1. A method, comprising:
identifying a landing page associated with sponsored content;
identifying intrusion features of the landing page;
generating a feature score for the landing page based on the identified intrusion features;
determining if the feature score for the landing page exceeds a feature threshold;
classifying the landing page as a candidate landing page if the feature score for the landing page exceeds the feature threshold;
submitting the candidate landing page to an intrusion detection engine;
receiving an intrusion score for the candidate landing page from the intrusion detection engine; and
precluding the serving of the sponsored content associated with the candidate landing page if the intrusion score exceeds an intrusion threshold.
2. The method of claim 1, wherein the sponsored content is an advertisement.
3. The method of claim 2, further comprising:
receiving an appeal request for the sponsor account and in response to receiving the appeal request:
re-submitting the candidate landing page to the intrusion detection engine;
receiving another intrusion score for the candidate landing page from the intrusion detection engine;
precluding the serving of the advertisement associated with the candidate landing page if the another intrusion score exceeds the intrusion threshold; and allowing the serving of the advertisement associated with the candidate landing page if the another intrusion score does not exceed the intrusion threshold.
4. The method of claim 3, further comprising:
identifying a sponsor account associated with the advertisement, the sponsor account including additional advertisements; and
precluding the serving of the additional advertisements associated with the sponsor account if the intrusion score of the candidate landing page exceeds the intrusion threshold.
5. The method of claim 4, further comprising:
receiving an appeal request for the sponsor account and in response to receiving the appeal request:
submitting the candidate landing page and additional landing pages associated with the additional advertisements to the intrusion detection engine;
receiving another intrusion score of the candidate landing page and additional intrusion scores for the additional landing pages from the intrusion detection engine; and
determining if the intrusion scores for the landing pages exceed the intrusion threshold.
6. The method of claim 5, further comprising precluding the serving of advertisements associated with landing pages having intrusion scores that exceed the intrusion threshold.
7. The method of claim 5, further comprising precluding the serving of advertisements associated with the sponsor account if an intrusion score for any of landing pages exceeds the intrusion threshold.
8. The method of claim 2, wherein the intrusion features comprise one or more iFrame features, one or more URL features, andor one or more script features, and wherein each intrusion feature has an associated feature weight, and generating an intrusion score for the landing page based on the identified intrusion features comprises summing the feature weights.
9. The method of claim 2, further comprising:
identifying a sponsor account associated with the advertisement, the sponsor account including additional advertisements;
identifying additional landing pages associated with the additional advertisements;
generating feature scores for the additional landing pages based on the identified intrusion features;
determining if the feature scores for the landing page exceeds a feature threshold;
classifying each additional landing page as an additional candidate landing page if the feature score for the additional landing page exceeds the feature threshold;
accumulating the feature scores of the candidate landing page and the additional candidate landing pages to obtain an account score;
assigning a risk category to the sponsor account based on the account score; and
initiating one of a plurality of account remediation processes for the sponsor account based on the risk category.
10-16. (canceled)
17. A system, comprising:
a scoring engine comprising software instructions stored in computer readable medium and executable by a processing system, and upon such execution causes the processing system to:
identify a landing page associated with sponsored content;
identify intrusion features of the landing page;
generate a feature score for the landing page based on the identified intrusion features;
determine if the feature score for the landing page exceeds a feature threshold;
classify the landing page as a candidate landing page if the feature score for the landing page exceeds the feature threshold; and
a malware evaluation engine comprising software instructions stored in computer readable medium and executable by a processing system, and upon such execution causes the processing system to:
submit the candidate landing page to an intrusion detection engine;
receive an intrusion score for the candidate landing page from the intrusion detection engine; and
preclude the serving of the sponsored content associated with the candidate landing page if the intrusion score exceeds an intrusion threshold.
18. The system of claim 17, wherein the sponsored content is an advertisement.
19. The system of claim 18, further comprising:
an appeal processing evaluation engine comprising software instructions stored in computer readable medium and executable by a processing system, and upon such execution causes the processing system to:
receive an appeal request for a sponsor account associated with the landing page and in response to the appeal request cause the malware evaluation engine to:
re-submit the candidate landing page to the intrusion detection engine;
receive another intrusion score for the candidate landing page from the intrusion detection engine;
preclude the serving of the advertisement associated with the candidate landing page if the another intrusion score exceeds the intrusion threshold; and
allow the serving of the advertisement associated with the candidate landing page if the another intrusion score does not exceed the intrusion threshold.
20. The system of claim 18, wherein the malware evaluation engine comprises further software instructions stored in computer readable medium and executable by a processing system, and upon such execution causes the processing system to:
identify a sponsor account associated with the advertisement, the sponsor account including additional advertisements; and
preclude the serving of the additional advertisements associated with the sponsor account if the intrusion score of the candidate landing page exceeds the intrusion threshold.
21. The system of claim 20, further comprising:
an appeal processing evaluation engine comprising software instructions stored in computer readable medium and executable by a processing system, and upon such execution causes the processing system to:
receive an appeal request for a sponsor account associated with the landing page and in response to the appeal request cause the malware evaluation engine to:
identify landing pages associated with the sponsor account;
submit the landing pages to the intrusion detection engine;
receive intrusion scores for the landing pages from the intrusion detection engine; and
allow the serving of the advertisement and the additional advertisements if each of the intrusion scores do not exceed the intrusion threshold.
22. The system of claim 21, further comprising:
a data store storing training landing pages associated with advertisements and testing landing pages associated with advertisements; and
a machine learning engine comprising software instructions stored in computer readable medium and executable by a processing system, and upon such execution causes the processing system to:
iteratively train a classification model on intrusion features of the training landing pages;
iteratively test the classification model on the intrusion features of the testing landing pages until the occurrence of a testing cessation event; and
store an association of feature weights and intrusion features in a classification model in response to the cessation event, the association of feature weights and intrusion features derived from the iterative training and testing;
wherein the generation of a feature score for the landing page is based on the feature weights associated with the identified intrusion features stored in the classification model.
23. A system, comprising:
means for generating a classification model associating feature weights with intrusion features;
means for identifying candidate landing pages associated with advertisements;
means for submitting the candidate landing pages to an intrusion detection engine, for receiving intrusion scores for the candidate landing pages, and for precluding the serving of sponsored content associated with the candidate landing pages; and
means for appealing the preclusion of the serving of sponsored content associated with the candidate landing pages that are determined to be associated with intrusive software.
24. A method, comprising:
identifying a landing page associated with sponsored content;
identifying intrusion features of the landing page;
evaluating the landing page to determine if a likelihood that the landing page includes malware exceeds a first threshold based on the presence of the intrusion features;
if the likelihood that the landing page includes malware exceeds a first threshold, further evaluating the landing page, the further evaluating including performing at least one additional evaluation process on the landing page; and
based on the further evaluation, classifying the landing page as one or including malware or not including malware.
25. The method of claim 24, further comprising:
iteratively training a classification model on intrusion features of training landing pages;
iteratively testing the classification model on the intrusion features of testing landing pages until the occurrence of a testing cessation event; and
storing an association of feature weights and intrusion features in the classification model in response to the cessation event, the association of feature weights and intrusion features derived from the iterative training and testing and for facilitating the evaluation of the landing page.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A method of covalently-stabilizing a trimeric coiled-coil structure that mimics all or a portion of an internal, trimeric coiled-coil motif of an enveloped virus membrane-fusion protein comprising:
(a) engineering a soluble chimeric peptide comprising an \u03b1-helical domain which comprises a soluble, trimeric form of a coiled-coil fused in helical phase to all or a portion of a NH2-terminal heptad repeat domain, or a modified form thereof, of said enveloped virus membrane-fusion protein; and,
(b) stabilizing three chimeric peptides from (a) in a trimeric coiled-coil structure by formation of covalent bonds between said peptides, wherein said covalent bonds are selected from the group consisting of disulfide bonds and covalent bonds resulting from chemoselective reactions.
2. A method of covalently-stabilizing a trimeric coiled-coil structure that mimics all or a portion of an internal, trimeric coiled-coil motif of an enveloped virus membrane-fusion protein comprising:
(a) engineering a soluble chimeric peptide comprising:
(i) an \u03b1-helical domain which comprises a soluble, trimeric form of a coiled-coil fused in helical phase to all or a portion of a NH2-terminal heptad repeat domain, or a modified form thereof, of said enveloped virus membrane-fusion protein; and,
(ii) at least two cysteine residues located outside of said \u03b1-helical domain;
(b) incubating a plurality of said soluble chimeric peptides at a concentration at which a trimeric coiled-coil structure forms; and,
(c) oxidizing said trimeric coiled-coil structure formed in (b) to covalently-stabilize said coiled-coil via formation of disulfide bonds between cysteine residues on juxtaposed chimeric peptides of said coiled-coil.
3. A method of covalently-stabilizing a trimeric coiled-coil structure of claim 2, wherein said soluble chimeric peptide in (a) comprises all or a portion of a N-peptide of HIV gp41.
4. A method of covalently-stabilizing a trimeric coiled-coil structure that mimics all or a portion of an internal, trimeric coiled-coil motif of an enveloped virus membrane-fusion protein comprising:
(a) engineering a soluble chimeric peptide comprising an \u03b1-helical domain which comprises a soluble, trimeric form of a coiled-coil fused in helical phase to all or a portion of a NH2-terminal heptad repeat domain, or a modified form thereof, of said enveloped virus membrane-fusion protein;
(b) incubating a plurality of said soluble chimeric peptides at a concentration at which a trimeric coiled-coil structure forms; and,
(c) stabilizing the trimeric coiled-coil structure in (b) by formation of covalent, chemical bonds between said chimeric peptides, wherein said chemical bonds are formed by a chemoselective reaction.
5. A method of claim 4, wherein said soluble chimeric peptide in (a) comprises all or a portion of a N-peptide of HIV gp41.
6. A method of claim 5, wherein said chemical bonds are thioether bonds.
7. A method of claim 6, wherein a plurality of said soluble chimeric peptides is incubated at a concentration at which the resulting trimeric coiled-coil structure is comprised of:
(a) one chimeric peptide which further comprises at least two cysteine residues located outside of the \u03b1-helical domain of said peptide; and,
(b) two chimeric peptides which are each derivatized to incorporate an electrophilic moiety;
wherein a nucleophilic sulfhydryl of each cysteine residue in the chimeric peptide in (a) forms a thioether bond with the electrophilic moiety of the derivatized chimeric peptides in (b).
8. A method of claim 7, wherein the two chimeric peptides in (b) are derivatized to incorporate an electrophilic moiety selected from the group consisting of an alkyl halide moiety and a Michael acceptor.
9. A soluble chimeric peptide which comprises:
(a) a scaffold portion comprising a soluble, trimeric form of a coiled-coil;
(b) a N-peptide portion comprising all or a portion of a N-peptide of HIV gp41, or a modified form thereof; and,
(c) a cysteine portion comprising at least two cysteine residues;
wherein said scaffold portion in (a) is fused in helical phase to said N-peptide portion in (b), forming an \u03b1-helical domain, and said cysteine portion in (c) is located outside of said a-helical domain.
10. (canceled)
11. A chimeric peptide of claim 9, wherein said N-peptide portion is fused to the COOH-terminus of the scaffold portion of said peptide.
12. A chimeric peptide of claim 11, wherein said N-peptide portion comprises a sufficient portion of the HIV gp41 N-peptide to include amino acid residues which form a N-helix coiled-coil hydrophobic pocket.
13. A chimeric peptide of claim 12, wherein said N-peptide portion comprises N17 (LLQLTVWGIKQLQARIL (SEQ ID NO:44)).
14. A chimeric peptide of claim 12, wherein said N-peptide portion is a modified, HIV-1 gp41 N-peptide that has been mutated to eliminate a potential immunodominant epitope located from about amino acid 577 to about amino acid 581 of HIV-1 gp41.
15. A chimeric peptide of claim 14, wherein said modified N-peptide comprises N17Ala4 (LLQLTVWGIKALAAAIA (SEQ ID NO:60)).
16. A chimeric peptide of claim 11, wherein the scaffold portion of said peptide comprises all or a portion of Suzuki-IZ coiled-coil motif (YGGIEKKIEAIEKKIEAIEKKIEAIEKKIEA (SEQ ID NO:29)), or a modified form thereof.
17. A chimeric peptide of claim 16, wherein the scaffold portion of said peptide comprises IZ coiled-coil motif (IKKEIEAIKKEQEAIKKKIEAIEK (SEQ ID NO:31)), a modified Suzuki-IZ coiled-coil motif.
18. A chimeric peptide of claim 16, wherein the scaffold portion of said peptide comprises EZ coiled-coil motif (IEKKIEEIEKKIEEIEKKIEEIEK (SEQ ID NO:32)) a modified Suzuki-IZ coiled-coil motif.
19. A chimeric peptide of claim 16, wherein the scaffold portion of said peptide comprises SZ coiled-coil motif (IEKKIEAIEKKIEAIEKKIEAIEK (SEQ ID NO:107)), a modified Suzuki-IZ coiled-coil motif.
20. A chimeric peptide of claim 11, wherein the cysteine portion of said peptide comprises at least two, consecutive cysteine residues located at the NH2-terminus of the \u03b1-helical domain of said peptide.
21. A chimeric peptide of claim 20, wherein said cysteine residues are separated from said \u03b1-helical domain by a spacer region.
22. A chimeric peptide of claim 11, wherein the cysteine portion of said peptide comprises at least two, consecutive cysteine residues located at the COOH-terminus of the \u03b1-helical domain of said peptide.
23. A chimeric peptide of claim 11, wherein the cysteine portion comprises three consecutive cysteine residues located at the NH2-terminus of the \u03b1-helical domain of said peptide.
24. A chimeric peptide of claim 23, wherein said cysteine residues are separated from said \u03b1-helical domain by a spacer region.
25. A chimeric peptide of claim 24, wherein the cysteine residue located at the NH2-terminus has a protected thiol group.
26. A chimeric peptide comprising an amino acid sequence selected from the group consisting of:
(a) SEQ ID NO:2, designated as CCIZN17;
(b) SEQ ID NO:7, designated as CCEZN17;
(c) SEQ ID NO:5, designated as CCIZN17Ala4;
(d) SEQ ID NO:9, designated as CCEZN17Ala4;
(e) SEQ ID NO:13, designated as CCIZN23;
(f) SEQ ID NO:15, designated as CCEZN23;
(g) SEQ ID NO:12, designated as Biotin-CCIZN23;
(h) SEQ ID NO:21, designated as SCCIZN17;
(i) SEQ ID NO:98, designated as CCSZN17;
(j) SEQ ID NO:100, designated as CCS17N17;
(k) SEQ ID NO:109, designated as CCE10N17;
(l) SEQ ID NO:116, designated as CCE17N17;
(m) SEQ ID NO:120, designated as CCE17GluN17; and,
(n) SEQ ID NO:18, designated as CCIZN36.
27. A covalently-stabilized, trimeric coiled-coil that mimics all or a portion of a HIV gp41 N-peptide coiled-coil comprising three chimeric peptides, wherein each chimeric peptide comprises:
(a) an \u03b1-helical domain comprising a soluble, trimeric form of a coiled-coil fused in helical phase to all or a portion of a HIV gp41 N-peptide, or a modified form thereof; and,
(b) at least two cysteine residues located outside of said \u03b1-helical domain;
and wherein said chimeric peptides are covalently-stabilized as a coiled-coil via disulfide bonds between said cysteine residues of individual peptides.
28. A covalently-stabilized, trimeric coiled-coil that mimics all or a portion of a HIV gp41 N-peptide coiled-coil which comprises:
(a) one chimeric peptide comprising:
(i) an \u03b1-helical domain which comprises a soluble, trimeric form of a coiled-coil fused in helical phase to all or a portion of a HIV gp41 N-peptide, or a modified form thereof; and,
(ii) at least two cysteine residues located outside of the \u03b1-helical domain of said peptide; and,
(b) two chimeric peptides comprising an \u03b1-helical domain which comprises a soluble, trimeric form of a coiled-coil fused in helical phase to all or a portion of a HIV gp41 N-peptide, or a modified form thereof, that are each derivatized with an electrophilic moiety capable of forming a thioether bond;
wherein a nucleophilic sulfhydryl of each cysteine residue in the chimeric peptide in (a) forms a thioether bond with the electrophilic moiety of each derivatized chimeric peptide in (b).
29. A covalently-stabilized, trimeric coiled-coil that mimics all or a portion of a HIV-1 N-peptide coiled-coil selected from the group consisting of:
(a) (CCIZN17)3 (SEQ ID NO:23);
(b) (CCEZN17)3 (SEQ ID NO:73);
(c) (CCIZN17Ala4)3 (SEQ ID NO:53);
(d) (CCEZN17Ala4)3 (SEQ ID NO:93);
(e) (CCIZN23)3 (SEQ ID NO:133);
(f) (CCEZN23)3 (SEQ ID NO:153);
(g) (Biotin-CCIZN23)3 (SEQ ID NO:123);
(h) (SCCIZN17)3 (SEQ ID NO:213);
(i) C(thioIZN17)3;
(j) C(thioS17N17)3;
(k) C(thioE10N17)3;
(l) C(thioEZN17)3;
(m) C(thioE17N17)3;
(n) C(thioE17GluN17)3;
(o) (CCIZN36)3 (SEQ ID NO:183).
30. An antigenic conjugate of a chimeric peptide of claim 9, wherein said peptide is covalently linked to an immunogenic carrier molecule.
31. An antigenic conjugate of claim 30, wherein said carrier molecule is OMPC.
32. An antigenic conjugate of a covalently-stabilized, trimeric coiled-coil of claim 27, wherein said trimeric coiled-coil is covalently linked to an immunogenic carrier molecule.
33. An antigenic conjugate of claim 32, wherein said carrier molecule is OMPC.
34. An antigenic conjugate of a covalently-stabilized trimeric coiled-coil of claim 28, wherein said trimeric coiled-coil is covalently linked to an immunogenic carrier molecule.
35. An antigenic conjugate of claim 34, wherein said carrier molecule is OMPC.
36. A pharmaceutical composition comprising an effective amount of an antigenic conjugate of claim 33 mixed with a biologically effective adjuvant, protein, or other agent capable of increasing the immune response, wherein said composition is useful as an immunogen capable of eliciting HIV-specific neutralizing antibodies in mammals.
37. A pharmaceutical composition comprising an effective amount of an antigenic conjugate of claim 35 mixed with a biologically effective adjuvant, protein, or other agent capable of increasing the immune response, wherein said composition is useful as an immunogen capable of eliciting HIV-specific neutralizing antibodies in mammals.
38. A method of inhibiting fusion of HIV to a cell comprising contacting said cell with an effective amount of a covalently-stabilized, trimeric coiled-coil of claim 27 to interfere with gp41 from adopting a structural conformation that mediates fusion of HIV to said cell by binding to an epitope on a gp41 fusion intermediate so as to thereby inhibit HIV infection of said cell.
39. A method of inhibiting fusion of HIV to a cell comprising contacting said cell with an effective amount of a covalently-stabilized, trimeric coiled-coil of claim 28 to interfere with gp41 from adopting a structural conformation that mediates fusion of HIV to said cell by binding to an epitope on a gp41 fusion intermediate so as to thereby inhibit HIV infection of said cell.
40. A method of identifying a HIV fusion inhibitor comprising:
(a) combining a test compound, a covalently-stabilized, trimeric coiled-coil of claim 27, and an antibody that recognizes a conformational epitope within the HIV gp41 N-peptide coiled-coil;
(b) measuring the effect the test compound has on the affinity of the covalently-stabilized, trimeric coiled coil and the antibody; and,
(c) comparing the effect the test compound has on the affinity of the antibody for the covalently-stabilized, trimeric coiled-coil versus the affinity of the antibody for the coiled-coil in the absence of the test compound;
wherein if said test compound decreases the affinity of the antibody for the covalently-stabilized, trimeric coiled-coil, the test compound is identified as a HIV fusion inhibitor.
41. A method of identifying a HIV fusion inhibitor comprising:
(a) combining a test compound, a covalently-stabilized, trimeric coiled-coil of claim 28, and an antibody that recognizes a conformational epitope within the HIV gp41 N-peptide coiled-coil;
(b) measuring the effect the test compound has on the affinity of the covalently-stabilized, trimeric coiled coil and the antibody; and,
(c) comparing the effect the test compound has on the affinity of the antibody for the covalently-stabilized, trimeric coiled-coil versus the affinity of the antibody for the coiled-coil in the absence of the test compound;
wherein if said test compound decreases the affinity of the antibody for the covalently-stabilized, trimeric coiled-coil, the test compound is identified as a HIV fusion inhibitor.
42. A soluble chimeric peptide which comprises:
(a) a scaffold portion comprising a soluble, trimeric form of a coiled-coil; and,
(b) a N-peptide portion comprising all or a portion of a N-peptide of HIV gp41, or a modified form thereof;
wherein said chimeric peptide is derivatized to incorporate an electrophilic moiety capable of forming a thioether bond.
43. A chimeric peptide of claim 42, wherein said peptide is derivatized to incorporate an electrophilic moiety selected from the group consisting of an alkyl halide moiety and a Michael acceptor.
44. A chimeric peptide of claim 43, wherein said alkyl halide moiety is a bromoacetyl moiety.