Griffithsin mutants
Inventors
O'Keefe, Barry R. • Moulaei, Tinoush • Palmer, Kenneth E. • Rohan, Lisa C. • Fuqua, Joshua L. • Kramzer, Lindsay F.
Assignees
University of Louisville Research Foundation ULRF • University of Pittsburgh • US Department of Health and Human Services
Publication Number
US-10501507-B2
Publication Date
2019-12-10
Expiration Date
2036-02-10
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Abstract
The invention provides modified griffithsin polypeptides comprising the amino acid sequence of SEQ ID NO: 1, as well as corresponding nucleic acids, vectors, cells, fusion proteins, constructs, conjugates, and methods of inhibiting viral infection.
Core Innovation
The invention provides modified griffithsin polypeptides with the amino acid sequence of SEQ ID NO: 1 comprising specific mutations at defined positions. These modifications avoid the amino acid sequence of wild-type griffithsin (SEQ ID NO: 2) and include variants where particular amino acid residues (X1, X2, X3, X4, X5, X6, and X7) can be substituted with certain amino acids. The invention also covers corresponding nucleic acids, vectors, cells, fusion proteins, constructs, conjugates, and methods of inhibiting viral infection, particularly HIV.
The problem being addressed is the need for additional griffithsin compounds with improved properties. Wild-type griffithsin has known antiviral activity but faces issues such as methionine oxidation, limited shelf-life, solubility problems, and suboptimal bioavailability across various physiological pH ranges. The invention modifies griffithsin to reduce methionine oxidation, increase shelf-life, improve solubility, and enhance bioavailability in diverse pH compartments.
The modified griffithsin polypeptides include mutations, for example at position 78, replacing methionine with amino acids lacking sulfur or charge to prevent oxidation. Further mutations at positions 61, 75, 106, 107, 116, and 119 modulate isoelectric point and solubility, facilitating better product properties such as stability and bioavailability in tissues with differing pH values. Importantly, these mutations preserve dimer formation of griffithsin, maintaining antiviral potency.
Claims Coverage
The patent includes one independent claim which covers a modified griffithsin polypeptide with defined amino acid substitutions and related entities. The claims disclose inventive features related to polypeptide composition, conjugates, nucleic acids, vectors, cells, compositions, and methods of inhibiting viral infection.
Modified griffithsin polypeptide comprising specified amino acid substitutions
A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 wherein X1 can be M or V, X2 is Q, X3 can be M, A, K, V, F, L, I, Q, R, or G, X4 can be S or R, X5 can be A or S, X6 can be I or F, and X7 can be E or Q; provided that the polypeptide does not comprise the wild-type SEQ ID NO: 2 sequence.
Polypeptide forms a dimer
The polypeptide retains the ability to form dimers, maintaining the enhanced potency associated with dimeric griffithsin.
Conjugates comprising the modified polypeptide and effector components
Conjugates comprising the polypeptide and at least one effector component selected from polyethylene glycol, albumin, dextran, toxin, immunological reagent, virus, viral envelope glycoprotein, antiviral agent, or solid support matrix.
Nucleic acid molecules encoding the modified polypeptide
Nucleic acid molecules comprising nucleotide sequences encoding the polypeptide, including sequences of SEQ ID NO: 4, 6, 8, or 10.
Vectors and cells comprising nucleic acids encoding the polypeptide
Vectors containing nucleic acids encoding the polypeptide and cells transformed or transfected with such nucleic acids or vectors.
Pharmaceutical compositions comprising the polypeptide
Compositions comprising the polypeptide and a carrier suitable for administration.
Methods for prophylactic or therapeutic inhibition of viral infection
Methods of administering the polypeptide to inhibit viral infections in cells or hosts, specifically including HIV infection.
Methods of virus inhibition on biological samples and inanimate objects
Methods of contacting biological samples or inanimate objects with the polypeptide to inhibit viruses, optionally combined with additional antiviral agents.
The claims collectively cover modified griffithsin polypeptides with defined amino acid substitutions that maintain dimer formation, conjugates with various effector components, corresponding nucleic acids, vectors, and cells, pharmaceutical compositions thereof, and methods of inhibiting viral infections including HIV by administering these inventive polypeptides and related compositions.
Stated Advantages
Reduced methionine oxidation, preventing protein degradation and increasing the shelf-life of griffithsin formulations.
Improved solubility across different pH ranges, enhancing bioavailability in various physiological environments.
Increased stability of griffithsin under oxidative conditions, such as exposure to biologically relevant hydrogen peroxide levels.
Retention of dimer formation which leads to approximately 1000-fold increased antiviral potency compared to monomeric griffithsin.
Ability to modify isoelectric point to optimize bioavailability in body compartments such as nasal cavity, lung, gut, blood, vagina, and rectum.
Documented Applications
Prophylactic and therapeutic inhibition of viral infections, especially HIV-1 and HIV-2, including multiple HIV groups and clades.
Inhibition of a broad range of viruses including retroviruses, herpes viruses, influenza viruses, Ebola, coronaviruses such as SARS and MERS, and Nipah virus.
Use as topical microbicide agents to prevent viral infections in humans, including application to mucosal surfaces and contraceptive devices.
Administration via various routes such as oral, intravenous, inhalation, transdermal, topical, rectal, and vaginal for viral infection prevention or treatment.
Use in formulations for virucidal sterilization of biological samples and inanimate objects like medical equipment.
Expression of griffithsin mutants in host cells including recombinant production in bacteria, yeast, mammalian cells, and plants for therapeutic applications.
Female-controllable prophylaxis via intravaginal administration of transformed lactobacilli producing griffithsin mutant polypeptides.
Combination therapy with other antiviral agents or immune response-inducing agents.
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