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HIV neutralizing peptides, sulfated HIV-1 envelope proteins and immunogenic fragments thereof are disclosed, as well as nucleic acids encoding these molecules and methods of producing these peptides, envelope proteins and fragments. Methods are also provided for the treatment or prevention of a human immunodeficiency type 1 (HIV-1) infection.
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The methods can include administering to a subject an HIV neutralizing peptide, sulfated HIV-1 envelope protein or immunogenic fragment thereof as disclosed herein. In several embodiments, administering the HIV neutralizing peptide, sulfated HIV-1 envelope protein or immunogenic fragment generates an immune response in a subject.
RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Application No.
61/736,350, filed Dec. 12, 2012, which is incorporated by reference in its entirety. FIELDThis disclosure relates to HIV neutralizing peptides, sulfated HIV-1 envelope proteins and immunogenic fragments thereof, for treatment and prevention of Human Immunodeficiency Virus (HIV) infection and disease. BACKGROUNDOver 30 million people are infected with HIV worldwide, and 2.5 to 3 million new infections have been estimated to occur yearly. Although effective antiretroviral therapies are available, millions succumb to AIDS every year, especially in sub-Saharan Africa, underscoring the need to develop measures to prevent the spread of this disease.An enveloped virus, HIV-1 hides from humoral recognition behind a wide array of protective mechanisms. The major envelope protein of HIV-1 is a glycoprotein of approximately 160 kD (gp160). During infection proteases of the host cell cleave gp160 into gp120 and gp41.
Gp41 is an integral membrane protein, while gp120 protrudes from the mature virus. Together gp120 and gp41 make up the HIV envelope spike, which is a target for neutralizing antibodies. Further, HIV envelope binds to CD4 and CC chemokine receptor 5 (CCR5) as co-receptors required for cellular entry and infection.It is believed that immunization with an effective immunogen based on the HIV-1 envelope glycoprotein can elicit a neutralizing response, which may be protective against HIV infection.
Further, it is believed that peptide therapeutics based on gp120 can neutralize gp120, and thus HIV. However, despite extensive effort, a need remains for immunogens and peptide therapeutics capable of such action. SUMMARYDisclosed herein is the surprising discovery that the second variable loop (V2) of gp120 contains previously unrecognized sulfated tyrosines that bolster its intramolecular interaction with the third variable loop (V3) loop, thereby constraining the HIV-1 envelope in its native, antibody-shielded conformation. Further, the sulfated region of V2 molecularly mimics the CCR5 N-terminal domain because both regions can adopt an α-helix conformation and lock onto the conserved base of V3.
Upon binding to V3, a peptide including the tyrosine-sulfated region of V2 competes with CCR5 binding to gp120, thereby inhibiting HIV infection. Surprisingly, this V2 peptide also binds to CD4, further contributing to inhibition of HIV infection. These discoveries led to the identification of novel HIV-1 therapeutic peptides, as well as sulfated HIV-1 envelope proteins, fragments thereof containing gp120 positions 173 and/or 177, and methods of making and using such molecules.In some embodiments, a method of making a sulfated HIV-1 envelope protein or immunogenic fragment thereof is provided.
The method can include providing a plurality of HIV-1 envelope proteins or immunogenic fragments thereof comprising tyrosine residues at gp120 positions 173, 177, or 173 and 177, and sulfating the tyrosine residues at gp120 positions 173, 177, or 173 and 177 on at least 90% of the HIV-1 envelope proteins or immunogenic fragments in the plurality of HIV-1 envelope proteins or immunogenic fragments. The method can further include purifying the plurality of HIV-1 envelope proteins or immunogenic fragments.In some embodiments, a method of making a sulfated HIV-1 envelope protein or immunogenic fragment thereof is provided, including co-expression of a first and a second heterologous nucleic acid molecule in a cell. The first heterologous nucleic acid molecule encodes a HIV-1 envelope protein or immunogenic fragment thereof, wherein the HIV-1 envelope protein or immunogenic fragment thereof comprises tyrosine residues at gp120 positions, 173, 177, or 173 and 177. The second heterologous nucleic acid molecule encodes a tyrosine sulfotransferase. The first and second nucleic acid molecules are expressed in the cell under conditions sufficient for efficient sulfation of the tyrosine residues at gp120 positions 173, 177, or 173 and 177 of the HIV envelope protein or immunogenic fragment thereof. Following expression, the sulfated HIV-1 envelope protein or immunogenic fragment thereof can be purified.In some embodiments, a method of making a sulfated HIV-1 envelope protein or immunogenic fragment thereof is provided, including incubating a HIV-1 envelope protein or immunogenic fragment thereof comprising tyrosine residues at gp120 positions 173, 177, or 173 and 177 with a purified tyrosine sulfotransferase under conditions sufficient for sulfation of the tyrosine residues.
Following incubation, the sulfated HIV-1 envelope protein or immunogenic fragment thereof can be purified.In several embodiments, the HIV-1 envelope protein or immunogenic fragment comprises gp160, gp140, or gp120.In some embodiments, the HIV-1 envelope protein or immunogenic fragment comprises both the V2 and the V3 loop combined or complexed within the same preparation. Thus, the sulfated tyrosine residues at gp120 positions 173, 177, or 173 and 177, can bind to the base of the V3 loop and recreate the physiological complex that is presented in the native envelope.Some embodiments include the sulfated HIV-1 envelope protein or immunogenic fragment thereof made by the disclosed methods.In further embodiments, HIV neutralizing peptides are disclosed that include gp120 positions 171-178 according to the HXB2 numbering system and correspond to the amino acid positions in the amino acid sequence set forth as SEQ ID NO: 1. In some embodiments, the HIV neutralizing peptide includes gp120 positions 168-185 according to the HXB2 numbering system and corresponding to the amino acid positions in the amino acid sequence set forth as SEQ ID NO: 1. The HIV neutralizing peptides include a first sulfated tyrosine at position 173, a second sulfated tyrosine at position 177, or both a first sulfated tyrosine at position 173 and a second sulfated tyrosine at position 177, at most four additional amino acid substitutions compared to a wild-type HIV-1 gp120, and they are at most 50 amino acids in length; and neutralize HIV. In several embodiments, the HIV neutralizing peptides compete with CCR5 for binding to gp120. In several embodiments, the HIV neutralizing peptides increase the sensitivity of HIV to neutralization with antibodies directed to the V3-loop, the CD4 binding site or other neutralization epitopes.In additional embodiments, the disclosed HIV neutralizing peptides or sulfated HIV-1 envelope proteins or immunogenic fragments are included in a pharmaceutical composition, such as an immunogenic composition. In several embodiments the composition includes an anti-retroviral agent.Additional embodiments include a composition comprising a plurality of HIV-1 envelope proteins or immunogenic fragments thereof comprising tyrosine residues at gp120 positions 173, 177, or 173 and 177, wherein at least 90% of the tyrosine residues at gp120 positions 173, 177, or 173 and 177 on the HIV-1 envelope proteins or immunogenic fragments in the plurality of HIV-1 envelope proteins or immunogenic fragments are sulfated.
The composition may be a pharmaceutical composition suitable for administration to a subject, and may also be contained in a unit dosage form. The HIV-1 envelope proteins or immunogenic fragments may also be conjugated to a carrier (such as a monomeric subunit of a protein nanoparticle) to facilitate presentation to the immune system.Methods of generating an immune response in a subject are disclosed, as are methods of treating, inhibiting or preventing a HIV-1 infection in a subject.
In such methods a subject, such as a human subject, is administered an effective amount of a disclosed HIV neutralizing peptide or sulfated HIV-1 envelope protein or immunogenic fragment. Several embodiments also include administering to the subject a therapeutically effective amount of an anti-retroviral agent in combination with the disclosed HIV neutralizing peptide.The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGSFIGS. 1A-1D depict a sequence alignment, a ribbon diagram and a graph illustrating the conservation and predicted structure within the V2 loop of HIV-1 gp120. (A) Consensus sequences of the V2 domain of gp120 in different HIV-1 genetic subtypes (A to F; SEQ ID NOs: 15-20, respectively). Within each subtype, amino acid (aa) conservation greater than 80% is shown by a grey background, between 60% and 80% by a light grey background, and less than 60% by a white background. Conservative substitutions were considered as conserved residues according to standard Clustal parameters.
Identical residues in all subtype consensus sequences are indicated by an asterisk; conserved substitutions by a colon; semi-conserved substitutions by a period. Residues are numbered using the HXB2 reference sequence. Since no consensus sequence was obtained for the C-terminal variable region, the average number of aa (±SD) is indicated in parenthesis for each subtype.
(B) Prediction of the secondary structure of the V2 loop of HIV-1 BaL (SEQ ID NO: 21) using 5 different algorithms (Agadir, GOR V, Jufo 9, Psi-Pred, NNPred). The grey background indicates a predicted α-helix. Residue numbering follows the HXB2 reference sequence. (C) Prediction of the three-dimensional structure of the V2 segment spanning aa 168-178 using Gromacs (grey) and ROSETTA (cyan). (D) Circular dichroism analysis of V2-derived peptides pV2α and pV2α-Pro spanning amino acids 168-185.FIGS.
2A-2K depict digital images and diagrams illustrating that the V2 domain of HIV-1 gp120 contains sulfated tyrosines and is predicted to interact with the CCR5-binding site at the base of V3. (A) Sequence alignment of the CCR5 N-terminal domain (SEQ ID NO: 22) and the V2 α-helix (residues 14-24 of SEQ ID NO: 21). The grey background highlights two conserved tyrosine residues in both sequences. The V2 residue numbering follows the HXB2 reference sequence. (B) Detection of sulfated tyrosines in gp120 from HIV-1 virions produced in primary human CD4 + T cells.
Gp120 was immunoprecipitated from whole HIV-1 virions produced in primary CD4 + T cells and analyzed by Western blot with a monoclonal antibody specific for sulfated tyrosines; an anti-gp120 antibody (IgG1-b12) was tested in parallel as a control. (C) Detection of sulfated tyrosines in cells expressing either wild-type HIV-1 BaL gp160 or a ΔV2 (Δ164-190) mutant using a vaccinia virus system. The protein was immunoprecipitated from the cell surface and analyzed by Western blot as described for (B). (D) Detection of sulfated tyrosines in cells expressing either wild-type HIV-1 BaL gp160 or phenylalanine-substituted mutants BaL Y173F, BaL Y177F, BaL Y173F/Y177F using a vaccinia virus system. The protein was immunoprecipitated from the cell surface and analyzed by Western blot as described for (B). (E) Interaction energy between sulfated (Tys) or non-sulfated (Tyr) tyrosine 177 and gp120. Nonbonded interaction energy, separated into van der Waals (vdW) and electrostatic terms, was measured in molecular dynamics simulations of 100 nsec.
As expected, the presence of the sulfo-group leads to a large increase in the electrostatic component of the interaction energy. (F to H) Predicted interaction of the V2 α-helix with the conserved base of the V3 loop. Three-dimensional representations of tyrosine-sulfated peptide pV2α-Tys interacting with the base of V3 after docking followed by molecular dynamics analysis. (F) Panoramic view of the interaction between the helix-containing peptides and the base of V3. (G) Top view of H-bond interactions of Tys177 inside the cavity at the base of V3. (H) Sectional view of H-bond interactions of Tys177 inside the cavity.
(I) Two-dimensional LigPlot representation of the interactions between the central region of pV2α-Tys (Tys173 to Tys177) with the base of V3. (J) The specificity of anti-sulfotyrosine mAb 1C-A2 used in Western blot analyses was validated in ELISA tests against a tyrosine-sulfated V2-derived synthetic peptide (pV2α-Tys) and its unsulfated counterpart (pV2α).FIGS. 2L-2O illustrate the different efficiency of V2 tyrosine sulfation in recombinant gp120 produced in epithelial cell lines versus virion-associated gp120 produced by primary CD4 + T cells. (L) Detection of sulfated tyrosines by Western blot in recombinant gp120 produced in CHO cells. (M) Lack of adverse effects of sodium chlorate treatment or TPST2 overexpression on the viability of HeLa and HEK293 cells.
The cell lines were treated with 30 mM sodium chlorate (NaClO 3) or transfected with a plasmid vector expressing TPST2 and cultured for 72 hrs. Cell viability was measured by quantitative counting using timed flow cytometry on a FACSCanto II and is expressed as percent viable cell recovery. (N) Side-by-side comparison of the extent of tyrosine sulfation detected by Western blot in a reference sulfated mAb (412d), in virion-associated gp120 produced by infected primary human CD4 + T cells (V-gp120), and in recombinant gp120 produced in HEK293 cells (R-gp120). Identical amounts of mAb 412d and purified gp120 were loaded on the gels. For V-gp120, the protein was immunoprecipitated directly from the infectious viral stocks. The antibodies used for Western blots and the methods used for relative quantification were the same as in (L). (O) Detection of sulfated tyrosines by Western blot in gp120 purified from HIV-1 virions produced by infected primary human CD4 + T cells as described for (N).FIGS.
3A-3H are a series of graphs illustrating that a tyrosine-sulfated peptide containing the V2 α-helix binds to gp120, competes with an anti-CCR5-binding site antibody for HIV-1 virion binding, and blocks HIV-1 entry, infection and fusion. (A) Effect of tyrosine-sulfated V2-loop (pV2α-Tys) and CCR5 N-terminus (pCCR5-Tys) mimetic peptides on HIV-1 virion capture by mAb 412d, a sulfated mAb directed to the CCR5-binding site at the base of V3. Unsulfated homologous peptides (pV2α and pCCR5) were tested in parallel as controls. Infectious viral stocks from HIV-1 BaL were pre-treated with the peptides (each at 50 μM) and then incubated with immunomagnetic beads armed with mAb 412d; beads armed with mAb 2G12 (directed to a glycan-dependent epitope on the gp120 outer domain) were used in parallel as a control. Asterisks denote significant differences with the untreated control (p6-25) between the half-maximal concentrations of trimer-preferring antibodies (PG9, PG16, CH01) required for binding to the synthetic trimer versus neutralization of the homologous virus.Similar to other post-translational modification like N-linked glycosylation, tyrosine sulfation of gp120 can be remarkably variable in different cells. Indeed, very inefficient gp120 sulfation was found in epithelial cell lines, most likely due to limited expression of tyrosyl sulfotransferases in these cells.
This observation has practical implications because epithelial cells lines such as HEK293 are widely used for the production of recombinant gp120 for both structural and biological studies, as well as for pre-clinical and clinical vaccine trials, and neutralization assays based on pseudotyped HIV-1 particles. The low sulfation efficiency detected in epithelial cell lines further emphasizes the need to utilize in vitro protein expression systems capable of producing gp120 with physiologically relevant post-translational modifications. Importantly, however, it was found that in the most important physiological target cells for HIV-1 infection, such as primary human CD4 + T lymphocytes, gp120 was efficiently sulfated, corroborating the biological relevance of this modification. Furthermore, the altered antigenic profile and neutralization sensitivity induced by modulation of gp120 tyrosine sulfation clearly demonstrates that the sulfated fraction of gp120 is functionally relevant. The results indicate that the sulfotyrosine-bolstered interaction between V2 and V3 represents a critical mechanism whereby HIV-1 constrains gp120 in its energetically unfavorable but antibody-shielded pre-fusion conformation, which facilitates immune evasion. Several considerations point to Tys177, analogous to Tys14 in CCR5, as the most important sulfated tyrosine in V2, while the role of Tys173, corresponding to Tys10 in CCR5, may be less critical.
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In agreement with this model, Tys177 is extremely conserved across all HIV-1 subtypes, whereas Tys173 shows a somewhat greater variability, being conserved in subtypes A, B and C, but often replaced by histidine in subtypes D, E and F. However, a hallmark of HIV-1 is an extraordinary degree of genetic and phenotypic diversity, which is reflected by a broad range of neutralization sensitivities among primary isolates.