An improved microtiter assay for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma

BACKGROUND: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. METHODS: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor(s), the tested sera or plasma was removed by a washout procedure after initial 3–6 h culture in the assay. RESULT: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. CONCLUSION: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma

Identification and Characterization of a New Class of Human Immunodeficiency Virus Type 1 Recombinants Comprised of Two Circulating Recombinant Forms, CRF07_BC and CRF08_BC, in China

We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on gag-reverse transcriptase sequences of 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new “second-generation” recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants

Evaluation of Cynanchum otophyllum glucan sulfate against human immunodeficiency virus and herpes simplex virus as a microbicide agent

Objective : The root of Cynanchum otophyllum -also known as Qing Yang Sheng-is a traditional ethnical Chinese medicine. The objective of this study was to evaluate in vitro activities and safety of C. otophyllum glucan sulfate (PS20) against Human Immunodeficiency Virus (HIV) and Herpes Simplex Virus (HSV). Materials and Methods : Anti-HIV activity was detected with syncytial formation assay and quantitative P24 Enzyme-Linked Immunosorbent Assay (ELISA). Anti-HSV activity was detected with plaque reduction assay; cytotoxicity was tested with MTT colorimetric assay; and anti-bacterial activity was tested with microdilution method. Anti-HIV mechanism was investigated with fusion inhibition, time of addition, and pretreatment. Results : The 50% Inhibition Concentration (IC 50 ) of PS20 for HIV-1 IIIB , HIV- Ada-M , HIV-1 Bal , HSV-I, and -II were 0.26 ± 0.02 mM, 0.46 ± 0.02 mM, 0.90 ± 0.04 mM, 3.45 ± 0.85 mM, and 0.70 ± 0.22 mM, respectively. Selectivity Indices (SI) were 653, 50, 39, 85, and 362, respectively. Studies on anti-HIV mechanism of PS20 showed that the target molecule should be the envelope protein. The 50% Cytotoxicity Concentrations (CC 50 ) of PS20 for HeLa and ME-180 cell lines and human foreskin fibroblast cells was more than 70 mM. The Minimum Inhibitory Concentration (MIC) for vaginal lactobacilli was more than 1000 mM. Conclusion : PS20 possesses anti-HIV and HSV effect and low cytotoxicity to epithelium cells and vaginal lactobacilli. It may be considered as a potential microbicide agent for further investigation