Antibody profiling and predictive modeling discriminate between Kaposi sarcoma and asymptomatic KSHV infection

Protein-level immunodominance patterns against Kaposi sarcoma-associated herpesvirus (KSHV), the aetiologic agent of Kaposi sarcoma (KS), have been revealed from serological probing of whole protein arrays, however, the epitopes that underlie these patterns have not been defined. We recently demonstrated the utility of phage display in high-resolution linear epitope mapping of the KSHV latency-associated nuclear antigen (LANA/ORF73). Here, a VirScan phage immunoprecipitation and sequencing approach, employing a library of 1,988 KSHV proteome-derived peptides, was used to quantify the breadth and magnitude of responses of 59 sub-Saharan African KS patients and 22 KSHV-infected asymptomatic individuals (ASY), and ultimately to support an application of machine-learning-based predictive modeling using the peptide-level responses. Comparing anti-KSHV antibody repertoire revealed that magnitude, not breadth, increased in KS. The most targeted epitopes in both KS and ASY were in the immunodominant proteins, notably, K8.129-56 and ORF65140-168, in addition to LANA. Finally, using unbiased machine-learning-based predictive models, reactivity to a subset of 25 discriminative peptides was demonstrated to successfully classify KS patients from asymptomatic individuals. Our study provides the highest resolution mapping of antigenicity across the entire KSHV proteome to date, which is vital to discern mechanisms of viral pathogenesis, to define prognostic biomarkers, and to design effective vaccine and therapeutic strategies. Future studies will investigate the diagnostic, prognostic, and therapeutic potential of the 25 discriminative peptides. Kaposi sarcoma-associated herpesvirus (KSHV) is the aetiologic agent of sub-Saharan African endemic Kaposi sarcoma (EnKS) and HIV-associated epidemic KS (EpKS). The humoral immune response against KSHV has been studied using infected cell immunofluorescence assays and immunoassays against recombinant proteins. The latency-associated nuclear antigen (LANA) has been established as the most antigenic KSHV protein, and seroreactivity to LANA and the glycoprotein K8.1 are common determinants of KSHV infection. While protein-level antigenicity of a near-complete KSHV proteome has been reported, the epitopes within those proteins have not yet been defined. We have recently demonstrated the utility of phage display in facilitating the first high-resolution epitope mapping of the highly antigenic LANA protein and now apply that same approach to the entire KSHV proteome. Our study presents the most comprehensive and highest-resolution antibody profiling of the KSHV peptidome to date and provides a direct comparison of responses in KS patients and asymptomatic KSHV-seropositive individuals with and without HIV-1 co-infection. The antibody repertoire and epitopes defined here are valuable to furthering the discovery of prognostic biomarkers as well as designing effective preventatives and therapeutics.