Development Of A Safe And Rapid Neutralization Assay Of HIV-1

As the AIDS epidemic continues to spread across the world,it is becamethe focus that the treatment strategies of it,s infection and the prevention of it,sspreading. We currently have no effective treatment to reverse the diseaseprocess or vaccines to prevent infection. It is generally believed that a potentneutralizing antibody response is required to prevent infection with HIV, as isthe case with many viruses;however, this is a highly controversial point withmuch conflicting data. Critical to developing a vaccine to prevent AIDS is anassay to measure the neutralizing antibody present in blood from patients,experimental vaccine recipients, and experimental animals.The text next be aims to describe the characteristics of a usefulneutralizing antibody assay, compare currently available methodologies forsuch an assay, report on new technology, and provide suggestions for thedirection to be taken to produce a standardized assay.The sensitivity with which antibody (Ab)-mediated virus neutralization isdetected is dependent on the assay conditions used. These conditions include,but are not limited to:the type of antibody or antiserum be used,the presence orabsence of complement,the type of virus used,the multiplicity of infection,theratio of antibody to virus,the length of exposure of antibody to virus,the lengthof exposure of the antibody-treated virus to target cells, the type of host cellused to produce the virus,the type of target cell used for infection in the assay,the kinetics of virus growth,the length of the assay, the type of read-outused,the method of data analysis,the criteria used to define neutralization.What are the characteristics of a good neutralizing antibody assay? Aneutralizing antibody assay should have the following characteristics:1. permit measurement of a variety of antibody samples, including patientserum and purified antibodies (monoclonals), and also samples derived fromexperimental animals;2. permit measurement of neutralization of a wide variety of virussamples including primary isolates, laboratory strains, macrophage-and Tcell-tropic strains, syncytia-inducing and non-syncytia inducing strains, andalso SIV and other primate lentivirus;3. provide a measurable, quantitative endpoint without the requirementfor long term culture and multiple rounds of infection which confoundinterpretation;4. employ the simplest and most cost-effective technology possible sincea large-scale vaccine trial would involve assaying many thousands of samples.Also, a standardized assay would need to be reproducible among differentlaboratories for comparison of results.Currently Used Assays:The typical neutralization assay currently in use consists of: premixingvirus sample and antibody sample, using this mix to inoculate either primaryperipheral blood mononuclear cells or a cell line, and culturing for a few toseveral days until a detectable endpoint is reached (up to 14 days if necessary).The level of infection is measured indirectly by assaying for viral replicationvia p24 or reverse transcriptase assay, or by detection of cytopathic effect inthe target cells by staining with a vital dye or syncytia formation. Controlslacking the antibody are run in parallel;a decrease in infectivity (lower p24 orRT values) or CPE (fewer cells staining as dead, fewer syncytia) is anindication of virus neutralization. hese assays are relatively simple;however,they can be costly, as in the case of RT and p24 endpoint determinations. Also,they require that infection proceed through multiple cycles to reach detectablelevels which confounds interpretation of the results and can take many days tocomplete. In addition, different target cells must be used depending onwhether the virus to be tested uses the fusin or CCR-5 co-receptors. In caseswhere primary cells are used as targets, there is a certain level of variabilityfrom donor to donor.Another type of assay uses the sensitivity of PCR to detect the earliestevents of the infection cycle and thereby avoids the complication of multiplerounds of infection. Antibody and virus samples are mixed and applied to cells.After several hours, cells are collected, DNA isolated and PCR performed todetect LTR or gag region DNA. The PCR products are quantitated andcompared to controls lacking antibody. Neutralization is defined as a decreasein the PCR signal, which correlates with fewer infection events.While thisassay has the advantage of single infection cycle, it uses PCR to measure theendpoint. PCR is costly for the large scale studies for which a neutralizingantibody assay is needed, and is wrought with reproducibility andcontamination problems and is labor intensive.Newer Techniques:Originally reported in 1992, the MAGI (multinuclear activation of agalactosidase indicator) assay was intended to quantitatively measure virusinfectivity. The heart of the assay is the target cell, HeLa cells stabilytransfected with CD4 and a reporter construct consisting of the b-galactosidasegene (modified to localize to the nucleus) driven by a truncated HIV-1 LTR.Expression of the b-galactosidase gene is Tat-dependent such that an incomingvirus must produce active Tat protein to drive expression of the reporter. If thisoccurs, the cells stain with a blue nucleus which is easily visualized with alight microscope. The MAGI assay is usually read on day 2 or 3 after initiationof infection;the number of blue cells equals the number of infectious virusparticles in the inoculum. Therefore, since the assay scores infectious particles,one can determine the number of defective particles as well. Recently, asimilar system for testing SIV infection (sMAGI) using a similarly modifiedrhesus macaque cell line was reported.In this study,our goal is to development of a safe and rapid Neutralizationassay using pseudovirus with HIV type 1 envelope glycoprotein prevalent inChina. Pseudotype viruses were prepared by cotransfection of 70% to80%-confluent 293T cells with VR-env-x-IRES-puro libraries plus an HIVgenomic vector that contains a firefly luciferase indicator genepNL4-3.luc.E-R—. Recombinant viruses pseudotyped were harvested 48 hposttransfection and incubated for 1 h at 37°C with serial 3-fold dilutions ofheat-inactivated serum samples (antibody). HOS cells that express CD4 plusthe CCR5 and CXCR4 coreceptors were inoculated with virus serum(antibody)dilutions.Virus infectivity was determined 72 h postinoculation by measuringthe amount of luciferase activity expressed in infected cells. Neutralizingactivity is displayed as the percent inhibition of viral.In addition , stable celllines are constructing to express human immunodeficiency pseudovirus orderto construct a system of neutralizing antibody assay which is moresafer,rapid , effective,reduplicate and stable.In this paper,the following studies we have been finished:all of thetransient transfection parameter have been optimized to ensure consistent andinfection virus particle production.And the stability of the pseudovirus havebeen measured in order to sure that the virus can be kept in -80℃ for a longtime.The stable cell lines are constructing still.In addition,wo have studied of the HIV-1 VLP vaccine's immuno-genicity,which one is constructed by our lab.We have assayed it's effect thatcause neutralizing antibody.In summary,the completion of this study is promised to construct asystem of HIV-1 prevalent in China neutralizing antibody assay which ismore safer,rapid , effective,reduplicate and stable .