Strengths The VirScan assay has several advantages over alte
The VirScan assay has several advantages over alternative multiplex serological assays for peptide discovery. These advantages include quantitative assessment of antibody binding to peptides that span all open reading frames in the HIV genome, including both structural and regulatory proteins; representation of a wide range of HIV subtypes and strains, including groups M, N, and O, and HIV-2 (Table S4); and fine resolution for epitope identification, which can be further refined with alanine scanning mutagenesis. The assay also provides information about antibody binding to >200 other human viruses. In this report, data from other viral peptides were used to normalize peptide binding measures and allowed us to compare the impact of ART on the antibody response to a prevalent non-HIV viral infection (EBV). Data from the same assay runs could be used to examine the evolution and fine specificity of Strengths to other viruses, and the impact of viral co-infections on the anti-HIV antibody response. Future studies could also explore use of the VirScan assay to identify serosignatures for estimating incidence of other viral infections, such as hepatitis C virus. Finally, future phage libraries composed of additional protein products, such as those from the gut microbiome, may be used to explore the impact of immune system pre-conditioning on the response to HIV infection. Further evaluation of serosignatures will be performed using sophisticated statistical and machine learning approaches that we used in previous studies to evaluate multi-assay algorithms for cross-sectional incidence estimation (e.g., algorithms included the LAg-Avidity assay, other serologic assays, CD4 cell count and HIV viral load) (Konikoff et al., 2013, Laeyendecker et al., 2018). Those studies used large datasets from individuals with HIV infection duration ranging from 1 month to >8 years (e.g., data from 2,442 samples from 278 adults with subtype C infection [Laeyendecker et al., 2018]; data from 1,782 samples from 709 adults with subtype B infection [Laeyendecker et al., 2013]). We will use the electrochemiluminescent assay described above to obtain peptide-binding data for the same sample sets. We will use those data to compare serosignatures that include different sets of peptides, weighting for individual peptides, and different cutoffs for antibody binding to each peptide included in the models; the analyses will also determine key performance characteristics of different serosignatures (e.g., the mean window period for recent infection). As a final step, performance of serosignatures will be validated by comparing cross-sectional HIV incidence estimates to HIV incidence observed in longitudinal trials and cohort studies. This study has several limitations. One limitation of the VirScan assay is that it does not allow evaluation of discontinuous or highly conformational epitopes, or epitopes that include post-translational modification, such as glycosylation. In this study, we used protein A/G-coated magnetic beads for immunoprecipitation of all IgG subclasses. Analyses of IgA or specific IgG subclasses may provide complementary information (Kadelka et al., 2018). Another limitation of this report is that the sample cohort included only women. The samples in the discovery set all had subtype C HIV; the validation set included subtype C and D samples. Further studies are needed to compare these data to data from samples from other geographic regions where other HIV subtypes and strains circulate. The preliminary studies described in this report also did not include viremic controllers who naturally suppress HIV infection, or individuals who initiated ART early in infection. It would be worthwhile to evaluate the serologic responses to HIV infection in those populations, since viremic control and early suppression of viral replication can impact the production and evolution of HIV antibodies (Koenig et al., 2013).