A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients

Abstract

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.

Antisense Transcripts and Antisense Protein: A New Perspective on Human Immunodeficiency Virus Type 1

“…The ability of HIV-1 to produce antisense transcripts and ASP in vitro has been well established; many studies have described the expression of antisense transcripts and ASP in various HIV-1 infected cells, including T-cells, monocyte-derived macrophages, and dendritic cells ( Ludwig et al, 2006 ; Landry et al, 2007 ; Clerc et al, 2011 ; Kobayashi-Ishihara et al, 2012 ; Torresilla et al, 2013 ; Affram et al, 2019 ). Moreover, CD8 + T cells and antibodies targeting ASP were detected in HIV-1 infected patients ( Vanhée-Brossollet et al, 1995 ; Berger et al, 2015 ; Bet et al, 2015 ; Savoret et al, 2020 ). Bioinformatic approaches demonstrated that a conserved asp gene was created, concomitant with the emergence of the HIV-1 pandemic group M ( Cassan et al, 2016 ), further supporting the idea that the asp gene might now be considered as the 10th gene of HIV-1.…”

“…The creation of de novo proteins appears to be a significant element in the evolution of viruses ( Li and Ding, 2006 ; Rancurel et al, 2009 ). Recent de novo creation of asp gave rise to a translation product which has been named ASP ( Miller, 1988 ; Vanhée-Brossollet et al, 1995 ; Clerc et al, 2011 ; Torresilla et al, 2013 ; Berger et al, 2015 ; Bet et al, 2015 ; Affram et al, 2019 ; Savoret et al, 2020 ), a small strongly hydrophobic protein consisting of 189 amino acids (reference sequence HXB2). ASP contains 14 conserved cysteine residues, seven of which are located in the N-terminal region (cysteine 7 C and two cysteine triplets 10 CCC 12 and 22 CCC 24 ), two SH3 domain-binding motifs ( 47 PXXPXXP 53 ), and two strongly hydrophobic putative transmembrane (TM) domains ( Figure 4 ).…”

“…Unfortunately, Western blotting did not allow the frequency of patients displaying antibodies against ASP to be accurately determined. Recently, a quantitative technique known as Luciferase Immunoprecipitation System (LIPS) was described to assess the antibody response targeting ASP in a panel of HIV-1-infected patients ( Savoret et al, 2020 ). LIPS is an assay that was initially developed to quantitatively detect antibodies targeting a particular antigen in patient biological samples ( Figure 5 ; Burbelo et al, 2005 ).…”

“…Using LIPS, the breadth of the antibody response directed against different viral antigens, including HIV-1 whole proteome, has previously been reported ( Burbelo et al, 2007 , 2014 ; Enose-Akahata et al, 2013 ; Furuta et al, 2015 ). Of note, the use of this technique allowed the quantitative study of the antibody response targeting ASP, which was detected in 10–15% of the HIV-1-infected patients ( Savoret et al, 2020 ).…”

“…The frequency of patients displaying antibodies to ASP is similar to those observed for both HBZ ( Enose-Akahata et al, 2013 ; Shiohama et al, 2016 ) and the auxiliary and regulatory proteins of HIV-1 ( Reiss et al, 1990 ; O’Neil et al, 1997 ; Rezza et al, 2005 ). The ASP-specific antibody response was sustained for at least 9 months and seemed to target both the 26–62 residues of ASP bearing the highly conserved proline-rich motif and the core residues 62–141 of ASP ( Figures 2 , 6A ; Savoret et al, 2020 ). Cytotoxic CD8 + T cells targeting a panel of ASPs overlapping peptides were also reported in 30% of the HIV-1 infected patients ( Berger et al, 2015 ; Bet et al, 2015 ).…”