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
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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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