Compositions and Methods for Determining Resistance to Inhibitors of Virus Enter Using Recombinant Virus Assays

Abstract

The invention provides a method for determining whether a human immunodeficiency virus is resistant to a viral entry inhibitor. The methods are particularly useful for determining resistance to inhibitors that act by a non-competitive mechanism. In certain aspects, the methods comprise determining whether an HIV population is resistant to an HIV entry inhibitor, comprising determining a log-sigmoid inhibition curve comprising data points for entry of the HIV population in the presence of varying concentrations of the HIV entry inhibitor, wherein if the entry of the HIV population cannot be completely inhibited by the HIV entry inhibitor, the HIV population is resistant to the HIV entry inhibitor.

1 - 16 . (canceled) 
     
     
         17 . A method for detecting mutations of a virus that are correlated with reduced susceptibility to a virus entry inhibitor, comprising:
 (a) obtaining individual biological samples from a subject at a plurality of different time points during infection, the samples each comprising virus nucleic acid molecules;   (b) cloning a plurality of nucleic acid molecules, each comprising a gene that encodes an envelope protein for one of the virus molecules in the sample of step (a);   (c) transfecting into a first cell: (i) a nucleic acid comprising one of the cloned envelope protein genes of step (b) or a population thereof; and (ii) a viral expression vector which lacks the viral envelope protein gene, such that a population of identical pseudoviruses or a population of representative pseudoviruses are produced by the first cell, and optionally, contacting the pseudoviruses produced by the first cell with a virus entry inhibitor;   (d) contacting the pseudoviruses of step (c) with a second cell, wherein the second cell expresses at least one cell surface receptor to which the virus binds;   (e) measuring the ability of the pseudoviruses to infect the second cell;   (f) comparing the infectivity of the pseudoviruses of step (e) when the pseudoviruses have been exposed to the virus entry inhibitor as compared to when the pseudoviruses have not been exposed to the virus entry inhibitor, wherein a partial decrease, no change, or an increase in infectivity for the pseudoviruses exposed to the virus entry inhibitor indicates that the pseudoviruses have reduced susceptibility to the virus entry inhibitor; and   (g) determining the nucleic acid sequence of at least some of the pseudoviruses of step (f) to determine whether a mutation or mutations in the gene encoding the envelope protein is correlated with the reduced susceptibility to the virus entry inhibitor.   
     
     
         18 . The method of  claim 17 , further comprising:
 h) using site-directed mutagenesis to generate a specific mutant pseudovirion having one of the mutations in the gene encoding the envelope protein correlated with reduced sensitivity to the virus entry inhibitor; and   i) testing the specific mutant pseudovirion for infectivity upon exposure to the virus entry inhibitor as compared to in the absence of exposure to the virus entry inhibitor.   
     
     
         19 . The method of  claim 17 , wherein the viral expression vector comprises an indicator nucleic acid that produces a detectable signal or the second cell comprises an indicator nucleic acid that produces a detectable signal upon infection with the virus. 
     
     
         20 . The method of  claim 17 , wherein the receptor is CD4, and wherein the cell optionally further comprises at least one coreceptor, wherein the coreceptor is at least one of CXCR4 and CCR5. 
     
     
         21 . The method of  claim 17 , wherein the nucleic acid encoding the envelope protein encodes the surface envelope glycoprotein (gp120), the transmembrane envelope glycoprotein (gp41), the envelope polyprotein (gp160), both gp120 and gp41, or fragments thereof. 
     
     
         22 - 25 . (canceled) 
     
     
         26 . The method of  claim 17 , wherein the patient has been treated with an HIV inhibitor between the first and second time points. 
     
     
         27 . The method of  claim 26 , wherein the HIV inhibitor is an entry inhibitor. 
     
     
         28 . The method of  claim 27 , wherein the entry inhibitor is an antibody, fragment thereof, or combination of antibody fragments. 
     
     
         29 . The method of  claim 28 , wherein the antibody binds to the HIV envelope surface glycoprotein (gp120). 
     
     
         30 . The method of  claim 28 , wherein the antibody binds to the CD4 binding site, the variable region 3 (V3), the variable region 2 (V2), or a conformational epitope of the HIV envelope surface glycoprotein. 
     
     
         31 . The method of  claim 28 , wherein the antibody binds to the HIV envelope transmembrane glycoprotein (gp41). 
     
     
         32 . The method of  claim 28 , wherein the antibody binds to the membrane proximal external region (MPER) of the HIV envelope transmembrane glycoprotein. 
     
     
         33 . The method of  claim 19 , wherein the indicator nucleic acid encodes luciferase. 
     
     
         34 . The method of  claim 17 , wherein the at least one cell surface receptor of the second cell is CD4. 
     
     
         35 . The method of  claim 34 , wherein the second cell further comprises at least one coreceptor, wherein the coreceptor is CXCR4 or CCR5. 
     
     
         36 . The method of  claim 17 , wherein step (f), step (g), or both are performed using computer software. 
     
     
         37 . The method of  claim 17 , wherein the infectivity is measured using a log-sigmoid inhibition curve. 
     
     
         38 . The method of  claim 37 , wherein the infectivity is measured by detecting the maximum inhibition percentage of the pseudoviruses.