WO2021067741A1 - Glycopeptides pour induire une réponse immunitaire et procédés d'utilisation - Google Patents

Glycopeptides pour induire une réponse immunitaire et procédés d'utilisation Download PDF

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WO2021067741A1
WO2021067741A1 PCT/US2020/053996 US2020053996W WO2021067741A1 WO 2021067741 A1 WO2021067741 A1 WO 2021067741A1 US 2020053996 W US2020053996 W US 2020053996W WO 2021067741 A1 WO2021067741 A1 WO 2021067741A1
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glycopeptide
cell
cells
hiv
gpepip
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PCT/US2020/053996
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Fikri Avci
Lina Sun
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University Of Georgia Research Foundation, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • HIV has been a major threat to human health and a protective AIDS vaccine does not exist.
  • Isolation and characterization of protective antibodies from virus infected individuals targeting the viral envelope glycoprotein was a milestone in fighting against this deadly virus. Since then, inducing the production of protective antibodies through vaccination has been a highly promising strategy to halt the spread of the disease.
  • the design of the current generation of vaccines does not maximize stimulation of helper T cells and induce T cell- dependent high-affinity, long-lasting and protective antibody responses against the viral envelope.
  • a glycopeptide includes an amino acid sequence having structural similarity to an amino acid sequence of at least 7 consecutive amino acids of a human HIV-gpl20 protein.
  • An example of a glycopeptide is one that includes an amino acid sequence having structural similarity to the amino acid sequence LDVVPIDNNNTSY (SEQ ID NO: 1), wherein the residue at position 10 of SEQ ID NO: 1 includes a glycan.
  • a glycopeptide having structural similarity to SEQ ID NO: 1 further includes a Lysine at the amino-terminal end of SEQ ID NO: 1, an arginine at the carboxy-terminal end of SEQ ID NO:l, or both lysine and arginine.
  • the glycopeptide includes no greater than 20 amino acids. In one embodiment, the glycopeptide includes a mannose, such as 2, 3, 4 or 5 mannose residues. In one embodiment, the glycopeptide reacts with mannose-specific lectin Concanavalin A. In one embodiment, the glycan does not include one or more of a fucose saccharide or a sialic acid. In one embodiment, the glycan is an N-linked glycosylation. In one embodiment, the glycopeptide is a multimer. In one embodiment, the glycopeptide is a fusion protein including a heterologous amino acid sequence, such as, but not limited to, a linker sequence. In one embodiment, the heterologous amino acid sequence includes a cleavable sequence, such as, but not limited to, an acid-labile sequence or a protease recognition sequence.
  • FIG. 2A-H shows gpl20 glycopeptide epitopes are recognized by CD4+ T cells.
  • B ALB/c mice were immunized with pooled gpl20 gly copeptides (prepared by protease digestion of gpl20). After booster immunization, CD4+ T cells were isolated and stimulated in vitro with either intact gpl20 or PNGase F-treated, deglycosylated gpl20 (DG-gpl20) in the presence of mitomycin C-treated APCs for five days.
  • FIG. 2A, B Flow cytometric analysis of T cell proliferation by CFSE division among CD4+ T cells.
  • FIG. 3C Binding of the GpepIP epitope to MHCII molecule was verified.
  • FIG. 3D Purified MHCII monomers (mouse allele I- Ad) were loaded with an equal amount of the indicated peptides. Peptide-loaded MHCII heterodimers were detected by running of complexes in IEF gel and immunoblotting with mouse MHCII antibody.
  • FIG. 7 shows full MS profile of N-glycans released from GpepIP expressed in 293-F cells.
  • the glycopeptides from 293-F cells were eluted between 20 and 50 min in the LC-MS run, and the deconvoluted MS spectrum was obtained from this time region.
  • Glycan composition of this glycopeptide showed that the recombinant glycopeptide carries more complex-type glycans with heavy fucosylation and sialylation, while high-mannose types were detected as minor components.
  • FIG. 8A-J shows the glycopeptide epitope GpepIP elicits a glycan-specific cellular and humoral immune response.
  • FIG. 8A, B CD4+ T cells obtained from mice immunized with gpl20 were stimulated in vitro with either GpepIP or pepIP, and T cell proliferation was examined by flow cytometry with use of CFSE fluorescence dilution through cell division.
  • FIG. 8C, D CD4+ T cells obtained from mice immunized with GpepIP expressed in GnTI-/- cells (FIG. 8C) or with pepIP (FIG.
  • FIG. 8H-J Serum from mice immunized with GpepIP expressed in GnTI-/- cells were collected 7 days after booster immunization. Titers of IgGl (FIG. 8H), IgG2a (FIG. 81), and IgG3 (FIG. 8J) for recognition of glycosylated gpl20 or deglycosylated gpl20 were measured by ELISA.
  • FIG. 9A-F shows transcriptomic analysis of GpepIP- and pepIP-stimulated CD4+ T cell populations.
  • FIG. 9A Gating strategy of sorting antigen specific CD4+ T cell populations by flow cytometry is shown.
  • FIG. 9B Dendrogram and hierarchical clustering heat map of genes from control, GpepIP and pepIP populations. The blue and red bands indicate low and high gene expression quantity, respectively. The vertical distances between branches of the dendrogram represent the similarity of gene expression profiles between samples. Biological replicates showed the highest degree of correlation followed by GpepIP or pepIP stimulated populations.
  • Heatmap colors represent the log2 fold change values relative to the control.
  • FIG. 9F Volcano plot showing the gene signature of GpepIP compared to pepIP specific CD4+ T cells.
  • X axis represents log2 transformed fold change.
  • Y axis represents -log 10 transformed significance. Red points represent upregulated DEGs. Blue points represent down-regulated DEGs. Gray points represent non- DEGs. Genes associated with Th cell differentiation were labeled and highlighted.
  • FIG. 10A-B shows GO analysis and gene expression signature of GpepIP specific CD4+ T cells.
  • FIG. 10A The most enriched GO of DEGs in GpepIP specific CD4+ T cells compared to control cells was represented. The number of genes of GO in biological process, cellular component and molecular function were shown.
  • FIG. 10B Volcano plot showing the gene signature of GpepIP specific CD4+ T cells compared control. DEGs (greater than twofold; P value ⁇ 0.05) were shown as colored dots. Genes associated with Thl and Th2 signaling, Thl7 signaling and activated T cell co-stimulatory molecules were labeled.
  • FIG. 10A The most enriched GO of DEGs in GpepIP specific CD4+ T cells compared to control cells was represented. The number of genes of GO in biological process, cellular component and molecular function were shown.
  • FIG. 10B Volcano plot showing the gene signature of GpepIP specific CD4+ T cells compared control.
  • FIG. 11 A-E shows cytokine profile of GpepIP and pepIP stimulation.
  • Splenic and lymph node cells isolated from GpepIP or pepIP immunized mice were stimulated with GpepIP or pepIP for 5 days.
  • Th-cell related cytokines in the supernatants from GpepIP (FIG. 11 A) or pepIP (FIG. 1 IB) stimulation compared to no stimulation (medium) were analyzed by a multiplex- based assay. (FIG.
  • FIG. 12A-B shows Th-cell related cytokine analysis of GpepIP- or pepIP-stimulated CD4+ T cells.
  • Splenic and lymph node cells isolated from GpepIP or pepIP immunized mice were stimulated with GpepIP or pepIP for 5 days.
  • Cytokines IFN-g, IL-5 and IL-17A on CD4+ T cells were assessed by intracellular cytokine staining and flow cytometry.
  • FIG. 13A-G shows GpepIP specific CD4+ T cells exhibit high potency on helping humoral immune responses to HIV trimer.
  • FIG. 13A Immunization scheme. BALB/c mice were primed twice by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Three weeks later, all groups were immunized with the clade A BG505 gpl40 NFL trimer emulsified in incomplete Freund’s adjuvant. Sera were collected at the indicated time points. Mice were euthanized 10 days after trimer immunization.
  • FIG. 13B Splenic and lymph node cells were isolated and stimulated with GpepIP or pepIP in vitro for five days.
  • FIG. 13C BG505-specific IgG production was examined in all three groups across the indicated time points by ELISA. Antibody titers were significantly higher in mice primed with GpepIP than in those primed with pepIP or adjuvant alone.
  • FIG. 13D GC response, defined by the percentage of GL7+Fas+ B cells, was evaluated in all three groups on day 10 after trimer immunization by flow cytometry.
  • FIG. f 3E-G Expression levels of activation marker CD69 (FIG. 13E), CD80 (FIG. 13F) and MHCII (FIG. 13G) were detected on splenic B cells after in vitro stimulation with the BG505 trimer for 3 days. MFI, mean fluorescence intensity.
  • FIG. 14A-E shows GpepIP primary immunization prior to trimer immunization elicits functional antibody production.
  • FIG. 14A Immunization scheme. BALB/c mice were primed three times (with a 3-week interval) by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Subsequently, all groups were immunized with the clade A BG505 gpl40 NFL trimer adjuvanted with Alum for three times (with a 3-week interval). Sera were collected 7 days after each trimer immunization (Post 1, 2 and 3). (FIG.
  • Antibody CHOI-31 was used as positive control (shown as antibody concentration).
  • BG505 was chemically labeled with fluorescein isothiocyanate (FITC) and incubated with BMDCs at 37°C for two hours. Cells were then collected and antigen uptake was measured by flow cytometry. To evaluate antisera for their function, fluorophore-labeled BG505 was pre-incubated with antisera used in (FIG. 14E) at different dilutions before adding into BMDCs. The uptake rate is defined as FITC positive cells compared to no BG505 (medium) group.
  • FITC fluorescein isothiocyanate
  • FIG. 15 shows trimer-specific IgG titers.
  • BG505-specific IgG production was examined in antisera from adjuvant, GpepIP and pepIP primed and post the third trimer boost by ELISA. All three groups have equivalent BG505 IgG titers.
  • glycoprotein is inclusive of a glycoprotein, a glycopolypeptide and a proteoglycan.
  • An “isolated” compound, such as a polynucleotide or glycopeptide, is one that has been removed from its natural environment. Polynucleotides and glycopeptides that are produced by recombinant, enzymatic, or chemical techniques are considered to be isolated and purified by definition, since they were never present in a natural environment.
  • a glycopeptide of the present disclosure includes an amino acid sequence that includes a series of consecutive amino acids derived from the amino acid sequence of a human HIV-1 gpl20 protein, is glycosylated with at least one glycosylation site and at least one linked glycan as described herein, and binds a class 2 major histocompatibility complex in such a way that it is presented to a T cell receptor, and/or induces the production of antigen-specific antibodies, and/or induces the production of T cells that are specifically stimulated by the glycopeptide
  • a glycopeptide described herein includes no greater than 20, no greater than 19, no greater than 18, no greater than 17, no greater than 16, no greater than 15, no greater than 14, no greater than 13, no greater than 12, no greater than 11, no greater than 10, no greater than 9, no greater than 8, or no greater than 7consecutive amino acids of a human HIV-1 gpl20 protein.
  • a glycopeptide described herein includes additional amino acids that are normally or naturally found flanking the sequence depicted at, for instance, SEQ ID NO:l, in a natural gpl20 molecule of HIV-1.
  • the natural gpl20 molecule is SEQ ID NO: 10 (encoded by reagent pSyngpl20JR-FL, NIH AIDS Reagent Program).
  • the additional amino acids do not include a naturally occurring full length gpl20 molecule.
  • the additional amino acids can be at the amino terminal end, the carboxy terminal end, or both.
  • a glycopeptide can be isolated from a cell or from an MHC II complex or can be produced using recombinant techniques, or chemically or enzymatically synthesized using routine methods. Methods for determining whether a protein has structural similarity with the amino acid sequence of SEQ ID NO: 1 are described herein.
  • amino acid sequence of a gly copeptide having structural similarity to SEQ ID NO: 1 or another glycopeptide described herein can include conservative substitutions of amino acids present in SEQ ID NO:l or another glycopeptide described herein.
  • a conservative substitution is typically the substitution of one amino acid for another that is a member of the same class.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic such as charge, hydrophobicity, and/or hydrophilicity
  • Whether a glycopeptide is structurally similar to a protein of SEQ ID NO: 1 can be determined by aligning the residues of the two proteins (for example, a candidate protein and any appropriate reference protein described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • a reference protein may be a protein described herein.
  • a reference protein is a protein described at SEQ ID NO: 1 or another glycopeptide described herein.
  • a candidate protein is the protein being compared to the reference protein.
  • a candidate protein can be produced using recombinant techniques, or chemically or enzymatically synthesized.
  • a pair-wise comparison analysis of amino acid sequences can be carried out using the Blastp program of the Blastp suite-2sequences search algorithm, as described by Tatusova et ah, (FEMS Microbiol Lett , 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website.
  • proteins may be compared using other commercially available algorithms, such as the BESTFIT algorithm in the GCG package (version 10.2, Madison WI).
  • structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions.
  • reference to an amino acid sequence disclosed at SEQ ID NO:l or another glycopeptide described herein can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference amino acid sequence.
  • reference to an amino acid sequence disclosed at SEQ ID NO: 1 or another glycopeptide described herein can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least
  • amino acid sequence of an identified SEQ ID NO includes variants having sequence similarity or sequence identity of at least 80%.
  • a glycopeptide having structural similarity to a glycopeptide described herein has immunogenic activity.
  • a glycopeptide having one or more conservative and/or nonconservative substitutions compared to a glycopeptide described herein has immunogenic activity.
  • a glycopeptide described herein can be a fusion protein, where the additional amino acids can be heterologous amino acids.
  • heterologous amino acids refers to amino acids that are not normally or naturally found flanking the sequence depicted at, for instance, SEQ ID NO: 1, in a natural gpl20 molecule of HIV-1.
  • the natural gpl20 molecule is SEQ ID NO: 10 (encoded by reagent pSyngpl20JR-FL, NET AIDS Reagent Program).
  • the additional amino acid sequence may be useful for purification of the fusion protein by affinity chromatography. Various methods are available for the addition of such affinity purification moieties to proteins.
  • a fusion protein is a series of two or more glycopeptides described herein covalently joined together as a multimer.
  • the number of glycopeptides joined together is not limiting and in one embodiment there is no upper number of glycopeptides that can be present in a multimer.
  • the number of multimers can be at least 2, at least 5, at least 10, at least 50, or at least 100.
  • the glycopeptides of a multimer are joined as a fusion protein where the carboxy -terminal end of one glycopeptide is attached to the amino-terminal end of the next one.
  • a multimer includes a spacer, e.g., one or more amino acids between the glycopeptides of a multimer.
  • a spacer is an amino acid sequence that joins protein domains in a fusion protein.
  • a spacer can be flexible or rigid, and in one embodiment is flexible.
  • a spacer can be at least 3, at least 4, at least 5, or at least 6 amino acids in length. It is expected that there is no upper limit on the length of a linker used in a multimer described herein; however, in one embodiment, a spacer is no greater than 10, no greater than 9, no greater than 8, or no greater than 7 amino acids in length.
  • a spacer sequence can be any amino acid sequence, and can have small R groups to reduce steric hindrance.
  • the carbohydrate component of a glycopeptide is commonly referred to as a “glycan.”
  • a glycan may contain one monosaccharide, or it may contain two or more monosaccharides linked by glycosidic bonds.
  • a glycan can include nonrepeating or repeating monosaccharides, or both.
  • glycan is interchangeable with the term saccharide, which includes a monosaccharide, a disaccharide, a trisaccharide, etc.; it can include an oligosaccharide or a polysaccharide.
  • An oligosaccharide is an oligomeric saccharide that contains two or more saccharides.
  • the structure of an oligosaccharide is typically characterized by a particular identity, order, linkage positions (including branch points), and linkage stereochemistry (a, b) of the monomers, and as a result has a defined molecular weight and composition.
  • a glycan can be branched or unbranched.
  • a complex glycan is a glycan that contains at least one branch point.
  • the monosaccharide at the branch point is covalently linked to two other saccharides at carbons other than Cl.
  • a branch point monosaccharide may be linked to other monosaccharides at C4 and C6, in addition to being linked to another monosaccharide or to an amino acid at Cl.
  • a complex glycan may be, without limitation, biantennary, triantennary, or tetraantennary.
  • a glycopeptide described herein includes at least one glycan attached to the protein component.
  • a glycopeptide includes at least 1, at least 2, at least 3, or at least 4 glycans.
  • a glycopeptide includes no greater than 4, no greater than 3, no greater than 2, or no greater than 1 glycans.
  • the glycan is an oligo mannose structure.
  • oligo mannose structure refers to an oligosaccharide that includes mannose residues.
  • An “oligo mannose structure” includes a common structure, referred to as a core, which typically contains two N-acetyl glucosamine residues and three or more mannose residues.
  • An oligo mannose structure includes at least 3, at least 4, at least 5, at least 6, or at least 7 mannose residues.
  • an oligo mannose residue includes no greater than 9, no greater than 8, no greater than 7, no greater than 6, no greater than 5, no greater than 4, or no greater than 3 mannose residues.
  • An oligo mannose structure typically terminates in mannose residues at the non-reducing end of the glycan.
  • One or more glycans of a glycopeptide may contain modifications such as sulfation or phosphorylation.
  • a glycan of a glycopeptide may be a complex type N-glycan or a hybrid N-glycan.
  • a glycopeptide described herein has immunogenic activity (IA).
  • IA includes ability to bind to a MHC II molecule, and/or induce the production of antigen-specific antibodies, and/or induce the production of T cells that are specifically stimulated by the glycopeptide.
  • Whether a protein has IA can be determined by in vitro or in vivo assays.
  • an assay determines glycopeptide binding to MHCII molecules, such a a mouse or a human MHCII molecule, and can be carried out as described in Example 1.
  • a glycopeptide described herein will bind to, and be presented by, MHC II molecules.
  • an assay determines the T cell response to glycopeptides and can be carried out as described in Example 1.
  • T cells from the subject will be stimulated by the gly copeptide to produce interleukin-4 and interferon- gamma.
  • an assay determines the production of antigen-specific antibodies and can be carried out as described in Example 1.
  • the antigen-specific antibodies can react with the gly copeptide used to immunize the subject, or react with a second antigen administered to the subject with the gly copeptide or following immunization of the subject with the glycopeptide.
  • the second antigen is one that shares at least one epitope with the glycopeptide.
  • An example of such a protein is one based on an HIV gpl40, such as a trimer envelope protein.
  • the present disclosure also includes isolated polynucleotides encoding a protein described herein.
  • a polynucleotide encoding a protein described herein can have a nucleotide sequence encoding a protein having the amino acid sequence shown in, e.g., SEQ ID NO: 1 or a glycopeptide disclosed herein, or a protein that is structurally similar.
  • a nucleotide sequence of a polynucleotide encoding a protein described herein can be readily determined by one skilled in the art by reference to the standard genetic code, where different nucleotide triplets (codons) are known to encode a specific amino acid.
  • the class of nucleotide sequences that encode any protein described herein is large as a result of the degeneracy of the genetic code, but it is also finite.
  • a polynucleotide encoding a glycopeptide described herein can be present in a vector.
  • a vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989).
  • a vector may provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector.
  • the term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors.
  • viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors.
  • a vector is capable of replication in a microbial host, for instance, a prokaryotic bacterium, such as E. coli.
  • the vector is a plasmid.
  • Suitable host cells for cloning or expressing the vectors herein include eukaryotic cells. Suitable eukaryotic cells include, but are not limited to, human Embryonic Kidney (HEK) 293 cells and Chinese hamster ovary (CHO) cells. Vectors may be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells.
  • HEK human Embryonic Kidney
  • CHO Chinese hamster ovary
  • Polynucleotides can be produced in vitro or in vivo.
  • methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer.
  • Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known.
  • An expression vector optionally includes regulatory sequences operably linked to the coding region.
  • the disclosure is not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region.
  • the promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to the host cell.
  • a vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium.
  • a marker sequence may render the transformed cell resistant to an antibiotic, or it may confer compound-specific metabolism on the transformed cell.
  • Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, and neomycin.
  • Proteins described herein may be produced using recombinant DNA techniques, such as an expression vector present in a cell (e.g., a genetically modified cell described herein). Such methods are routine and known in the art.
  • the present disclosure also includes genetically modified cells that have an exogenous polynucleotide encoding a glycosylated protein described herein.
  • genetically modified cell refers to a cell into which has been introduced an exogenous polynucleotide.
  • a cell is a genetically modified cell by virtue of introduction into a suitable cell of an exogenous polynucleotide.
  • exogenous refers to a compound, such as a polynucleotide or glycopeptide, that is not normally or naturally found in a specific cell.
  • a genetically modified cell can exhibit production of a glycopeptide described herein.
  • a polynucleotide encoding a glycopeptide can be present in the organism as a vector or integrated into a chromosome.
  • a genetically engineered cell can stably express a glycopeptide, or the expression can be transient.
  • Examples of cells include, for instance, prokaryotic (e.g., microbial) and eukaryotic.
  • eukaryotic cells include yeast, insect, and animal cells.
  • Examples of animal cells include vertebrate cells, such as mammalian cells.
  • An example of a mammalian cell includes, but is not limited to, HEK293 N-acetylglucosaminyltransferase I (GnTI)-deficient cells.
  • An animal cell can be an in vitro cell (e.g., a cell that is capable of long term culture in tissue culture medium), or an ex vivo cell (e.g., a cell that has been removed from the body of a subject and capable of limited growth in tissue culture medium).
  • a genetically modified cell useful herein is able to glycosylate proteins to include oligomannose N-glycans but prevent the production of complex N-glycans.
  • a suitable cell is one that includes a mutation in N- acetylglucosaminyltransferase I (GnTI), an enzyme used for the conversion of oligomannose N- glycans to complex N-glycans.
  • GnTI N- acetylglucosaminyltransferase I
  • An example of a cell having a mutation in N- acetylglucosaminyltransferase I (GnTI-/-) is HEK293S GnTT, available from the ATCC® (Manassas, VA) as CRL-3022TM.
  • Kifunensine a mannosidase inhibitor of the ER and Golgi mannosidase I, can be added during expression in 293-F cells to produce glycopeptides.
  • the glycan or gly cans attached to the glycopeptide - whether the glycopeptide is a monomer or multimer - of a composition is the same (e.g., the oligosaccharide is M5N2), and in another embodiment the oligosaccharides attached vary (e.g., the oligosaccharides can be M5N2 and M4N2 or other glycopeptides as endolysososomal processing products ).
  • a composition can include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Solutions or suspensions can include the following components: a sterile diluent such as water for administration, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; electrolytes, such as sodium ion, chloride ion, potassium ion, calcium ion, and magnesium ion, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, phosphate buffered saline (PBS), and the like.
  • PBS phosphate buffered saline
  • a composition is typically sterile and, when suitable for injectable use, should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • Sterile solutions can be prepared by incorporating the active compound (e.g., a glycopeptide described herein) in the required amount in an appropriate solvent with one or a combination of ingredients routinely used in pharmaceutical compositions, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and any other appropriate ingredients.
  • preferred methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterilized solution thereof.
  • the active compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and/or fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the active compounds may be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants.
  • a controlled release formulation including implants.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • a composition can also include another compound useful in eliciting an immune response.
  • a useful compound is one the elicits an immune response to HIV.
  • One such compound includes a stabilized envelope trimer. Stabilized envelope trimers are available as immunogens (Sanders, et al., 2013, PLoS pathogens, 9, el 003618, doi:10.1371/journal.ppat.l003618).
  • Toxicity and therapeutic efficacy of the active compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Recombinant glycopeptides exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration used.
  • the therapeutically effective dose can be estimated initially from animal models.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of signs of disease). Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured using routine methods.
  • a composition is administered in an amount sufficient to provide an immunological response to a glycopeptide described herein.
  • the amount of glycopeptide present in a composition can vary.
  • the dosage of glycopeptide can be between 0.01 micrograms (pg) and 3000 milligrams (mg), typically between 10 pg and 2000 pg.
  • the glycopeptide can be present in the composition in an amount such that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0-3.0 ml.
  • the compositions can be administered one or more times per day to one or more times per week, including once every other day.
  • treatment of a subject with an effective amount of an active compound can include a single treatment or, preferably, can include a series of treatments. Such factors can be determined by one skilled in the art.
  • a composition can include a biological response modifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha, IFN-gamma, and other cytokines that effect immune cells.
  • a composition can include an inhibitor of degradation of the glycopeptide.
  • a method is for making glycopeptide described herein.
  • the method includes incubating a genetically modified cell under suitable conditions for expression of a glycopeptide.
  • the cell can be, but is not limited to, a genetically modified cell that includes an exogenous coding region encoding the protein component of the glycopeptide and includes the cellular machinery to add the carbohydrate component and optionally process the carbohydrate component.
  • a genetically modified cell can be used to produce a glycopeptide monmer.
  • a genetically modified cell can be used to make a multimer that is then cleaved into shorter glycopeptides.
  • a method includes administering to a subject an effective amount of a composition including a glycopeptide described herein.
  • an “effective amount” of a composition including a glycopeptide described herein is the amount able to elicit the desired response in the recipient.
  • the subject can be, for instance, murine (e.g., a mouse or rat), or a primate, such as a human.
  • a method includes inducing an immune response to the glycopeptide by administering to a subject an effective amount of a composition including a glycopeptide described herein.
  • an “effective amount” is an amount effective to result in the production of an immune response in the subject.
  • the immune response can be humoral, cell-based, or a combination thereof.
  • a humoral immune response includes the production of antibodies that are antigen-specific and bind the glycopeptide used to induce the immune response. Methods for determining whether a subject has produced antibodies that specifically bind a glycopeptide described herein can be determined using routine methods.
  • a cell-based response includes the production of T cells that produce interleukin-4 and/or interferon-gamma after stimulation by the glycopeptide used to induce the immune response.
  • an antibody that can “specifically bind” a protein is an antibody that interacts with the epitope of the glycopeptide that induced the synthesis of the antibody or interacts with a structurally related epitope. It is expected that some of the epitopes present a glycopeptide described herein are epitopes that are conserved in glycopeptides of different clades of HIV.
  • a subject “at risk” of infection by a virus is a subject that is a member of a population at increased risk of being exposed to the virus. Accordingly, administration of a composition can be performed before, during, or after the subject has first contact with the virus. Treatment initiated after the subject’s first contact with the virus may result in decreasing the severity of symptoms and/or clinical signs of infection by the virus, completely removing the virus, and/or decreasing the likelihood of experiencing a clinically evident infection.
  • the term “symptom” refers to subjective evidence of a disease or condition experienced by a subject and caused by infection by HIV.
  • the term “clinical sign” or, simply, “sign” refers to objective evidence of disease or condition caused by infection by HIV. Symptoms and/or clinical signs associated with conditions referred to herein and the evaluations of such symptoms are routine and known in the art.
  • the method includes administering an effective amount of a composition described herein to a subject having, or at risk of having, an HIV infection.
  • whether the viral load has decreased is determined.
  • an “effective amount” is an amount effective to reduce the viral load in a subject, or reduce the likelihood that the subject experiences a clinically-evident infection.
  • a method in another embodiment, includes treating one or more symptoms or clinical signs of certain conditions in a subject that may be caused by infection by HIV.
  • the method includes administering an effective amount of a composition described herein to a subject having or at risk of having a condition, or exhibiting symptoms and/or clinical signs of a condition, and determining whether at least one symptom and/or clinical sign of the condition is changed, preferably, reduced.
  • a method of the present disclosure can further include additional administrations (e.g., one or more booster administrations) of the composition to the subject to enhance or stimulate a secondary immune response.
  • a booster can be administered at a time after the first administration, for instance, one to eight weeks, such as two to four weeks, after the first administration of the composition. Subsequent boosters can be administered one, two, three, four, or more times annually.
  • one or more other antigen can be administered.
  • suitable secondary antigens include, but are not limited to, other proteins and/or glycopeptides that are produced during an HIV infection, and proteins and/or glycopeptides that include epitopes present in the glycopeptide administered to result in an immune response. Examples include, but are not limited to, a protein and/or glycopeptide based on an HIV gpl40, such as a trimer envelope protein.
  • the secondary antigen is administered at the same time as a glycopeptide described herein, in the same composition or separately.
  • the secondary antigen is administered after the glycopeptide is administered.
  • the secondary antigen can be administered before, at the same time as, or after a booster of the glycopeptide is administered.
  • HIV targets the CD4+ population of T cells in a subject, and infection typically results in the subject being unable to mount a cell mediated immune response to foreign antigens.
  • a glycopeptide of the present disclosure is administered after the subject has received therapy to increase the CD4+ population of T cells.
  • Therapeutic agents and regimes that can be used to increase the CD4+ population of T cells in a subject infected with HIV are known to the skilled person.
  • kits are provided.
  • a kit is for using a glycopeptide described herein, such as using a glycopeptide to induce an immune response, or treat an infection, condition, symptom or sign.
  • a kit is for making a glycopeptide described herein.
  • the kit includes at least one of the glycopeptides described herein (e.g., one, at least two, at least three, etc.) or a multimer of at least one of the glycopeptides, or a genetically engineered cell that can express a glycopeptide described herein in a suitable packaging material in an amount sufficient for at least one assay or use.
  • suitable packaging material e.g., one, at least two, at least three, etc.
  • other reagents such as buffers and solutions are also included.
  • Instructions for use of the packaged glycopepide or cell are also typically included.
  • packaging material refers to one or more physical structures used to house the contents of the kit.
  • the packaging material is constructed by routine methods, generally to provide a sterile, contaminant-free environment.
  • the packaging material may have a label which indicates how a glycopeptide described herein can be used.
  • the packaging material can contain instructions indicating how the materials within the kit are employed to administer a glycopeptide to an animal.
  • the term “package” refers to a container such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the proteins, and other reagents, for instance a secondary antibody.
  • “Instructions for use” typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
  • Embodiment 1 An isolated glycopeptide comprising an amino acid sequence having structural similarity to the amino acid sequence LDVVPIDNNNTSY (SEQ ID NO:l), wherein the residue at position 10 of SEQ ID NO:l comprises a glycan.
  • Embodiment 2 An isolated glycopeptide comprising an amino acid sequence having structural similarity to an amino acid sequence of at least 7 consecutive amino acids of a human HIV-gpl20 protein.
  • Embodiment 4 The glycopeptide of any of embodiments 1-3 wherein the glycan comprises a mannose.
  • Embodiment 5 The glycopeptide of any of embodiments 1-4 wherein the glycan comprises 2, 3, 4 or 5 mannose residues.
  • Embodiment 6. The gly copeptide of any of embodiments 1-5 wherein the gly copeptide reacts with mannose-specific lectin Concanavalin A.
  • Embodiment 8 The glycopeptide of any of embodiments 1-7 wherein the glycan is an N- linked glycosylation.
  • Embodiment 9 The glycopeptide of any of embodiments 1-8 wherein the glycopeptide is a multimer.
  • Embodiment 10 The glycopeptide of any of embodiments 1-9 wherein the glycopeptide is a fusion protein comprising a heterologous amino acid sequence.
  • Embodiment 11 The glycopeptide of any of embodiments 1-10 wherein the heterologous amino acid sequence comprises a linker sequence.
  • Embodiment 12 The glycopeptide of any of embodiments 1-11 wherein the heterologous amino acid sequence comprises a cleavable sequence.
  • Embodiment 13 The glycopeptide of any of embodiments 1-12 wherein the cleavable sequence comprises an acid-labile sequence or a protease recognition sequence.
  • Embodiment 14 The glycopeptide of any of embodiments 1-13, the glycopeptide further comprising a lysine at the amino-terminal end of SEQ ID NO: 1.
  • Embodiment 15 The glycopeptide of any of embodiments 1-14, the glycopeptide further comprising an arginine at the amino-terminal end of SEQ ID NO: 1.
  • Embodiment 16 A composition comprising the glycopeptide any of embodiments 1-15.
  • Embodiment 17 The composition of embodiment 16 further comprising a pharmaceutically acceptable carrier.
  • Embodiment 18 The composition of any of embodiments 16-17 further comprising an adjuvant.
  • Embodiment 19 A genetically engineered cell comprising an exogenous polynucleotide comprising a coding region encoding the protein component of the glycopeptide of any of embodiments 1-18.
  • Embodiment 20 The genetically engineered cell of embodiment 19 wherein the coding region is expressed and the protein is processed to comprise a glycan.
  • Embodiment 21 The cell of any of embodiments 19-20 wherein the cell comprises a N- acetylglucosaminyltransferase I mutation (GnTI-/-).
  • Embodiment 22 The cell of any of embodiments 19-21 wherein the cell stably expresses the glycopeptide.
  • Embodiment 28 The method of any of embodiments 25-27 wherein the subject has or is at risk of having an infection caused by HIV.
  • Embodiment 29 A method for treating an infection in a subject, the method comprising: administering an effective amount of the composition of any of embodiments 16-18 to a subject having or at risk of having an infection caused by HIV.
  • Embodiment 30 A method for treating a symptom or a sign in a subject, the method comprising: administering an effective amount of the composition of any of embodiments 16-18 to a subject having or at risk of having an infection caused by HIV.
  • Embodiment 31 A method for treating a condition in a subject, the method comprising: administering an effective amount of the composition of any of embodiments 16-18 to a subject having or at risk of having a condition caused by HIV.
  • Embodiment 32 The method of any one of embodiments any of embodiments 25-31 wherein the HIV is a member of clade A.
  • Embodiment 33 The method of any one of embodiments any of embodiments 25-31 wherein the HIV is a member of clade B.
  • Embodiment 34 The method of any one of embodiments 25-33 further comprising a booster administration.
  • Embodiment 35 The method of embodiment 34 wherein the booster comprises an envelope trimer.
  • Gly copeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help
  • Glycopeptide-induced CD4+ T cell response prior to Env trimer immunization elicits neutralizing antibody development and production of antibodies facilitating uptake of immunogens by antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • the identification of gpl20 glycopeptide-induced, T cell-specific immune responses offers a foundation for developing future knowledge-based vaccines that elicit strong and long-lasting protective immune responses against HIV infection. This Example is also available as Sun et ak, 2020, Nature Communications, 11:2550 (doi: 10.1038/s41467-020- 16319-0).
  • GO Gene Ontology
  • DEGs differentially expressed genes
  • Fig. 10A Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis of DEGs identified the enriched pathways were highly associated with immune functions, such as pathways in cancer, inflammatory bowel disease (IBD), hematopoietic cell lineage, cytokine-cytokine receptor interaction and leishmaniasis.
  • IBD inflammatory bowel disease
  • hematopoietic cell lineage cytokine-cytokine receptor interaction
  • leishmaniasis leishmaniasis
  • transcriptomes of pepIP Comparing transcriptomes of pepIP to control T cells, 2620 genes were differentially expressed with 1950 genes upregulated and 670 genes downregulated (Fig. 9C middle and data not shown). KEGG pathways analysis of DEGs of pepIP-specific CD4+ T cells also exhibited an enrichment of Thl, Th2 and Thl7 cell differentiation (Fig. 9 D middle). However, when comparing transcriptomes between GpepIP and pepIP specific CD4+ T cells, only 620 genes were differentially expressed (615 up-regulated and 205 downregulated) (Fig. 9C right and data not shown). Notably, IL-17 signaling pathway was favorably enriched in GpepIP induced CD4+ T cells (Fig. 9D right).
  • BG505-specific IgG titers were observed in mice primed with GpepIP than with adjuvant or pepIP (P ⁇ 0.0001) (Fig. 13C).
  • the help from pepIP priming was different from adjuvant priming only at the experimental endpoint (Fig. 13C).
  • Env trimer specific B-cell responses were enhanced in GpepIP -primed mice as well.
  • GpepIP primary immunization induced superior germinal center (GC) response defined by a significantly increased percentage of GL7+Fas+ B cells (Fig. 13D).
  • gpl20 glycan moieties survive the antigen processing, yielding glycopeptide epitopes presented by MHCII to CD4+ T cells. Therefore, gpl20 glycan shield will significantly skew CD4+ T cell epitope “hot spots”. Thus, it is plausible to evaluate the importance of gpl20 glycopeptidespecific CD4+ T cells. Additionally, the view that carbohydrates serve directly as nonconventional epitopes to induce CD4+ T cell responses have received increasing appreciation (21, 23, 26, 52). Here, we isolated and characterized a MHCII-presented glycopeptide epitope (GpepIP) eliciting T cell-mediated humoral immune responses to the HIV envelope glycoprotein.
  • GpepIP MHCII-presented glycopeptide epitope
  • RNA-seq analysis and Th-associated cytokine profiles demonstrate that a peptide epitope (pepIP) induces a Thl dominant immune response, whereas recognition of a glycopeptide epitope (GpepIP) by CD4+ T cells drives the induction of Th differentiation towards Th2 and Thl7 features. Further investigation is needed to elucidate the mechanisms underlying regulation of CD4+ T cell differentiation and function by their glycan recognition.
  • mice Eight-week-old female BALB/c mice were obtained from Taconic Bio-sciences (Hudson, NY) and housed in the Coverdell Rodent Vivarium at the University of Georgia. Mice were kept in microisolator cages and handled under a laminar flow hood. All mouse experiments were conducted in compliance with the University of Georgia Institutional Animal Care and Use Committee under an approved animal use protocol.
  • the codon-optimized pSyn gpl20 plasmid encoding JR-FL (clade B) gpl20 was obtained from Dr. Eun-Chung Park and Dr. Brian Seed through the NIH AIDS Reagent Program, Division of AIDS, NTATD, NIH (60, 61).
  • the HIV-1 BG505 gpl40 NFL expression vector was obtained from Dr. Richard T. Wyatt through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (30).
  • gpl20 and BG505 trimer were expressed in a serum-free medium by transient transfection of wild-type FreeStyleTM 293-F suspension cells (Life Technologies); gpl20 and BG505 trimer were purified from supernatant by affinity chromatography using Galanthus nivalis lectin- agarose (Vector Labs, Burlingame, CA) and then further purified on a Superdex S200 size exclusion column (Bio-Rad) to isolate gpl20 and trimer fraction as described previously (24,
  • gpl20 was also expressed and purified from HEK293S GnTI-/- cells (ATCC).
  • gpl20 was digested by sequencing-grade Endoproteinase Glu-C (Promega) in ammonium bicarbonate buffer. Digested products were separated on a Superdex Peptide 10/300 GL column (GE Healthcare) in order to selectively isolate glycopeptides from non-glycosylated peptides. Glycopeptide-containing fractions were identified and pooled together on the basis of biotinylated ConA (Vector Labs) reactivity in a lectin dot blot. Pooled glycopeptides were desalted on a HiPrep 26/10 Desalting column (GE Healthcare) and lyophilized overnight.
  • the gpl20 GpepIP expression vector was generated by cloning of the synthetic nucleotide sequence (GENEWIZ) that codes for the signal sequence-6xHistidine-GpepIP peptide into the pGEn2 restriction-site mammalian expression vector (provided by Dr. Kelley Moremen, University of Georgia) via Notl and BamHI restriction sites. GpepIP expression vector containing a His tag was generated. Recombinant GpepIP was expressed in transiently transfected 293-F cells or HEK293S GnTI-/- cells (ATCC) as described previously (24).
  • mice were primed three times (with a 3-week interval) by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Three weeks later, all groups were boosted by intraperitoneal injection of 20ug of the clade A BG505 gpl40 NFL trimer adjuvanted with 2% alhydrogel (Invivogen) for three times (with a 3-week interval). Sera were collected 7 days after each trimer boost. T cell proliferation, B cell activation and flow cytometry
  • CD4+ T cells were isolated from lymph nodes of mice three weeks after booster immunization with a mouse CD4 T lymphocyte enrichment set (BD Biosciences) according to the manufacturer’s protocol.
  • CD4+ T cells were stimulated in vitro in the presence of mitomycin C-treated (25 pg/ml, Sigma-Aldrich) splenic mononuclear cells pulsed with 10 pg/ml of indicated antigens (20 pg/ml for glycopeptide and peptide) for 5 days. In some experiments, total splenic and lymph node cells were used.
  • CD4+ T cells were incubated with 2 mM CFSE solution (Sigma-Aldrich) at 37 °C for 8 minutes before stimulation. CFSE dilution was measured by flow cytometry as an indication of the T cell proliferation rate.
  • splenic and lymph node cells were isolated from GpepIP and pepIP immunized mice and stimulated in vitro with GpepIP and pepIP respectively for three or five days. GolgiPlug (BD Biosciences) was added at the recommended concentration in the last 6 hours of stimulation. Cells were collected and stained for surface marker CD4 (Biolegend).
  • Cytokine production resulting from T cell stimulation was measured by ELISA.
  • 96-well plates (Costar) were coated overnight with antibody to IFN-g or IL-4 (1 : 1000 dilution; Biolegend) and blocked with 1% BSA/PBS. Plates were washed with 0.05% PBS-Tween and incubated with cell supernatants for 2 hours at room temperature. After washing, biotinylated detection antibodies to IFN-g or IL-4 (1 : 1000 dilution; Biolegend) were added for 2 hours at room temperature, after which HRP-conjugated Avidin (1 : 1000 dilution; Biolegend) was added for 1 hour at room temperature.
  • BMDCs were generated after GM-CSF (PeproTech) induction for 8 days as described previously (24) and were incubated with gpl20 protein (250 pg/ml) at 37°C for 18 hours.
  • Cell lysate was prepared with the following lysis buffer (pH 8.0): 20 mM Tris-HCl, 137 mM NaCl, 1% Nonidet P-40 (NP-40), and 2mM EDTA with a protease inhibitor cocktail (AEBSF, Aprotinin, Bestatin, E-64, EDTA, Leupeptin) (Sigma-Aldrich).
  • AEBSF protease inhibitor cocktail
  • the protein concentration was measured by bicinchoninic acid analysis (Pierce).
  • Lysate was pre-cleared by incubation with isotype antibody and bead slurry at 4°C for 1 hour. Affinity purification of MHCII molecules was performed as described previously (65). In brief, MHCII molecules from cleared lysate were immunoprecipitated by incubation with purified anti-mouse I-A/I-E (20 pg/mg of lysate) (Biolegend) at 4°C overnight; protein G agarose bead slurry (200 m ⁇ ) was then added (Invitrogen), with incubation for 5 hours at 4°C.
  • MHII molecules were eluted by addition of 1 ml of 10% acetic acid and incubation at room temperature for 4 min with rotation.
  • MHCII-peptide complexes were boiled at 70°C for 10 minutes.
  • Peptides and glycopeptides were separated from MHCII by ultrafiltration through a 30 kDa-cutoff membrane filter (Sigma-Aldrich).
  • the eluted MHCII-bound peptides and glycopeptides were subjected to LC-MS/MS analysis in order to assess peptide identity and glycan heterogeneity, as described below.
  • LC-MS analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with an EASY nanospray source and an Ultimate3000 autosampler LC system (Thermo Fisher Scientific). Sample separation was performed on a nano-C18 column (Acclaim pepMap RSLC, 75 pm c 150 mm; C18, 2 pm) via an 80-minute gradient of increasing mobile phase B (80% acetonitrile, 0.1% formic acid in distilled H20) at a flow rate of -300 nl/min into the mass spectrometer.
  • the minimal and maximal peptide lengths were set at 5 and 30, respectively, with unspecific search (no-enzyme group). Differential modifications of 57.02146 Da, 15.9949 Da, and 2.98826 Da were allowed for alkylated cysteine, oxidated methionines, and 18 0-labeled aspartic acid, respectively.
  • the search was performed against the pSyn gpl20 sequence. Any peptide identified from the preliminary software search with a false discovery rate (FDR) >1 % was filtered out. The filtered peptides were further validated manually.
  • glycopeptides expressed in 293-F cells were digested with trypsin because of the complexity of the glycosylation, whereas the glycopeptides expressed in GnTI-/- cells were profiled without tryptic digestion.
  • An Orbitrap-Fusion Lumos Tribrid mass spectrometer equipped with an EASY nanospray source and an Ultimate 3000 autosampler LC system was used. LC separation was performed on a nano-C18 column with use of a water/acetonitrile gradient with formic acid.
  • Protein bands were visualized by the addition of IRDye secondary antibodies (LI-COR Biosciences), incubation at room temperature for 1 hour, and scanning with the Odyssey CLx Imaging System (LICOR Biosciences). For MS analysis, gel was visualized by Coomassie staining (Bio-Rad). Corresponding bands from IEF gel were excised into smaller pieces, destained, and in-gel tryptic digested. The extracts were purified by passage through a Cl 8-spin column (Nest group) and profiled by LC-MS for peptide/glycopeptide identification as mentioned above. RNA-seq analysis.
  • Neutralizing activity of antisera against tier 1 and tier 2 HIV-1 viruses was determined using a luciferase-based TZM-bl cell assay as described elsewhere 33. Antisera from adjuvant, GpepIP and pepIP primed and three BG505 boosts were tested against Murine leukemia virus (MLV)-pseudotyped virus, tier 1 A MN.3, tier IB JR-FL and tier 2 BG505/T332N pseudoviruses produced in 293T cells. Neutralization titers (50% inhibitory dose, ID50) were calculated as the serum dilution at which relative luminescence units (RLUs) were reduced 50% compared to virus control wells (no test sample). MLV-pseudotyped virus was used as negative control for non-HIV-specific inhibitory activity in the assay. Monoclonal antibody CHOI-31 was used as positive control (shown as antibody concentration).
  • MLV Murine leukemia virus
  • BMDC induction and trimer uptake BMDCs were generated from bone marrow as described previously 24. Briefly, bone marrow was flushed out from the tibiae and femurs of 6-8-week-old female BALB/c mice (Taconic Biosciences). Macrophages were removed by incubating bone marrow cells at 37°C for two hours. The suspension cells were cultured in BMDC induction media (RPM1 1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml stereptomycin, 1% (v/v) MEM non-essential amino acids, 1 mM sodium pyruvate, 2mM LGlutamine (Thermo
  • BMDCs were incubated with 20ug/ml FITC-labeled BG505 with or without pre-incubation with indicated antisera at 37°C for 2 hours. Cells were collected and washed with cold PBS before staining for surface CD1 lc using anti -mouse CD1 lc antibody (Biolegend, clone N418). Fluorescent signal was detected using flow cytometry'.
  • Virus envelope protein of HTLV-III represents major target antigen for antibodies in AIDS patients. Science 228, 1094-1096 (1985).
  • van Montfort, T. et al. HIV-1 N-glycan composition governs a balance between dendritic cell-mediated viral transmission and antigen presentation. J. Immunol 187, 4676-4685 (2011). 51. Li, H. et al. Proximal glycans outside of the epitopes regulate the presentation of HIV-1 envelope gpl20 helper epitopes. J. Immunol 182, 6369-6378 (2009).

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Abstract

La présente invention concerne un répertoire de lymphocytes T CD4+ qui reconnaît un épitope de glycopeptide sur Gp120 présenté par une voie MHCII. La présente invention concerne des glycopeptides gp120 qui sont capables d'induire des réponses immunitaires humorales et cellulaires spécifiques. L'invention concerne également des compositions, des cellules génétiquement modifiées codant pour les glycopeptides, et des procédés d'utilisation des glycopepitdes.
PCT/US2020/053996 2019-10-02 2020-10-02 Glycopeptides pour induire une réponse immunitaire et procédés d'utilisation WO2021067741A1 (fr)

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