| Pseudovirus technology has been widely employed in vaccine and anti-viral drugevaluation, among which the most well-known were the non-enveloped virus humanpapillomavirus and the enveloped virus human immunodeficiency virus. However,their use in large sero-epidemiological studies or clinical trials is challenging sincethese assays are laborious and difficult to perform. In this study, replacement ofreporter genes, intrduction of novel result-reading method and modification ofbackbone were employed to improve the established methods.The pseudoviron-based neutralization assay is accepted as the gold standard toevaluate the functional humoral immune response against HPV. Two neutralizationassay were developed and optimized, both of which were based on pseudovirus, oneusing Gaussia luciferase (Gluc) and the other using fluorescence proteins as thereporter genes. For this purpose, high-titers pseudovirons were generated bycotransfecting293T cells with HPV structural genes and reporter gene expressingplasmids. For the Gluc assay, six types of neutralizing monoclonal antibodies,vaccine-immunized serum samples and WHO international antibody standard wereused to validate the new developed assay. The ideal circumstances of the assay wereidentified for cell counts (30,000/well for96-well plate), pseudoviron inoculating size(100times RLU above background for the virus control) and incubation time (72hours). The sensitivity of the Gluc assay was comparable to secreted alkalinephosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay.The non-specific background for different types of sample were significantly different(rabbit sera>human sera>mouse sera, p<0.01). The non-specific neutralization effectswere not attributed to IgG antibody. The cutoff value for this assay was determined as50%inhibition at a dilution of1:40. Without requirements of sample dilution anddifferent incubation times at different temperatures before processing, the detectiontime was shortened from more than90min to less than5min for a96-well platecompared with the SEAP-based assay. For the fluorescence assay, the Fluorospottechnology was employed to read the results of the assay. And the noval Fluorospotwere compared to the classical FACS method, the results of which showed goodcorrelation with R2of great than0.98. Based on the Fluorospot, two-colorneutralization assay were developed to test two type of neutralizing antibodiessimultaneously, which further enhanced the efficiency of the assay. The two-color neutralization assay is more suitable for large sero-epidemiological studies or clinicaltrials and more ameNAble to automation.Fifty HPV16/18vaccine-immunized samples were tested using the Gluc method.Cross-neutralizaiton was observed between HPV16-HPV31and HPV18-HPV45. Butthe titers for the non-vaccine types were significantly lower than that for the vaccinetypes. For the natural infection samples, the positive rate of neutralizing antibody was84%in the332HPV DNA positive samples. HPV16-HPV31, HPV16-HPV33,HPV31-HPV45, HPV33-HPV35, HPV52-HPV6, HPV18-HPV45andHPV59-HPV68were significantly more likely to exist in the same sample. The reasonfor this phenomenon might not be the cross-neutralization between the geneticallyrelated HPV types but the coinfeciton or superinfection of multiple types. No specificassociation was observed between the HPV neutralizing antibody and cytologicalresults.Similar to HPV neutralizaition assay, pseudovirus was also employed in theanalyses of HIV neutralization and drug resistance. To develop a high throughputhuman immunodeficiency virus (HIV) phenotypic drug resistance assay, the originalbackbone plasmid was modified. The parental backbone plasmid pNL4-3.Lac wasconstructed, of which the luciferase was inactivated and bearing the LacZ geneinstead of protease and reverse transcriptase genes. The factors that may affect themethod were optimized, including cell numbers (10,000/well), virus inoculation (200TCID50/well) and DEAE-dextran quantity (15μg/ml). The reproducibility of thisassay was confirmed by testing12anti-HIV drugs against the pseudovirus. Sixpseudoviruses were constructed and tested against the12anti-HIV drugs, the resultsof which were compared with the pSG3pseudovirus assay established previously inour laboratory. The two methods presented good consistency. So, a high throughputhuman immunodeficiency virus phenotypic drug resistance assay was developed,which not only could be used to analyze the drug resistance patterns of HIV-1infectors but only screened new drugs for antiretroviral therapy quickly andeffectively.In addition, pseudovirus platform was introdued to the eiptope analysis of themonoclonal neutralizing antibodiies. Neutralizing monoclonal antibodies IgG1b12(b12) could recognize conformational epitopes that overlap the CD4bs of Env, whichwas an important conserved target for anti-human immunodeficiency virus type1(HIV-1) neutralizing antibodies. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the keyamino acid residues affecting b12neutralization susceptibility using single genomeamplication and pseudovirus neutralization assay in this study. Eleven amino acidresidues were identified to affect the sensitivity of the Env to b12. Throughsite-directed mutagenesis, an amino acid substitution at position182in the V2regionof Env was conformed to play a key role in regulating the b12neutralizationsusceptibility. The introduction of V182L to a resistant strain enhanced its sensitivityto b12more than two fold. Correspondingly, the introduction of L182V to a sensitivestrain reduced its sensitivity to b12more than tenfold. Amino acid substitution atpositions267and346could enhance the sensitivity to b12more than two foldrespectively. CRF07_BC is a major circulating recombinant form of HIV-1in China.Our data may provide important information to understand the molecular mechanismregulating the neutralization susceptibility of CRF07_BC viruses to b12and may behelpful for the vaccine design targeting the CD4bs epitopes. |
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