The existence of an antisense Open Reading Frame (ORF) that encodes a putative AntiSense Protein (ASP) on the proviral genome of Human Immunodeficiency Virus type 1 (HIV-1) was a source of debate for 30 years.
During the last years, some progresses have been made to characterize the cellular immune response against ASP in HIV-1 seropositive patients. However, no tools were available for the detection of antibodies to ASP in the plasma of HIV-1-infected patients during the natural course of the infection.
The aim of our study was to develop a Luciferase Immuno-Precipitation System (LIPS) to monitor the quantitative detection of ASP-specific antibodies in the plasma of HIV-1-infected patients [antiretroviral therapy (ART) naive-patients, patients under ART and HIV-1 controllers], patients who discontinued antiretroviral drugs (ARV). We further used this approach to delineate the epitopes of ASP targeted by antibodies.
Antibodies directed against ASP were detected in 3 out of 19 patients who discontinued ARV (15%) and in 1 out of 10 ART-naive patients (10%), but were neither detected in HIV-1 infected patients under ART nor in HIV-1 controllers. Individual variations in levels of ASP-specific antibodies were detected overtime.
Both the conserved prolin-rich motif and the core 60-189 region of ASP were found to be essential for antibody recognition in the four patients tested positive for anti-ASP antibodies, who were all untreated at the time of sampling.
Moreover, for two of these patients, increased levels of ASP-specific antibodies were observed concomitantly to viremia declines. Overall, our method may represent a useful tool to detect a humoral response to ASP in HIV-1-infected patients, which allowed us to confirm the expression of ASP during the course of HIV-1 infection. Further studies will be needed to fully characterize the humoral response to ASP in HIV-1-infected patients.
Comparison of two Immunoassays for Concurrent Detection of HCV Antigen and Antibodies among HIV/HCV Co-infected Patients in Dried Serum/Plasma Spots.
Hepatitis C virus (HCV) antigen/antibody (Ag/Ab) assays offer the benefit of reducing the window period compared to assays that detect only HCV-Ab. In this study the performance of the Murex Ag/Ab (Murex, Abbott) and Monolisa Ag/Ab Ultra (Monolisa, Bio-Rad) ELISAs was compared for the use of filter dried serum/plasma spots (DS/PS) with a focus on the sensitivity and the percentage of correct positive test results.
Correct positive ELISA results were assumed for samples that subsequently tested positive for HCV RNA by RT-qPCR, or RNA negative samples that tested positive in a Western blot (confirmed ELISA results).
Sensitivity was evaluated from DS/PS eluates using HCV seroconversion panels [plasma samples of subtypes-(St) 1a, 2b)] and longitudinal HCV antibody-positive serum panels (St 1b, 2b, 3a, and 4d). The proportion of correct positive test results was evaluated using 1102 newly-diagnosed HIV-positive clinical dried serum spots DSS eluates for screening of potential HCV co-infection.
For the plasma HCV seroconversion samples, which were used as a reference for DSS eluates, the Murex became reactive earlier for antigen-positive bleeds. However, for the HCV antibody-positive eluates and dilutions thereof, the Monolisa demonstrated a superior sensitivity.
Of the clinical DSS 22.8% (28/123) of samples reactive in the Murex were negative in a subsequent RT-qPCR and Western blot, while only 1.9% (2/105) of the samples reactive in the Monolisa were negative in these confirmatory assays. Our results indicate that the Monolisa provides fewer false positive results for HCV detection in DSS, whereas for undiluted plasma or serum samples, the Murex can serve as an additional diagnostic tool to narrow the window period.