br Conclusion br Disclosure of

Conclusion
Disclosure of interest
Introduction The Joint United Nations Program on HIV and AIDS (UNAIDS) proposed the ambitious global goal of achieving the "90-90-90" target by 2020 and ending the HIV epidemic by 2030 (Barton-Knott, 2014). One important measure is to monitor the incidence of HIV-1 infection by detecting recent HIV-1 infection with HIV-1 incidence assays. In practice, the incidence assays are used to distinguish ‘recent’ from ‘long-term’ infection in a cross-sectional study based on the evolving nature of anti-HIV antibody with respect to their quantity (Janssen et al., 1998), proportion (Parekh et al., 2002), avidity (Chawla et al., 2007; Suligoi et al., 2002), or isotope (Viana et al., 2018). Since the 1990s, a number of incidence assays have been reported, such as the BED-capture-enzyme immunoassay (BED-CEIA) to detect the proportion of the specific anti-HIV gp41 IgG antibodies relative to the total IgG (Parekh et al., 2002), and the limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) to measure the avidity of antibodies against the immunodominant epitopes (IDEs) of HIV-1 gp41 (Duong et al., 2012). However, the test properties of currently reported incidence assays did not meet the criteria indicated in the ‘target product profile’ (TPP) such as large mean duration of recent infection (MDRI) and small false-recent rate (FRR) for tests of recent infection (Anon, 2019). A profound variation has been reported among the populations tested due to the differences in HIV-1 subtypes or genotypes, epidemic phases, levels of total IgG, HIV-1 viral loads and CD4+ cell counts as well as the extent of antiretroviral therapy (ART) use (Guy et al., 2009). Therefore, the performance of HIV-1 subtype or genotype-specific incidence test would require further investigation. The development of an incidence assay that is cross-reactive to the dominant genotypes of HIV-1 or refinement of the incidence assays according to the predominant and prevalent HIV-1 strains would be highly desirable for its application. We have previously reported a 57-mer peptide, gp41-p57, located at the loop region of HIV-1 gp41 that can differentiate recent and long-term HIV-1 infections (Li et al., 2016; Chong et al., 2015). In this study, we further determined the major IDEs within the gp41-p57 peptide and the role of the amino JQ1 sequences surrounding the gp41 IDEs, as well as the impact of HIV-1 genotype or subtype on the performance of HIV-1 genotype specific peptide-based immunoassay in distinguishing recent and long-term HIV-1 infections.
Materials and methods
Results
Discussion In this study, we confirmed the important role of the 57-mer HIV-1 gp41 peptide with two major IDEs of GKIIC and QKFLG motifs located in the loop region of HIV-1 gp41 in distinguishing recent and long-term HIV-1 infection. We also found that both the upstream and downstream amino acid residues of the major HIV-1 gp41 IDEs could block the binding of anti-gp41 antibody with the 57-mer gp41 peptide in particular for the immature and low avidity anti-HIV antibody from HIV-1 recently infected subjects. These results could explain why the relative high concentration (2–8 μg/ml) of the 57-mer gp41 peptide was used in our assays. In contrast, the current HIV-1 incidence assays, such as LAg-Avidity EIA, mainly depends on limiting the concentration of the coated antigen to restrict the binding with low avidity anti-HIV antibody (Duong et al., 2012; Wei et al., 2010). HIV incidence assays have been proved to be HIV-1 genotype specific (Young et al., 2003; Parekh et al., 2001). Their sensitivity and FRR vary across HIV-1 strains and geographical regions (Hallett et al., 2009; Karita et al., 2007). For example, HIV-1 subtype D has been repeatedly reported to be associated with misclassification of HIV-1 recent infections (Longosz et al., 2014). To overcome the problems associated with testing different HIV-1 subtypes, Parekh et al. developed a BED-CEIA assay by using branched synthetic peptide that incorporates immunodominant gp41 sequences from multiple subtypes including HIV-1 subtype B, E, and D (Parekh et al., 2002). Unfortunately, BED-CEIA was not available due to the lack of the specific branched peptides. Subsequently, Parekh et al. developed the LAg-Avidity EIA by using the recombinant fusion protein including the IDEs of HIV-1 gp41 from three major HIV-1 variants (Wei et al., 2010). In our study, we further addressed the effect of HIV-1 genotypes on the performance of HIV-1 incidence testing. We found that misclassification could occur if a single peptide was used for distinguishing recent HIV-1 infection (Table 1). Therefore, we adapted the gp41 peptides from multiple dominant HIV-1 genotypes CRF01_AE, CRF07_BC/CRF08_BC and subtype B in China. As expected, both the MP3 and MP4-based immunoassays using antigens from multiple HIV-1 genotypes increased the testing sensitivity by 5–30% (Table 1). These results suggest that the development of HIV-1 incidence assay must include antigens covering the local predominant HIV-1 strains in order to improve the testing sensitivity.