Stable peptide mimetics of the HIV-1 GP41 pre-hairpin intermediate

A technology of peptidomimetic, bromide, applied in the field of trivalent gp41 peptidomimetic and its use as an immunogen to elicit neutralizing antibodies against HIV

Inactive Publication Date: 2014-12-10
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although effective treatments for AIDS are available, the development of effective prophylactic vaccines for preventing HIV-1 infection has been hampered by the inability to identify and optimize immunogens capable of inducing broadly neutralizing antibodies to prevent viral entry

Method used

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  • Stable peptide mimetics of the HIV-1 GP41 pre-hairpin intermediate
  • Stable peptide mimetics of the HIV-1 GP41 pre-hairpin intermediate
  • Stable peptide mimetics of the HIV-1 GP41 pre-hairpin intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Immunogen production and characterization

[0178] Immunogen Production: Synthetic Peptides

[0179] 1. (CCIZN36) 3

[0180] Peptide monomer CCIZN36

[0181] Synthesized on an automated peptide synthesizer using solid-phase Fmoc / t-Bu chemistry. The resin used was H-Rink Amide ChemMatrix (Matrix-Innovation Inc., St. Hubert, Quebec, Canada). Acylation was carried out using double coupling for 30 minutes, using a 5-10 fold excess of amino acid over the free amino groups of the resin per cycle. With an equimolar amount of HATU [2-(1H-9-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-urea (aminum) hexafluorophosphate] and 2 times A molar excess of DIEA (N,N-diisopropylethylamine) activates the amino acid. The side chain protecting groups used are as follows: trityl for cysteine, glutamine, asparagine, and histidine; tert-butoxycarbonyl for lysine and tryptophan; tert-butyl for for glutamic acid, threonine and serine; and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-su...

Embodiment 2

[0254] Serology

[0255] 1. ELISA

[0256] Serum endpoint dilutions were determined by testing immune serum samples against biotinylated peptides added directly to streptavidin-coated 96-well plates (Thermo Fisher Scientific, Inc., Pittsburg, PA). Biotinylated peptides were coated at a concentration of 4 μg / ml / well in PBS overnight at 4°C. Plates were washed six times with PBS containing 0.05% Tween-20 (PBST) and blocked with PBST containing 3% (v / v) nonfat dry milk (PBST-milk). Test samples, pre-immune and immune samples were diluted, starting at 1:100, and serially diluted 4-fold, eight times in a final volume of 100 μl / well. Plates were incubated for 2 hours at room temperature, followed by six washes with PBST. Fifty microliters of HRP-conjugated goat anti-guinea pig (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or goat anti-human (Invitrogen) secondary antibodies were diluted 1:5000 or 1:2000 in PBST-milk, respectively, and Add to each well and i...

Embodiment 3

[0267] HIV 350 and 365: immunogenicity in guinea pigs

[0268] Duncan-Hartley guinea pigs (HIV-350, n=8 / group*) were immunized three times intramuscularly with 100 micrograms of the peptide immunogen at 0, 4 and 8 weeks. Peptides reconstituted at neutral pH in 20 mM Hepes buffer were formulated into 180 μg aluminum hydroxyphosphate sulfate (Merck & Co., Inc.) plus 40 μg Iscomatrix Adjuvant™ (CSL, Inc.) per dose. Serum samples were collected via whole blood in serum separator tubes for each animal at 7 and 1 week, as well as several serum collections prior to the first immunization (pre-bleed).

[0269] Studies of HIV-350 have tested the peptide construct SZN51. Table 3a The immunization schedule used in the study for this group is shown.

[0270] Duncan-Hartley guinea pigs (HIV-365, n=6 / group) were immunized three times intramuscularly with 30 μg of peptide immunogen at 0, 4 and 8 weeks. Peptides reconstituted at neutral pH in 20 mM HEPES buffer were formulated into 180 ...

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Abstract

The present invention relates to a gp41 trivalent peptide mimetic having three gp41 N-peptides on a chemical scaffold which conformationally constrains the N-peptides into a trimeric coiled-coil to mimic gp41 presentation. The present invention also relates to N-peptides having the entire HIV gp41 NH2-terminal heptad repeat region and which are capable of forming gp41 peptide mimetics. Such peptide mimetics of HIV-1 gp41 pre-hairpin intermediates can be utilized in a vaccine for the treatment or prevention of HIV-1 infection through eliciting neutralizing antibodies.

Description

Area of invention [0001] The invention involves a limited tertiary GP41 peptide simulation and it is used as a neutral antibody that caused HIV to cause HIV.The invention also involves the entire HIV GP41 NH 2 The end seven -peptide duplicate area or its modification form, and the N peptide of the GP41 peptide simulation can be formed. Invention background [0002] Human immune defect virus (HIV) is a pathogenic factor in obtaining sexual immune defect syndrome (AIDS) and related diseases.Although the effective treatment of AIDS is obtained, the development of effective prevention of vaccine for HIV-1 infection has been hindered by failing to identify and optimize immunogenic originals.Essence [0003] A considerable amount of research has been conducted to evaluate HIV-1 envelope glycoprotein as the purpose of immunogenicism.HIV-1 envelope glycoprotein is synthesized as a 160-kDA foretard, and it is cut into 120 KDA receptors (GP120) and 41 KDA membrane anchor sub-tadbs (GP41)....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/21C12N7/00G01N33/53A61K38/00
CPCA61K2039/64C12N2740/16134A61K38/16A61K39/12C12N7/00A61K38/00A61K2039/55505A61K2039/55577A61K2039/70A61P31/18A61P37/04A61K39/21C07K14/005C12N2740/16122
Inventor J.G.乔伊斯C.吴E.A.奥蒂格尔V.加斯基
Owner MERCK & CO INC
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