| Broadly neutralizing antibody(bnAb)responses may be required for an efficacious vaccine against global diverse HIV-1 strains.To accurately and quickly measure such neutralizing activity will depend on standardized high-throughput assays that can reliably determine the potency and breadth of nAb responses elicited during natural HIV-1 infection or by vaccines.A neutralization assay based on HIV-1 Env-pseudotype viruses and TZM-bl cells has been widely used with Envs from a variety of subtypes.TZM-bl is a HeLa cell derivative that expresses CD4,CCR5 and CXCR4 for HIV/SIV infection and contains Tat-responsive reporter genes: firefly luciferase(Luc)and Escherichia coli β-galactosidase.TZM-bl cells are more permissive to infection by a wide variety of HIV-1 strains and can generate more reproducible results than peripheral blood mononuclear cells.With its sensitive,quantitative and high-throughput linear dynamic range of several orders of magnitude,this assay has been widely used for measuring neutralization activities in HIV-1-infected individuals,human vaccine trials and in vaccinated animals.Neutralization results were generally similar between the methods based on TZM-bl cells and PBMCs.However,the genetically engineered TZM-bl cells express high levels of CCR5 and CXCR4.This makes the TZM-bl cells more susceptive to HIV-1 infection and may not fully reflect the natural infection process that may lead to possible neutralization differences.Neutralization based on PBMCs in vitro is considered to be the closest to physiological conditions.However,neutralization assays using PBMCs are cumbersome,expensive and less high-throughput.In addition,the large variability of PBMCs from different donors in the sensitivity to the HIV-1 infection and the limited numbers of PBMCs from donors greatly limit the use of PBMCs in standard neutralization assays.Infection can only be determined using expensive p24 or RT assays when live viruses or Env-pseudotype viruses are used to infect PBMCs or other cells without engineered reporter genes.To address this issue,HIV-1 backbone genomes containing a reporter gene(luciferase or human placental alkaline phosphatase)are used to determine viral replication and neutralization activities in cells without a reporter gene.However,due to the inconvenience and high background in the uninfected control cells,they have not been widely used for determination of neutralization activities.Humanized Gaussia luciferase(GLuc)can generate over 1000-fold higher bioluminescent signals in live cells in vivo in experimental animals and over 100-fold higher from live cells in vitro than humanized firefly(FLuc),renilla(RLuc)and vargula(VLuc)luciferases.GLuc has been engineered into the influenza viral genome and used to detect and identify sites of infection in live animals and cell cultures as well as to determine neutralization activities.In this experiment,we generated a new viral backbone by cloning the Gaussia luciferase gene into the SG3Δenv genome,and then infected the TZM-bl cells and T cell lines with the pseudotype virus produced by the HIV-1 Env clone.The infection can be detected by measuring the luciferase activity in the cell culture supernatant,and the detection system can be applied to any cell that HIV can infect.Next,we applied the Gaussia detection system to the HIV neutralization activity test.By detecting the activity in the serum of HIV patients,the neutralization titer obtained showed no statistics compared with the commonly used TZM-bl pseudotype virus neutralization test results.Learn the difference.Simultaneously,this strategy is very sensitive to the detection of neutralizing antibodies with lower titers,and the method is also easy to detect neutralizing activity in HIV-1 infected individuals,human vaccine trials and sera of vaccinated animals. |
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