被洗脱，收集不同洗脱体积下的洗脱峰，即为不同聚体形式的目的蛋白。
分子筛纯化结果如图3，可见发明人设计的BG505/NL4-3TSTIP的组分相较于 BG505/NL4-3更加单一，而BG505/NL4-3TSTIP的分子筛纯化图谱上可见明显的单体和多聚体峰，表明本发明的TSTIP蛋白TST蛋白三聚体含量更多。
分子筛纯化样品的SDS-PAGE:
将分子筛纯化过程中收取的不同洗脱体积下的样品于Vivaspin20ml，100KD浓缩管浓缩至1ug/ul，按洗脱先后顺序标记1、2、3、4.取各部分样品50ul，加入10ul非还原6Loading Buffer，混匀，取10ul非还原样品于8％SDS-PAGE中以80V电压电泳120min，经考马斯亮蓝染色后显示电泳条带，电泳结果如图4A-4C，可见经superdex200-16/600纯化后的BG505/NL4-3TSTIP其主要组分为gp140三聚体，而BG505/NL4-3SOSIP则含有大量gp140单体.
差式扫描量热法(DSC)热稳定性分析
仪器系统：GE Healthcare公司生产的VP-Capillary
样品为实施例2纯化浓缩蛋白
用酸性或弱碱性洗液对上样槽清洗1次，再用去离子水清洗3次；将样品及其对应缓冲液各吸取400μL于EP管中，离心去除沉淀及气泡；分别吸取300μL缓冲液于样品槽及对照槽中，润洗3次，再加入300μL缓冲液至样品槽及对照槽，加样过程要避免产生气泡；启动DSC软件和仪器，设置扫描循环次数，当扫描循环中DP值稳定在±0.2之间时即可让其循环扫描，基线平衡不少于3次；当最后一次循环扫描结束，温度降至30℃-10℃之间时将样品槽中的缓冲液吸出，迅速加入300ul待测蛋白样品，并继续扫描测试；扫描速率设置为：90℃/h.扫描完成后用Origin7.0对数据进行处理。结果如图5。发明蛋白BG505-TSTIP的Tm为74.51℃，对照蛋白BG505-SOSIP的Tm明显低于BG505-TSTIP的Tm,为68.18℃；NL4-3-TSTIP的Tm为63.41℃，NL4-3-SOSIP的Tm为62.00℃，在NL4-3毒株上其Tm虽未见明显提升，但相较于报道的优化设计NL4-3SOSIP有一定程度提高。这些结果说明，我们的设计能一定程度提高蛋白质的热稳定性，在部分毒株中其热稳定性可能得到极大的提升。
实施例4：BG505/NL4-3TSTIP抗原性及免疫原性鉴定
酶联免疫吸附测定(ELISA)
选择PGT121、PGT125、VRC01、2G12、B12等广谱中和抗体以及17b、447-52D、F105、F240等非中和抗体对BG505/NL4-3TSTIP以及BG505/NL4-3SOSIP进行了ELISA抗原性分析，具体过程如下：
(1)将BG505/NL4-3TSTIP与BG505/NL4-3SOSIP三聚体蛋白用1×CB稀释至1μg/Ml，按100μL/孔包被于96孔板，37℃恒温箱静置2h；
(2)洗板1次，甩干。用ED(180μL/孔)37℃恒温箱封闭2h；
(3)洗板1次，甩干。取96孔u底板，将抗体按1ug/ml或10ug/ml稀释后加于U底板首孔，每种抗体进行双孔重复，首孔150ul，3倍倍北稀释11个梯度。取100ul稀释好的抗体转移至包被的BG505/NL4-3TSTIP与BG505/NL4-3SOSIP96孔ELISA板中，37℃反应1h。
(4)洗板5次，甩干。将二抗GAH-HRP(1:5000)加入96孔板，100μL/孔，37℃反应45min；
(5)洗板5次，室温显色10min，终止，酶标仪450nm波长处检测；使用GraphPad Prism 5(GraphPad,USA)软件进行数据分析。结果如图6。
可见BG505/NL4-3TSTIP与PGT121、PGT125、VRC01、2G12、B12等广谱中和抗体具有强反应性，而与17b、F105、F240等非中和抗体的反应性较弱。17b、F105、F240分别是靶向CDi、CD4bs、gp41等非中和表位的抗体，这表明BG505/NL4-3TSTIP没有暴露这类非中和表位，而很好地呈现了诸如CD4bs、out glycan、V3等关键表位。总体而言，BG505/NL4-3TSTIP与各类广谱中和抗体以及非中和抗体的反应性与对照蛋白BG505/NL4-3SOSIP保持相当。
BG505/NL4-3TSTIP动物的免疫评价
小白鼠：雌性，6周龄，购自上海斯莱克实验动物有限责任公司。设置四组免疫小鼠，每组6只小鼠，实施例2所制备的BG505/NL4-3TSTIP以及BG505/NL4-3SOSIP作为免疫蛋白。将蛋白用生理盐水稀释，按体积比1：1的比例与氢氧化铝佐剂混合，使蛋白吸附于佐剂上，对小鼠进行腹腔免疫。小鼠免疫方案设置如表1。
表1：小鼠免疫方案
Figure PCTCN2021130414-appb-000014
在免疫前采集小鼠眼球血，按表1的免疫方案进行免疫，每次免疫前采集小鼠眼球血，免疫至第六针后采集眼球血后对小鼠进行断颈处理。血样于37℃放置30min后， 13300rpm离心10min，收集血清用于HIV-1假病毒中和和抗体滴度测定。
我们采集第六针血清进行了病毒中和实验，结果如图7所示，四组小鼠均检测到较强的NL4-3自体中和应答，而却未检测到同亚型tier2毒株TRO11的中和应答。结果表明本发明的BG505/NL4-3TSTIP可诱导产生较强的自体中和抗体应答，而难以产生针对其他毒株的中和应答。总体而言，本发明蛋白在免疫原性上保持与对照蛋白SOSIP相当的水平。本领域人员知晓，以本发明的蛋白为基础通过引入交叉免疫表位的氨基酸突变是可能诱导病毒交叉中和抗体。
实施例5：Bal.26-TSTIP假病毒颗粒的生产及鉴定
Bal.26-TSTIP和NL4-3-TSTIP假病毒的构建
本发明人生产的Bal26 TSTIP假病毒由HIV-1骨架质粒pfNL43-dGPE-EGFP(Addgene)及包膜质粒VRC8400-Bal26-TSTIP-gp160共转染293FT(Invitrogen)细胞获得。
包膜基因和骨架基因的获得
骨架质粒为pfNL43-dGPE-EGFP，购自addgene。该质粒由NL4-3感染性克隆改造而成，包含病毒包装所必须的gag、pol、tat、rev等基因，但其Env基因已部分被EGFP基因替换，导致Env基因的沉默，因此只需共转一个表达Env的表达质粒即进行假病毒生产。本发明中使用的包膜基因的表达载体为VRC8400,VRC8400载体为本实验室保藏。发明人依据BG505 TSTIP蛋白的设计思路对Bal.26的Env基因全长进行了TSTIP的设计，将携带病毒自身信号肽的全长Bal.26 TSTIP基因(其编码SEQ ID NO:16)克隆至VRC8400载体的EcoRV和BglII酶切位点之间，构建VRC8400-Bal.26-TSTIP-gp160表达质粒。获得的假病毒称为BaL.26-SD-FS。此外，基于Bal.26毒株的野生型gp160序列获得假病毒作为对照(BaL.26-WT)。
假病毒生产：
假病毒的生产采用PEI瞬时转染的方法，pfNL43-dGPE-EGFP质粒与包膜质粒VRC8400-Bal.26 TSTIP gp160质粒的混合物以及PEI分别稀释于90ul的生理盐水中，并将两者充分混匀静置18min形成质粒PEI复合物，以PEI:质粒＝2：1，pfNL43-dGPE-EGFP：VRC8400 Bal.26 TSTIP gp160＝1：1的比例共转染293FT细胞，质粒用量为20ug/板，待转染6h后更换新鲜的DMEM完全培养基，37℃，5％CO 2恒温培养48h后收获上清即为病毒液。
假病毒包装能力验证
收获上述转染的293FT细胞及细胞转染上清，用细胞裂解液裂解1h后，离心收取细胞裂解上清，与上述细胞裂解液一起进行western blot，具体步骤如下：
(1)取20ul细胞裂解液及病毒液上清于8％SDS聚丙烯酰胺凝胶中以80V电压进行电泳。
(2)2h后将凝胶转移至硝酸纤维素膜上，其上其下各放置2层滤纸，以1xTrans-
Figure PCTCN2021130414-appb-000015
Turbo TM Transfer Buffer(SDS)为转膜液进行半干转，转膜时间30min。
(3)去离子水洗膜，以封闭液1(购自厦门万泰)封闭膜2h。
(4)以实验室筛选到的单克隆抗体3A7为一抗，以1ug/ml浓度将一抗稀释于ED11(购自厦门万泰)，摇床室温孵育1h。
(5)以1×PBST洗液洗膜3次，5min/次，去离子水清洗1次，将二抗GAM-HRP按1:5000的比例稀释于ED11(购自北京万泰)，室温摇床孵育45min。
(6)以1×PBST洗液洗膜3次，5min/次，去离子水清洗1次后显色曝光。Western blot的结果如图8A，在细胞裂解液以及病毒液中均检测到Env蛋白。
同时，将病毒液进行5倍倍比稀释6个梯度，通过ELISA方法检测上清病毒液中p24含量，以梯度稀释的p24标准品的反应制作拟合曲线，拟合曲线为Y＝3618*X+151.3(图8B)，R 2为0.996，具有良好的线性关系，p24的定量结果如表2，同时对收获的假病毒液制样进行了负染电镜的观察，如图9。综合以上结果，本发明生产的假病毒其p24的含量有一定程度下降，但在可接受范围内，上清中能检测到Env蛋白的表达，且负染电竞下可以观察到明显的假病毒颗粒，表明本发明的假病毒能够正常地包装形成假病毒颗粒。
表2：p24含量测定
Figure PCTCN2021130414-appb-000016
假病毒感染能力验证
将收获的病毒液在U型底96孔板中进行稀释，首孔100ul病毒液，依次进行5次5倍倍比稀释，每孔中均含15ug/mlDEAE以促进假病毒对细胞的感染能力。并提前在96孔细胞培养板中培养TZM-b1细胞，37℃，5％CO2培养至细胞密度达80％时，将稀释好的假病毒液转移至TZM-b1细胞中，待病毒感染细胞40-48h之后，采用化学发 光法和ELISPOT两种方法检测生产的假病毒对靶细胞的感染能力。结果如图10所示，两种检测方法皆显示，经过TSTIP改造的假病毒几乎完全丧失了感染能力，而天然的假病毒则能正常地感染TZM-b1细胞。
NL4-3TSTIP及NL4-3WT假病毒颗粒的生产、包装和感染能力的验证依据上述Bal26假病毒的生产、包装和感染能力的验证方法进行。
实施例6：假病毒颗粒免疫原性鉴定
假病毒颗粒生产和纯化
根据实施例5的生产假病毒的方法，通过大量转染293FT细胞以生产假病毒颗粒(如NL4-3-TSTIP及未突变的NL4-3-WT假病毒颗粒)，收获病毒上清进行密度梯度离心，纯化获得假病毒颗粒，具体过程如下：
(1)提前制备20％蔗糖溶液，并用0.22um针头滤器过滤至无菌管中；
(2)将内外套管以及管盖用75％酒精清洗擦拭，扣干并于安全柜中用紫外灯照射30min；预浓缩的病毒混匀后3000rpm离心7min；
(3)采用0.45um针头滤器和10ml注射器将病毒上清过滤到超离内管中(30ml/管病毒液)，后使用10ml注射器吸取5ml/管20％蔗糖溶液并连接5cm针头插入装有病毒液的套管底部缓慢加入蔗糖溶液；
(4)将套管以1-4，2-5，3-6相对应排好，并做好样品标记，加样完成后，采用电子分析天平平衡，使对应的两管误差小于0.0005g之内；
(5)将套管盖子确认无盖错并拧紧，在sw28转头上对应的位置挂上套管；按下腔体抽真空，并设置25000rpm，2.5h，4℃，升速最大，降速可设带刹车；
(6)离心完，按下真空键放入空气，轻轻取出离心管，至安全柜中倒弃上清液，加入无菌PBS溶解过夜，收集液体即为浓缩的病毒样品。
将获得的HIV-1病毒样颗粒重悬于PBS，通过如下方法对病毒的p24和Env进行定量。
P24定量
1.抗体包被：以PB7.4稀释16G12抗体，以100ng/孔(100μL)16G12包被于96孔板，4℃包被过夜。
2.封闭：PBST洗一次96孔板，甩干后，加入180μL ED溶液，37℃封闭2h或4°过夜。
3.病毒孵育：以PBS为缓冲液在U底板中梯度稀释病毒(p24标准品按照350、700、1400和2800pg/mL加入板中)，每孔100μL体积，加入裂解液，每孔20ul体积。转移至96孔板，37℃孵育1h。
4.二抗反应：PBST洗液洗板5次，甩干96孔板。每孔加入100μL 2F2-HRP二抗溶液(1:10000稀释于ED11溶液)。37℃温箱中孵育45min。
5.显色：PBST洗液洗板5次，甩干96孔板每孔加入100μL等体积混合的A/B显色液。37℃孵育10min。
6.终止：每孔加入50μL硫酸终止液，96孔板放入酶标仪中读取OD650-450nm。
7.以p24标准品为参考，制作标准曲线计算病毒中p24含量。
Env定量
1.GNL包被：以500ng/孔(100ul)包被GNL于96孔板，缓冲液为PBS。4℃包被过夜。
2.封闭：PBST洗板一次，甩干后，加入180μL ED溶液，37℃封闭2h或4°过夜。
3.病毒包被：以ED为稀释液稀释病毒液(以首孔为1ug/ml的gp140作为标准品)，转移至96孔板，封板后，37℃孵育1h。
4.一抗反应：PBST洗液清洗5次，甩干。每孔加入100ul VRC01(1ug/ml稀释于ED11溶液)。封板后，37℃孵育1h。
4.二抗反应：PBST洗液清洗5次，甩干。每孔加入100μL GAH-HRP二抗溶液(1:5000稀释于ED11溶液)。封板后，37℃孵育45min。
5.显色：PBST洗液清洗5次，甩干96孔板每孔加入100μL等体积混合的A/B显色液。放入37℃温箱中孵育反应10min。
6.终止：每孔加入50μL硫酸终止液，96孔板放入酶标仪中读取OD650-450nm。
7.以gp140标准品为参考，制作标准曲线计算病毒中Env含量。
假病毒免疫原性鉴定
获得NL4-3假病毒颗粒的p24及Env定量信息后，设计实验方案进行NL4-3假病毒颗粒的小鼠免疫实验，免疫方案设置如表3。上海斯莱克实验动物有限责任公司购买6周龄的磁性小白鼠，设置A/B/C/D/E共五组实验，每组5只小鼠。联合上述铝佐剂共进行五针腹腔免疫，免疫周期为2周/针。免疫前采取小鼠0/2/4/6/8周及第10周眼球血，后对小鼠进行断颈处理。血样于37℃放置30min后，13300rpm离心10min，收集 血清进行Env及P24特异性的结合滴度的测定及HIV-1假病毒中和和抗体滴度测定。实验结果显示本发明的假病毒可诱导产生中和抗体应答，展现出良好的免疫原性。
表3：NL4-3假病毒颗粒免疫方案
Figure PCTCN2021130414-appb-000017
实施例7：NL4-3真病毒颗粒的生产和鉴定
参考TSTIP蛋白的设计对NL4-3毒株的Env全长序列进行设计，获得的全长TSTIP gp160基因(其编码SEQ ID NO:17所示的氨基酸序列)经密码子优化为适合哺乳动物细胞表达的碱基后送上海生工进行基因合成，并由其克隆替换到两种毒株基因组野生型Env基因的位置。分别取1ul合成的质粒转化Stbl3感受态(购自上海唯地生物技术有限公司)，涂布于氨苄青霉素抗性的固体培养基，30℃静置培养12-14小时至单菌落清晰可见，挑取单菌落至含有3ml氨苄青霉素抗性LB培养基的试管中，30℃220转/分振荡培养12小时，取500ul菌液与500ul50％甘油混合后冻存于-20℃。并接种菌液至500mlLB培养基中进行培养，以进行质粒提取，质粒提取过程参考实施例1中pcDNA3.1 NL4-3/BG505 TSTIP质粒提取过程。提取得到的质粒瞬时转染293FT贴壁细胞，转染试剂为PEI，转染方法参考实施例5 Bal.26-TSTIP假病毒颗粒生产及鉴定中假病毒生产过程。转染48小时后收取转染上清，进行NL4-3 TSTIP真病毒颗粒的纯化，并对收取的真病毒颗粒进行包装能力验证以及感染能力验证，具体方法参考实 施例5中所述的方法。实验结果显示通过如上方法获得的真病毒能够正常包装形成真病毒颗粒，并且经过TSTIP改造的真病毒几乎完全丧失了感染能力。
实施例8：真病毒颗粒免疫原性鉴定
根据实施例7的方法获得足够量的真病毒颗粒，并通过实施例6中的p24及Env定量方法获得纯化的真病毒颗粒的p24及Env含量信息。设计实验方案进行NL4-3 TSTIP真病毒颗粒免疫实验，免疫方案如表4所示。从上海斯莱克实验动物有限责任公司购买6周龄磁性小白鼠，分别设置A/B两组实验，每组五只小鼠。以Env含量为参考，A组免疫含2ug-Env的真病毒颗粒，B组免疫2ug纯化的gp140蛋白。联合铝佐剂进行腹腔免疫，免疫周期为2周/针，共计免疫5针。免疫前采取小鼠0/2/4/6/8周及第10周眼球血，后对小鼠进行断颈处理。血样于37℃放置30min后，13300rpm离心10min，收集血清进行Env及P24特异性的结合滴度的测定及HIV-1假病毒中和抗体滴度测定。实验结果显示本发明的真病毒可诱导产生中和抗体应答，展现出良好的免疫原性。
表4：NL4-3真病毒颗粒免疫方案
Figure PCTCN2021130414-appb-000018
实施例9：基于TSTIP的多种蛋白设计及性质鉴定
以BG505 TSTIP的氨基酸序列(SEQ ID NO:3)为基础，在BG505 TSTIP的β27与gp120连接处以及gp120与α8的连接处引入了由于TSTIP的设计需要删除的WNSSWSN和AKRRVVGREKR，获得SEQ ID NO:5(BGTSTIP-Full)。
以NL4-3TSTIP的氨基酸序列(SEQ ID NO:4)为基础，在NL4-3TSTIP的β27与gp120连接处以及gp120与α8的连接处引入了由于TSTIP的设计需要删除的WNSSWSN和AKRRVVGREKR，获得SEQ ID NO:6(NL4-3TSTIP Full)。
在BG505 TSTIP的β27与gp120连接处以及gp120与α8的连接处分别引入了GGGGS(SEQ ID NO:28)，获得SEQ ID NO:7(BG505-TSTIP G1)。
在NL4-3TSTIP的β27与gp120连接处以及gp120与α8的连接处引入了GGGGS，获得SEQ ID NO:8(NL4-3-TSTIP G1)。
在BG505 TSTIP的β27与gp120连接处以及gp120与α8的连接处分别引入了GGGGSGGGGS(SEQ ID NO:29)，获得SEQ ID NO:9(BG505-TSTIP G2)。
在NL4-3TSTIP的β27与gp120连接处以及gp120与α8的连接处引入了GGGGSGGGGS，获得SEQ ID NO:10(NL4-3-TSTIP G2)。
依据实施例1，2，3，4的步骤对上述获得的六种蛋白进行了一系列的研究。六种蛋白的SDS聚丙烯酰胺凝胶电泳结果如图11A，BGTSTIP-Full、NL4-3TSTIP Full和8NL4-3-TSTIP G1的蛋白经一步Ni柱纯化后的纯度有所降低；BGTSTIP-G1和NL4-3 TSTIP G2在还原条件下的考染显示>180KD的条带，可能为其二聚体形式，而BG505-TSTIP G2和则表现出与BG505 TSTIP相似的纯度和分子量大小。
发明人对四种纯度较高的蛋白进行了分析超离，结果如图11B，这一结果与上一步SDS聚丙烯酰胺凝胶电泳结果相吻合(14A)，BGTSTIP G1、NL4-3TSTIP G2分子量大于单体gp140分子量，而BG505 TSTIP的纯度较BGTSTIP有明显下降。这说明β27，gp120的连接处，及gp120，α8的连接处对Env蛋白的表现形式有明显的影响。其次在同一毒株上的不同改造，其表达量也存在一定差异，559位点Ile→Pro的突变能有效提高蛋白产量，具体地，表达量水平汇总如表3。
六种蛋白与已报道的多种人单克隆抗体的酶联免疫测定实验结果如图12，即便部分蛋白纯度不高，纯化得到的六种蛋白均能与多种抗体较好的识别，证明了其活性。
我们按上述免疫方案，将纯化的六种蛋白免疫BABL/C小鼠，动物免疫实验结果表明，六种蛋白均可以诱导小鼠产生中和抗体应答，如图13。此外基于这些蛋白进一步构建假病毒颗粒和真病毒颗粒，并进行小鼠免疫实验，检测其免疫原性。
表3：蛋白表达量汇总
Figure PCTCN2021130414-appb-000019
Figure PCTCN2021130414-appb-000020
实施例10：TSTIP改造在多亚型毒株中的应用
为了进一步确认TSTIP的设计在不同毒株上的作用效果，本发明人将类似于BG505/NL4-3-TSTIP的设计方法运用于多种毒株的Env蛋白，表4为12株全球代表株，因已完成了A亚型、B亚型毒株的表达及鉴定工作，我们在12株全球假病毒盘中选择了除A、B亚型以外的其他亚型毒株各1株，并进行Env蛋白TSTIP的改造，选择的毒株包括25710(C),X1632(G)，CH119(BC),CNE8(AE)，246F3(AC)五种毒株的gp140蛋白中。并依据如上所述的克隆，表达纯化，性质鉴定等一系列实验步骤和方法，对五种毒株的TSTIP-gp140蛋白进行了研究，系列结果如图14A-14D所示。
图14A中SDS聚丙烯酰胺凝胶电泳结果表明，五种毒株的TSTIP蛋白经一步Ni柱纯化后均得到了较高的纯度，还原SDS PAGE下分子量约为140KD，表明发明蛋白不同亚基之间如预期通过稳定的共价作用连接在一起。
图14B为五种TSTIP蛋白分子筛superdex200 16/600层析结果,洗脱峰较为单一，表明五种发明蛋白在在天然状态下组分较为均一，且以三聚体含量居多。
图14C为五种蛋白与多种已报道抗体的酶联免疫测定结果(ELISA)，不同毒株对同类抗体的识别结合能力存在差异，该结果表明，本发明人生产的五种毒株TSTIP蛋白都具有较好的活性。
图14D为五种蛋白免疫BALB/C小鼠后血清的中和实验，结果表明不同毒株的TSTIP蛋白免疫能够刺激小鼠产生中和应答。
表4：12株全球盘假病毒
Figure PCTCN2021130414-appb-000021
Figure PCTCN2021130414-appb-000022
表5：五种亚型TSTIP蛋白表达量汇总
Figure PCTCN2021130414-appb-000023
实施例11：基于TSTIP的其他蛋白设计及活性鉴定
11.1蛋白设计
1、BG-B1(1/1)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入GGGGS，将WNSSWSN补充到α8前，并在其前加入GGGGS。基于BG505毒株改造的序列如SEQ ID NO:30所示。
2、BG-B1(1/2)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入GGGGS，将WNSSWSN补充到α8前，并在其前加入(GGGGS) 2。基于BG505毒株改造的序列如SEQ ID NO:31所示。
3、BG-B1(2/2)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入(GGGGS) 2，将WNSSWSN补充到α8前，并在其前加入(GGGGS) 2。基于BG505毒株改造的序列如SEQ ID NO:32所示。
4、BG-B1(2/1)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入(GGGGS) 2，将WNSSWSN补充到α8前，并在其前加入GGGGS。基于BG505毒株改造的序列如SEQ ID NO:33所示。
5、BG-B1(2/3)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入 (GGGGS) 2，将WNSSWSN补充到α8前，并在其前加入(GGGGS) 3。基于BG505毒株改造的序列如SEQ ID NO:34所示。
6、BG-B2(1-1)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入GGGGS，将WNSSWSN补充到α8前，在其前加入GGGGS，并参考SOSIP的设计在gp120和gp41亚基间引入cys突变，以期待形成二硫键，具体地将第501位的Ala突变为cys，将第605位的Thr突变为cys。基于BG505毒株改造的序列如SEQ ID NO:35所示。
7、BG-B2(1-2)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入GGGGS，将WNSSWSN补充到α8前，在其前加入(GGGGS) 2，并参考SOSIP的设计在gp120和gp41亚基间引入cys突变，以期待形成二硫键，具体地将第501位的Ala突变为cys，将第605位的Thr突变为cys。基于BG505毒株改造的序列如SEQ ID NO:36所示。
8、BG-B2(1-3)
补全原来删除的β26后面的氨基酸序列AKRRVVG(不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将AKRRVVG补充到β26后面，并在其后引入GGGGS，将WNSSWSN补充到α8前，在其前加入(GGGGS) 3，并参考SOSIP的设计在gp120和gp41亚基间引入cys突变，以期待形成二硫键，具体地将第501位的Ala突变为cys，将第605位的Thr突变为cys。基于BG505毒株改造的序列如SEQ ID NO:37所示。
9、BG-C1(1/1)
补全原来删除的β26后面的氨基酸序列AKRRVVG(或其同源序列，不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将WNSSWSN补充到β27后面，并在其后引入GGGGS，将AKRRVVG补充到α8前，并在其后加入GGGGS。基于BG505毒株改造的序列如SEQ ID NO:38所示。
10、BG-C1(1/2)
补全原来删除的β26后面的氨基酸序列AKRRVVG(或其同源序列，不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将WNSSWSN补充到β27后面， 并在其后引入GGGGS，将AKRRVVG补充到α8前，并在其后加入(GGGGS) 2。基于BG505毒株改造的序列如SEQ ID NO:39所示。
11、BG-C1(1/3)
补全原来删除的β26后面的氨基酸序列AKRRVVG(或其同源序列，不包含furin酶切位点)及β27后的序列WNSSWSN(或同源序列)，将WNSSWSN补充到β27后面，并在其后引入GGGGS，将AKRRVVG补充到α8前，并在其后加入(GGGGS) 3。基于BG505毒株改造的序列如SEQ ID NO:40所示。
11.2 SDS-PAGE：
使用上述实施例1-2中所描述的方法制备并纯化这些蛋白，将浓缩样品稀释至1ug/ul后取两管50ul样品，分别加入10ul还原Loading buffer和10ul非还原Loading buffer，制备还原样品和非还原样品，还原样品于100℃沸水浴10min。取10ul还原样品及非还原样品于8％SDS PAGE中以80V电压电泳120min，经考马斯亮蓝染色后显示电泳条带。
电泳结果见图15A-15K中的图(I)所示。经SDS-PAGE分析显示经一步Ni-EXCEL纯化后，上述设计均可获得高纯度蛋白；在添加还原性Loading buffer的条件下，除BG-C1(1/1)以外，其他优化蛋白显示的主条带均为140KD，在非还原Loading buffer的条件下为多聚体条带。说明经优化设计的蛋白依旧如初始TSTIP蛋白一样采用稳定的共价连接方式将gp120和gp41胞外段相连。
11.3分子筛纯化：
仪器系统：AKTA Pure型制备型液相色谱仪；
层析柱：superdex 200 10/300
柱体积：24ml
缓冲液：PBS(20mM磷酸缓冲液，pH7.5，150mMNacl)
检测器波长：280nm
流速：0.5ml/min
样品为经纯化浓缩的蛋白。
纯化程序为：1倍柱体积的PBS平衡superdex200 10/300，500ul上样环上样纯化目的蛋白，inject模式下进行纯化样品的分子筛纯化，样品会按不同组分的分子量大小，由高到低，依次被洗脱，保存纯化后峰图。分子筛纯化结果如图15A-15K中的图(II)所示。 各蛋白均在分子筛图谱上显示出明显的单一的洗脱峰。其中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)的主洗脱峰的提托体积均在9.5ml附近，为三聚体峰，而BG-C1(1/1)的洗脱体积为14ml，为二聚体峰。
11.4分析型超速离心(AUC)蛋白均一性分析及分子量预测
发明人对上述纯化蛋白进行了分析型超离分析，AUC结果如图15A-15K中的图(III)所示。该结果显示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)主要为三聚体，BG-C1(1/1)主要为二聚体，这一结果与分子筛结果相吻合。
11.5酶联免疫吸附测定(ELISA)
使用上述实施例4中的酶联免疫吸附测定方法，对上述纯化蛋白的抗原性进行鉴定。结果如图15A-15K中的图(IV)所示。结果表明，这些蛋白具备抗原性，并且修改连接处linker的方式可以调整抗原活性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (34)

  1. 重组蛋白，其包含gp120和gp41胞外结构域(gp41 ectodomain,gp41ECTO)，其中，所述gp120位于所述gp41ECTO的β27和α8之间；
    优选地，所述重组蛋白从N端至C端方向包含：gp41ECTO的α6、α7、β27；gp120；gp41ECTO的α8、α9。
  2. 权利要求1所述的重组蛋白，其中，所述gp41ECTO的β27和α8之间的连接区域中的一个或多个(例如，1-12个，5-12个，5-10个；例如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个，11个，或12个)连续氨基酸被替换为gp120；
    优选地，所述连接区域对应于分离株HXB2的gp160序列的氨基酸位置607-618。
  3. 权利要求2所述的重组蛋白，其中，所述gp41ECTO在对应于分离株HXB2的gp160序列的氨基酸位置610-616的区域中的一个或多个(例如，1-7个，例如5-7个；例如1个，2个，3个，4个，5个，6个，或7个)连续氨基酸被替换为gp120；
    优选地，所述gp120位于对应于分离株HXB2的gp160序列的氨基酸位置606和619之间。
  4. 权利要求1所述的重组蛋白，其中，所述gp120插入在gp41ECTO的β27和α8之间的连接区域中的相邻氨基酸之间；
    优选地，所述连接区域对应于分离株HXB2的gp160序列的氨基酸位置607-618；
    优选地，所述gp120位于对应于分离株HXB2的gp160序列的氨基酸位置609和610之间；
    优选地，所述gp120位于对应于分离株HXB2的gp160序列的氨基酸位置616和617之间。
  5. 权利要求1-4任一项所述的重组蛋白，其中，所述gp120是经修饰的gp120，其与天然gp120相比，弗林蛋白酶识别位点包含突变以防止弗林蛋白酶位点被切割；
    优选地，所述突变选自氨基酸的置换、插入或缺失；
    优选地，所述弗林蛋白酶位点对应于分离株HXB2的gp160序列的氨基酸位置508- 511；
    优选地，与天然gp120相比，所述经修饰的gp120中的弗林蛋白酶识别位点被删除。
  6. 权利要求1-4任一项所述的重组蛋白，其中，所述gp120是经修饰的gp120，其与天然gp120相比，C端截短了1-11个(例如4-11个；例如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个，或11个)氨基酸；
    优选地，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置501-511的区域中包含一个或多个(例如，1-11个，4-11个；例如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个，或11个)连续氨基酸的缺失；
    优选地，所述经修饰的gp120在对应于分离株HXB2的gp160序列的氨基酸位置501-511的区域被删除。
  7. 权利要求1-6任一项所述的重组蛋白，其中，所述重组蛋白的gp120和gp41ECTO之间包含二硫键；
    优选地，所述重组蛋白在对应于分离株HXB2的gp160序列的氨基酸位置37和605之间具有二硫键；
    优选地，所述重组蛋白在对应于分离株HXB2的gp160序列的氨基酸位置37和605上的氨基酸残基为Cys。
  8. 权利要求1-4任一项所述的重组蛋白，其中，所述gp120是天然gp120。
  9. 权利要求1-8任一项所述的重组蛋白，其中，所述gp120的N端和/或C端任选地通过肽接头与所述gp41ECTO连接；
    优选地，所述肽接头是(GmS)n，其中m选自1-4的整数，n选自1-3的整数。
  10. 权利要求1-9任一项所述的重组蛋白，其还具备以下特征中的一项或多项：
    (1)所述gp41ECTO包含下列氨基酸置换：I559P；
    (2)所述gp120包含下列氨基酸置换：T332N；
    (3)所述gp120包含下列氨基酸置换：E64K和H66R；
    (4)所述gp120包含下列氨基酸置换：A316W；
    (5)所述gp120的CD4bs表位附近的N-连接糖基化位点(PNGS)被置换以防止糖基化；优选地，所述PNGS选自N276、N301、N360、N463；
    (6)所述gp120包含内部二硫键；优选地，所述gp120包含I201C和A433C之间的内部二硫键；
    (7)所述gp120和gp41ECTO之间还包含二硫键；例如，所述重组蛋白在E49C和L555C之间具有二硫键；
    以上位置的编号是根据HIV-1分离株HXB2的gp160中的编号。
  11. 权利要求1-10任一项所述的重组蛋白，其中，所述gp41ECTO和gp120来自相同或不同的HIV-1毒株；
    优选地，所述gp41ECTO和gp120来自相同的HIV-1毒株。
  12. 权利要求1-11任一项所述的重组蛋白，其包含选自下列的氨基酸序列：
    (1)由SEQ ID NO:1所示序列的第40位至第651位氨基酸残基构成的氨基酸序列；
    (2)由SEQ ID NO:2所示序列的第40位至第652位氨基酸残基构成的氨基酸序列；
    (3)由SEQ ID NO:3所示序列的第40位至第651位氨基酸残基构成的氨基酸序列；
    (4)由SEQ ID NO:4所示序列的第40位至第652位氨基酸残基构成的氨基酸序列；
    (5)由SEQ ID NO:5所示序列的第40位至第665位氨基酸残基构成的氨基酸序列；
    (6)由SEQ ID NO:6所示序列的第40位至第678位氨基酸残基构成的氨基酸序列；
    (7)由SEQ ID NO:7所示序列的第40位至第661位氨基酸残基构成的氨基酸序列；
    (8)由SEQ ID NO:8所示序列的第40位至第666位氨基酸残基构成的氨基酸序列；
    (9)由SEQ ID NO:9所示序列的第40位至第671位氨基酸残基构成的氨基酸序列；
    (10)由SEQ ID NO:10所示序列的第40位至第676位氨基酸残基构成的氨基酸序列；
    (11)由SEQ ID NO:11所示序列的第36位至第607位氨基酸残基构成的氨基酸序列；
    (12)由SEQ ID NO:12所示序列的第36位至第646位氨基酸残基构成的氨基酸序列；
    (13)由SEQ ID NO:13所示序列的第36位至第648位氨基酸残基构成的氨基酸序列；
    (14)由SEQ ID NO:14所示序列的第36位至第638位氨基酸残基构成的氨基酸序列；
    (15)由SEQ ID NO:15所示序列的第36位至第639位氨基酸残基构成的氨基酸序列；
    (16)由SEQ ID NO:16所示序列的第36位至第836位氨基酸残基构成的氨基酸序列；
    (17)由SEQ ID NO:30所示序列的第36位至第673位氨基酸残基构成的氨基酸序列；
    (18)由SEQ ID NO:31所示序列的第36位至第678位氨基酸残基构成的氨基酸序列；
    (19)由SEQ ID NO:32所示序列的第36位至第683位氨基酸残基构成的氨基酸序列；
    (20)由SEQ ID NO:33所示序列的第36位至第678位氨基酸残基构成的氨基酸序列；
    (21)由SEQ ID NO:34所示序列的第36位至第688位氨基酸残基构成的氨基酸序列；
    (22)由SEQ ID NO:35所示序列的第36位至第670位氨基酸残基构成的氨基酸序列；
    (23)由SEQ ID NO:36所示序列的第36位至第676位氨基酸残基构成的氨基酸序列；
    (24)由SEQ ID NO:37所示序列的第36位至第680位氨基酸残基构成的氨基酸序列；
    (25)由SEQ ID NO:38所示序列的第36位至第673位氨基酸残基构成的氨基酸序列；
    (26)由SEQ ID NO:39所示序列的第36位至第678位氨基酸残基构成的氨基酸序列；
    (27)由SEQ ID NO:40所示序列的第36位至第683位氨基酸残基构成的氨基酸序列；或
    (28)(1)-(27)任一项所述序列的变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)或者具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且所述变体保留了其所源自的序列的特性。
  13. 权利要求1-12任一项所述的重组蛋白，在其N端或C端任选地包含一种或多种选自下列的序列：信号肽、翻译起始序列(例如Kozak共有序列)、标签序列；
    优选地，所述重组蛋白在其N端任选地包含信号肽和/或翻译起始序列(例如Kozak共有序列)；
    优选地，所述重组蛋白在其C端任选地包含标签序列。
  14. 融合蛋白，其包含权利要求1-13任一项所述的重组蛋白以及与其C端连接的gp41的跨膜区和胞内区序列；
    优选地，所述gp41的跨膜区和胞内区序列与所述重组蛋白中的gp41ECTO来自相同的HIV-1毒株。
  15. 权利要求14所述的融合蛋白，其包含选自下列的氨基酸序列：
    (1)由SEQ ID NO:16所示序列的第33位至第836位氨基酸残基构成的氨基酸序列；
    (2)由SEQ ID NO:17所示序列的第34位至第837位氨基酸残基构成的氨基酸序列；
    (3)(1)-(2)任一项所述序列的变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)或者具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且所述变体保留了其所源自的序列的特性。
  16. 包含多个单体的多聚体，其中，各单体各自独立地选自权利要求1-13任一项所述的重组蛋白，或者各自独立地选自权利要求14或15所述的融合蛋白；
    优选地，所述各单体彼此相同；
    优选地，所述多聚体是三聚体或二聚体。
  17. 分离的核酸分子，其包含编码权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体的核苷酸序列。
  18. 载体，其包含权利要求17所述的分离的核酸分子。
  19. 宿主细胞，其包含权利要求17所述的分离的核酸分子或权利要求18所述的载体；
    优选地，所述宿主细胞是哺乳动物细胞。
  20. 制备权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体的方法，其包括，在合适的条件下培养权利要求19的宿主细胞，和从细胞培养物中回收所述重组蛋白、融合蛋白或多聚体；
    优选地，所述重组蛋白或融合蛋白以多聚体(例如三聚体或二聚体)形式存在。
  21. 粒子，在其表面上展示权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体；
    优选地，所述粒子是脂质体或纳米粒子。
  22. 假病毒颗粒，在其表面包含权利要求1-13任一项所述的重组蛋白、权利要求14 或15所述的融合蛋白或权利要求16所述的多聚体；
    优选地，所述假病毒颗粒通过在宿主细胞中共表达(i)包含权利要求17所述的核酸分子的载体和(ii)包装载体(例如骨架质粒)获得。
  23. 用于产生权利要求22所述的假病毒颗粒的包装系统，其包括：(i)包含权利要求17所述的核酸分子的表达载体、(ii)包装载体(例如骨架质粒)。
  24. 经改造的HIV病毒，其表达权利要求14或15所述的融合蛋白作为其包膜蛋白；
    优选地，所述经改造的HIV病毒的基因组包含以下改造：野生型env基因被替换为编码权利要求14或15所述的融合蛋白的核苷酸序列；
    优选地，所述HIV是HIV-1。
  25. 分离的核酸分子，其包含编码权利要求24所述的经改造的HIV病毒的基因组的核苷酸序列。
  26. 载体，其包含权利要求25所述的分离的核酸分子。
  27. 组合物，其包含权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体、权利要求17所述的分离的核酸分子、权利要求18所述的载体、权利要求19所述的宿主细胞、权利要求21所述的粒子、权利要求22所述的假病毒颗粒、权利要求23所述的包装系统、权利要求24所述的经改造的HIV病毒、权利要求25所述的分离的核酸分子、或权利要求26所述的载体；
    优选地，所述组合物还包含药学上可接受的载体和/或赋形剂。
  28. 权利要求27所述的组合物，其中，所述组合物是免疫原性组合物或疫苗；
    优选地，所述组合物包含佐剂。
  29. 权利要求28所述的组合物，其中，所述组合物是蛋白疫苗，其包含权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体、或权利要求21所述的粒子。
  30. 权利要求28所述的组合物，其中，所述组合物是病毒疫苗，其包含权利要求22所述的假病毒颗粒或权利要求23所述的经改造的HIV病毒。
  31. 权利要求28所述的组合物，其中，所述组合物是核酸疫苗，其包含权利要求17所述的分离的核酸分子、权利要求18所述的载体、权利要求25所述的分离的核酸分子、或权利要求26所述的载体；
    优选地，所述核酸疫苗包含DNA或RNA；
    优选地，所述DNA或RNA可以是裸露的或可以包裹于具有传递或/和保护功能的外壳内。
  32. 权利要求27-31任一项所述的组合物，其任选地包含抗逆转录病毒药剂，例如核苷逆转录酶抑制剂，非核苷逆转录酶抑制剂，蛋白酶抑制剂，或融合蛋白抑制剂。
  33. 权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体、权利要求17所述的分离的核酸分子、权利要求18所述的载体、权利要求19所述的宿主细胞、权利要求21所述的粒子、权利要求22所述的假病毒颗粒、权利要求23所述的包装系统、权利要求24所述的经改造的HIV病毒、权利要求25所述的分离的核酸分子、权利要求26所述的载体、或权利要求27-32任一项所述的组合物，用于在受试者中诱导针对HIV的免疫应答和/或用于在受试者中预防和/或治疗HIV感染的用途，或者在制备用于在受试者中诱导针对HIV的免疫应答和/或用于在受试者中预防和/或治疗HIV感染的药剂中的应用；
    优选地，所述药剂为疫苗；
    优选地，所述受试者是人；
    优选地，所述HIV是HIV-1；
    任选地，所述重组蛋白、融合蛋白、多聚体、分离的核酸分子、载体、宿主细胞、粒子、假病毒颗粒、包装系统、经改造的HIV病毒、组合物与抗逆转录病毒药剂联合施用，例如同时、分开或相继施用；优选地，所述抗逆转录病毒药剂选自核苷逆转录酶抑制剂，非核苷逆转录酶抑制剂，蛋白酶抑制剂，或融合蛋白抑制剂。
  34. 用于在受试者中诱导针对HIV的免疫应答或用于在受试者中预防和/或治疗HIV感染的方法，其包括向有此需要的受试者施用免疫学有效量的权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体、权利要求17所述的分离的核酸分子、权利要求18所述的载体、权利要求19所述的宿主细胞、权利要求21所述的粒子、权利要求22所述的假病毒颗粒、权利要求23所述的包装系统、权利要求24所述的经改造的HIV病毒、权利要求25所述的分离的核酸分子、权利要求26所述的载体、或权利要求27-32任一项所述的组合物；
    优选地，所述方法包括施用包含权利要求1-13任一项所述的重组蛋白、权利要求14或15所述的融合蛋白或权利要求16所述的多聚体、或权利要求21所述的粒子的免疫原性组合物或疫苗(例如蛋白疫苗)；
    优选地，所述方法包括施用包含权利要求22所述的假病毒颗粒或权利要求23所述的经改造的HIV病毒的免疫原性组合物或疫苗(例如病毒疫苗)；
    优选地，所述方法包括施用包含权利要求17所述的分离的核酸分子、权利要求18所述的载体、权利要求25所述的分离的核酸分子、或权利要求26所述的载体的免疫原性组合物或疫苗(例如核酸疫苗)；
    优选地，所述受试者是人；
    优选地，所述HIV是HIV-1；
    任选地，所述重组蛋白、融合蛋白、多聚体、分离的核酸分子、载体、宿主细胞、粒子、假病毒颗粒、包装系统、经改造的HIV病毒、组合物与抗逆转录病毒药剂联合施用，例如同时、分开或相继施用；优选地，所述抗逆转录病毒药剂选自核苷逆转录酶抑制剂，非核苷逆转录酶抑制剂，蛋白酶抑制剂，或融合蛋白抑制剂。
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003022869A2 (en) 2001-09-06 2003-03-20 Progenics Pharmaceuticals, Inc. Human immunodeficiency virus envelope clycoprotein mutants and uses thereof
WO2011082087A2 (en) 2010-01-04 2011-07-07 Kj Biosciences, Llc Dps fusion proteins for use in vaccines and diagnostics
US20140212458A1 (en) * 2012-11-05 2014-07-31 International Aids Vaccine Initiative Novel hiv-1 envelope glycoprotein
WO2014124301A1 (en) 2013-02-07 2014-08-14 University Of Washington Through Its Center For Commercialization Self-assembling protein nanostructures
CN103992396A (zh) * 2014-04-17 2014-08-20 南开大学 一种潜在的高效重组HIV-1 CRF07-BC gp140免疫原的制备方法
US20160122392A1 (en) 2014-11-03 2016-05-05 University Of Washington Polypeptides for use in self-assembling protein nanostructures
US20170233441A1 (en) * 2014-09-04 2017-08-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Recombinant hiv-1 envelope proteins and their use
CN109153704A (zh) * 2016-05-02 2019-01-04 斯克里普斯研究学院 与hiv-1免疫原相关的组合物和方法
CN109689091A (zh) * 2016-09-15 2019-04-26 扬森疫苗与预防公司 三聚体稳定性hiv包膜蛋白突变
CN110248954A (zh) * 2016-10-14 2019-09-17 开普敦大学 在植物中产生可溶性hiv包膜三聚体

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017055522A1 (en) * 2015-09-29 2017-04-06 Academisch Medisch Centrum Stabilized env proteins of hiv

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003022869A2 (en) 2001-09-06 2003-03-20 Progenics Pharmaceuticals, Inc. Human immunodeficiency virus envelope clycoprotein mutants and uses thereof
WO2011082087A2 (en) 2010-01-04 2011-07-07 Kj Biosciences, Llc Dps fusion proteins for use in vaccines and diagnostics
US20140212458A1 (en) * 2012-11-05 2014-07-31 International Aids Vaccine Initiative Novel hiv-1 envelope glycoprotein
WO2014124301A1 (en) 2013-02-07 2014-08-14 University Of Washington Through Its Center For Commercialization Self-assembling protein nanostructures
CN103992396A (zh) * 2014-04-17 2014-08-20 南开大学 一种潜在的高效重组HIV-1 CRF07-BC gp140免疫原的制备方法
US20170233441A1 (en) * 2014-09-04 2017-08-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Recombinant hiv-1 envelope proteins and their use
US20160122392A1 (en) 2014-11-03 2016-05-05 University Of Washington Polypeptides for use in self-assembling protein nanostructures
CN109153704A (zh) * 2016-05-02 2019-01-04 斯克里普斯研究学院 与hiv-1免疫原相关的组合物和方法
CN109689091A (zh) * 2016-09-15 2019-04-26 扬森疫苗与预防公司 三聚体稳定性hiv包膜蛋白突变
CN110248954A (zh) * 2016-10-14 2019-09-17 开普敦大学 在植物中产生可溶性hiv包膜三聚体

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
"Immunology-A Synthesis", 1991, SINAUER ASSOCIATES
"Protection by Live, Attenuated Simian Immunodeficiency Virus against Heterologous Challenge", JOURNAL OF VIROLOGY, 1999
BALE S ET AL., J VIROL., vol. 91, no. 16, 2017
E. MEYERSW. MILLER, COMPUT. APPL. BIOSCI., vol. 4, 1988, pages 11 - 17
F. M. AUSUBEL ET AL.: "Short protocols in Molecular Biology", 1995, JOHN WILEY & SONS, INC.
GUTTMAN, GARCIA ET AL.: "CD4-induced activation in a soluble HIV-1 Env trimer", STRUCTURE, 2014
HE L ET AL., NAT COMMUN., vol. 7, no. 1, 28 June 2016 (2016-06-28), pages 2041
HUCRAMPTON ET AL.: "Murine Antibody Responses to Cleaved Soluble HIV-1 Envelope Trimers Are Highly Restricted", SPECIFICITY, 2015
J. SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
KOZAK M., NUCLEIC ACIDS RESEARCH, vol. 12, 1984, pages 857 - 872
KOZAK M., NUCLEIC ACIDS RESEARCH, vol. 15, 1987, pages 8125 - 8148
LOPEZ-SAGASETA J ET AL., COMPUTATIONAL AND STRUCT BIOTECHNOL J, vol. 14, 2016, pages 58 - 68
M. PANCERA, S. MAJEED, Y.-E. A. BAN, L. CHEN, C.-C. HUANG, L. KONG, Y. D. KWON, J. STUCKEY, T. ZHOU, J. E. ROBINSON, W. R. SCHIEF,: "Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 107, no. 3, 19 January 2010 (2010-01-19), pages 1166 - 1171, XP055084839, ISSN: 0027-8424, DOI: 10.1073/pnas.0911004107 *
NEEDLEMAN ET AL., J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
SANDERS, R.W.J.P. MOORE: "Native-like Env trimers as a platform for HIV-1 vaccine design", IMMUNOL REV, 2017
SHEN HONGLIN, ZHANG ZHIQING, LI SHAOWEI, GU YING: "Progress in HIV-1 Env trimer design", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 36, no. 1, 25 January 2020 (2020-01-25), CN , pages 25 - 32, XP055930958, ISSN: 1000-3061, DOI: 10.13345/j.cjb.190162 *
YANG CHAO ,ZHANG ZHIQING , SHEN HONGLIN ,GAO SHUANGQUAN , LI SHAOYONG , LI SHAOWEI , XIA NINGSHAO , GU YING: "Expression and Characterization of Trimeric HIV-1 gp140 Membrane Protein from Mammalian Cells", CHINESE JOURNAL OF VIROLOGY, vol. 33, no. 4, 1 April 2017 (2017-04-01), pages 541 - 549, XP055930968, ISSN: 1000-8721, DOI: 10.13242/j.cnki.bingduxuebao.003180 *
ZHAO L ET AL., VACCINE, vol. 32, 2014, pages 327 - 337

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