Method of evaluating a patient antibody response to envelope proteins of a virus
View Patent ↗The invention provides a method for identifying whether a compound inhibits entry of a virus into a cell which comprises: (a) obtaining nucleic acid encoding a viral envelope protein from a patient infected by the virus; (b) co-transfecting into a first cell (i) the nucleic acid of step (a), and (ii) a viral expression vector which lacks a nucleic acid encoding an envelope protein, and which comprises an indicator nucleic acid which produces a detectable signal, such that the first cell produces viral particles comprising the envelope protein encoded by the nucleic acid obtained from the patient; (c) contacting the viral particles produced in step (b) with a second cell in the presence of the compound, wherein the second cell expresses a cell surface receptor to which the virus binds; (d) measuring the amount of signal produced by the second cell in order to determine the infectivity of the viral particles; and (e) comparing the amount of signal measured in step (d) with the amount of signal produced in the absence of the compound, wherein a reduced amount of signal measured in the presence of the compound indicates that the compound inhibits entry of the virus into the second cell.
1. In a patient infected with a virus, a method for detecting an antibody response capable of blocking virus infection comprising:
a) transfecting into a first sample of cells
i. nucleic acids encoding envelope proteins of a population of virus infecting the patient, and
ii. a viral expression vector which lacks a nucleic acid encoding an envelope protein, and which comprises an indicator nucleic acid which produces a detectable signal,
such that the first sample of cells produces viral particles comprising envelope proteins from the population of virus infecting the patient,
b) contacting the viral particles of step (a) with an antibody preparation from the patient;
c) contacting the viral particles and antibody preparation of step (b) with a second sample of cells, wherein the second sample of cells express a cell surface receptor and/or co-receptor which binds to the virus;
d) measuring the amount of detectable signal produced by the second sample of cells in step (c) in order to determine the infectivity of the viral particles; and
e) contacting the viral particles of step (a) with a second sample of cells, wherein the second sample of cells express a cell surface receptor and/or co-receptor which binds to the virus;
f) measuring the amount of detectable signal produced by the second sample of cells in step (e) in order to determine the infectivity of the viral particles; and
g) comparing the amount of signal measured in step (d) with the amount of signal produced in step (f), wherein a decrease in the amount of signal with the antibody treated viral particles indicates that the patient has developed an antibody response capable of blocking virus infection.
2. The method of claim 1 , wherein steps (a)–(f) are repeated with varying concentrations of antibody preparations, and the amounts of signal generated for each of the concentrations are compared in step (g).
3. The method of claim 2 , further comprising generating a plot of viral infectivity based on antibody concentrations.
4. The method of claim 3 , further comprising generating an IC50 from the plot.
5. The method of claim 1 , wherein the indicator nucleic acid comprises an indicator gene.
6. The method of claim 1 , wherein the indicator gene is a luciferase gene.
7. The method of claim 1 , wherein the virus is HIV-1.
8. The method of claim 1 , wherein the receptor is CD4.
9. The method of claim 1 , wherein the co-receptor is CXCR4 or CCR5.
10. The method of claim 9 , wherein the co-receptor is CXCR4.
11. The method of claim 9 , wherein the co-receptor is CCR5.
12. The method of claim 1 , wherein the nucleic acids encoding envelope proteins comprise nucleic acids encoding the envelope polyprotein (gp160).
13. The method of claim 1 , wherein the nucleic acids encoding envelope proteins comprise nucleic acids encoding the surface envelope protein (gp120) and the transmembrane envelope protein (gp41).
14. The method of claim 1 , wherein the viral expression vector comprises an HIV nucleic acid.
15. The method of claim 1 , wherein the viral expression vector comprises an HIV gag-pol gene.
16. The method of claim 1 , wherein the first and/or second sample of cells are mammalian.
17. The method of claim 16 , wherein the mammalian cells are human.
18. The method of claim 17 , wherein the human cells are human embryonic kidney cells.
19. The method of claim 18 , wherein the human embryonic kidney cells are 293 cells.
20. The method of claim 17 , wherein the human cells are human T cells.
21. The method of claim 17 , wherein the human cells are peripheral blood mononuclear cells.
22. The method of claim 1 , wherein the first and/or second sample of cells are astroglioma cells.
23. The method of claim 22 , wherein the astroglioma cells are U87 cells.
24. The method of claim 1 , wherein the first and/or second sample of cells are human osteosarcoma cells.
25. The method of claim 24 , wherein the osteosarcoma cells are HT4 cells.
26. The method of claim 1 , wherein the antibody preparation binds to the receptor or co-receptor.
27. The method of claim 26 , wherein the antibody preparation binds the receptor CD4.
28. The method of claim 26 , wherein the antibody preparation binds the co-receptor CXCR4 or CCR5.
29. The method of claim 28 , wherein the antibody preparation binds the co-receptor CXCR4.
30. The method of claim 28 , wherein the antibody preparation binds the co-receptor CCR5.
31. The method of claim 1 , wherein the antibody preparation binds the viral envelope.