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Neutralization of HIV-1 Infection of Human Peripheral Blood Mononuclear Cells (PBMC) : Infectivity Reduction Method
1Division of Retrovirology, Walter Reed Army Institute of Research, Rockville, MD.
Abstract:
The serum titration neutralization assay described in Chapter 33 utilizes a constant amount of infectious virus and indirectly estimates the antibody-mediated reduction in infectious virus by measuring p24 antigen expressed by human peripheral blood mononuclear cells (PBMC). The assay assumes a direct relationship between expressed p24 antigen and the number of target PBMC infected. Therefore, the optimal time of p24 antigen measurement may vary due to different growth kinetics among viruses. An infectivity reduction assay (IRA) directly measures the effect of antibody on virus endpoint infectious titer. The IRA is more labor-intensive and requires more serum than the antibody titration assay, and it is generally done with a single dilution of antibody. However, it has the important advantage of directly measuring the effect of antibody on virus median tissue culture infectious dose (TCID(50)). In addition, the IRA is unaffected by variation in virus growth kinetics and therefore is theoretically more suitable for comparing neutralization among diverse virus isolates. The assay described below calculates virus TCID(50) by serially diluting virus stock in quadruplicate wells with phytohemagglutinin (PHA)-stimulated PBMC. Wells are scored positive or negative for HIV-1 infection by measuring expressed p24 antigen after eight days in culture. Results are expressed as the antibody-mediated reduction in virus TCID(50) (i.e., ratio of virus TCID(50) in control sera to TCID(50) in test sera).
Insights
This study compares two methods for measuring HIV-1 neutralization: a serum titration assay and an infectivity reduction assay (IRA). The IRA directly measures antibody effects on virus infectivity, offering a more reliable comparison across different virus strains.
Area of Science:
- Virology
- Immunology
- Assay Development
Background:
- Serum titration assays indirectly measure antibody neutralization by quantifying p24 antigen expression in infected cells.
- This indirect method assumes a direct correlation between p24 antigen levels and the number of infected cells, which can be influenced by virus growth kinetics.
Purpose of the Study:
- To evaluate the Infectivity Reduction Assay (IRA) for directly measuring antibody-mediated neutralization of HIV-1.
- To compare the IRA with traditional serum titration assays, highlighting the IRA's advantages in handling diverse virus isolates.
Main Methods:
- The IRA quantifies the reduction in virus median tissue culture infectious dose (TCID(50)) by serially diluting virus stocks with stimulated peripheral blood mononuclear cells (PBMC).
- HIV-1 infection is detected by measuring p24 antigen expression after eight days in culture.
- Results are reported as the ratio of virus TCID(50) in control sera versus test sera.
Main Results:
- The IRA directly measures the impact of antibodies on virus infectivity, providing a more direct assessment of neutralization.
- Unlike titration assays, the IRA is not affected by variations in virus growth kinetics, making it suitable for diverse viral isolates.
- The assay quantifies antibody-mediated reduction in virus TCID(50).
Conclusions:
- The Infectivity Reduction Assay (IRA) provides a direct and robust method for assessing HIV-1 neutralization by antibodies.
- The IRA's independence from virus growth kinetics makes it superior for comparing neutralization potency across a wide range of HIV-1 isolates.
