Methods for determining the presence of antibodies blocking viral infection

A method for determining whether one or more viral species from a population of Human Immunodeficiency Virus-1 (HIV-1) present in a sample from an HIV-1-infected patient is susceptible to antibody neutralization comprising:

• (a) simultaneously, and in a single reaction vessel, amplifying nucleic acid molecules comprising the HIV-1 envelope gene of the population of HIV-1 present in the HIV-1-infected patient sample to obtain a plurality of nucleic acid molecules, each encoding an HIV-1 envelope protein from the population of HIV-1 present in the HIV-1-infected patient sample;
• (b) simultaneously, and in a single reaction vessel, inserting the plurality of nucleic acid molecules from step (a) into a first expression vector engineered to express the HIV-1 envelope protein in cells resulting in production of a plurality of distinct expression vectors, each member of which comprises only one of the plurality of nucleic acid molecules, wherein the plurality of distinct expression vectors express a plurality of variants of the HIV-1 envelope protein that is representative of the HIV-1 species found in the HIV-1-infected patient sample;
• (c) transfecting into a plurality of first cells (i) the plurality of expression vectors from step (b) and (ii) a viral expression vector which lacks the nucleic acid encoding the HIV-1 envelope protein from step (a) but includes nucleic acid encoding other proteins required for replication of the HIV-1, such that the plurality of first cells produce viral particles comprising the HIV-1 envelope proteins representative of the HIV-1 species present in the HIV-1-infected patient sample;
• (d) contacting a first portion of the viral particles produced in step (c) with an antibody preparation;
• (e) contacting the viral particles and antibody preparation of step (d) with a first plurality of second cells, wherein the second cells each expresses a cell surface receptor and at least one co-receptor to which the HIV-1 binds for entry, and wherein the second cells each comprise an indicator nucleic acid molecule which produces a detectable signal following infection by the viral particles of (c);
• (f) contacting a second portion of the viral particles of step (c) with a second plurality of the second cells in the absence of antibody;
• (g) measuring the amount of the detectable signal produced by the first and second plurality of second cells from step (e) and step (f) in order to determine the infectivity of the viral particles in the presence and the absence of antibody, respectively;
• and
• (h) comparing the amount of signal measured for viral particles contacted with the first plurality of second cells in the presence of antibody in step (e) with the amount of signal produced for viral particles contacted with the second plurality of second cells in the absence of antibody in step (f), wherein a reduced amount of signal measured for the viral particles of step (e) as compared to the viral particles of step (f) indicates that at least one species from the population of HIV-1 from the HIV-1-infected patient sample is susceptible to antibody neutralization.