Development Of The Rapid Identification Diagnosis Kit For Distinction HIV Natural Infections Generated Antibodies From That Induced By HIV Vaccine

Most of the current human immunodeficiency virus (HIV) vaccine candidates contain multiple viral components and generate antibodies that correspond with HIV infections. Vaccinees having antibodies that react with licensed HIV diagnostic kits may result in a false positive diagnosis. Understandably, false diagnoses could lead to long-term social and economic harms for the vaccinee, and indirectly impede the research and development of the HIV vaccine. To avoid the false diagnosis, Khurana et al. screened out three short peptides by biopanning using gene fragment phage display library (GFPDL) with HIV-1 early-phase seroconversion sera. These three peptides have been identified to differentiate vaccine from virus-induced antibodies. In the study, we attempted to obtain these three peptides by way of recombinant method and chemical synthesis, and compared the specificity and sensitivity of these peptides that produced in the two methods. The well performance peptides were used to prepare HIV collocidal gold rapid flow-through kit to effectively distinguish antibodies induced by vaccine from that induced by virus.We obtained the recombinant polypeptides by way of prokaryotic and proved that some of these polypeptides could react with HIV positive serum but didn't with HIV negative serum and DNA-tiantan vaccine induced serum. Purified recombinant peptides were obtained by affinity chromatography, but these short peptides were liable to be degradation. The results of ELISA kits that based on these peptides in detection with clinical sample were unfavourable. The result of dot-blot and ELISA detection showed that all the three synthesized peptides shared the excellent recactivity and specificity, and these peptides were applied to develop a novel flow-through kit. The novel kit based these three peptides showed 92% sensitivity and 100% specificity in the detection of clinical samples. The flow-through kit and ELISA kit showed a good coincidence (P>0.05). After combined with recombinant protein gp41 and gp36, the kit showed 100% sensitivity in diagnosis.In conclusion, a novel rapid flow-through kit was developed and it provided an effective method to detect HIV infection and to differentiate true HIV infections from vaccinees. Objective: To obtain HIV-1 antigen peptides that could differentiate vaccine-induced antibodies and those from virus-inducedMethods: 1. prokaryotic expression: The genes of sk1, sk2, sk3 and sk1-sk2-sk3 were amplified by PCR and overlap extension PCR,then respectively inserted the target genes into the expression vectors pET32a(+) and pGEX4T-2. The recombinant vectors were transformed into E.coli BL21(DE3)and induced by IPTG. The fusion peptides contained His or GST tag were purified by Ni-affinity chromatography or GST-affinity chromatography and analysed by SDS-PAGE. The antigenicity of these products was assayed by immunoblot using HIV positive serum. The ELISA kits were prepared with these recombinant peptides and the performances of the kits were evaluated by clinical samples. 2. chemical synthesis: HIV-1 peptides were obtained by way of chemical synthesis and analysed for antigenicity by dot-blot, and the specificity and sensitivity of ELISA kits were analysed by clinical samples.Result: 1. prokaryotic expression: His-sk1,His-sk123,His-sk213,His-sk231 and GST-sk3,GST-sk123,GST-sk213 were respectively expressed in pET32a(+) and pGEX4T-2. HIV-1 positive serum and HIV DNA-Tiantan vaccine serum western bloting proved that all of these expression products have the biological activity. When these fusion peptides used in ELISA kits, the sensitivity was less than 50% and the specificity was less than 90%. 2.chemical synthesis: The result of dot-blot showed that chemical synthesis peptide sk1 well reacted with HIV-1 positive serum, sk2 and sk3 have lower reactivity. Synthesis peptides didn't react with HIV negative serum sample and HIV Tiantan vaccines induced serum sample. The ELISA results showed that three peptides had 92% sensitivity and 100% specificity in detection of clinical samples.Conclusion: Three HIV peptides were obtained which have excellent biological activity and could be hopefully applied in HIV differential diagnosis kit. Objective: To develop a novel rapid flow-through test kit using the peptides that shared well sensitivity and specificity for detection HIV infection and differentiation HIV vaccine from virus elicited antibodies in HIV vaccine clinical trial.Methods: Developed a novel rapid flow-through test kit using chemical synthesis peptides combined with recombinant proteins of gp41 and gp36. The sensitivity of kit was obtained by detecting 100 HIV-1 positive samples, and the specificity was evaluated by detecting 30 HIV-1 Tiantan vaccines samples and 190 HIV-uninfected samples (100 normal, 44 HBV, 36 HCV and 10 TB). The sensitivity and specificity of the flow-through kit were compared with ELISA kit prepared in the study.Result: The novel kit based on these three peptides in detection clinical samples showed 92% sensitivity and 100% specificity. After being combined with gp41 and gp36, the kit showed 100% sensitivity in diagnosis. The novel kit and ELISA kits showed the good coincidence (P>0.05).Conclusion: The novel flow-through kit is a simple, time-conserving and effective method to detect HIV infection and differentiate true HIV infection from vaccine induced antibodies, thus the kit may be suitable for use in clinical settings in the future.