Antibody epitope profiling of the KSHV LANA protein using VirScan

The humoral antibody response against Kaposi sarcoma-associated herpesvirus (KSHV) in infected individuals has been characterized demonstrating the latency-associated nuclear antigen (LANA) as the most antigenic KSHV protein. Despite the antigenicity of the protein, specific LANA epitopes have not been systematically characterized. Here, we utilized a bacteriophage T7 library, which displays 56-amino acid KSHV LANA peptides with 28-amino acid overlap (VirScan), to define those epitopes in LANA targeted by antibodies from a cohort of 62 sub-Saharan African Kaposi sarcoma (KS) patients and 22 KSHV-infected asymptomatic controls. Intra- and inter-patient breadth and magnitude of the anti-LANA responses were quantified at the peptide and amino acid levels. From these data, we derived a detailed epitope annotation of the entire LANA protein, with a high-resolution focus on the N- and C-termini. Overall, the central repeat region was highly antigenic, but the responses to this region could not be confidently mapped due to its high variability. The highly conserved N-terminus was targeted with low breadth and magnitude. In a minority of individuals, antibodies specific to the nuclear localization sequence and a portion of the proline-rich regions of the N-terminus were evident. In contrast, the first half of the conserved C-terminal domain was consistently targeted with high magnitude. Unfortunately, this region was not included in LANA partial C-terminal crystal structures, however, it was predicted to adopt predominantly random-coil structure. Coupled with functional and secondary structure domain predictions, VirScan revealed fine resolution epitope mapping of the N- and C-terminal domains of LANA that is consistent with previous antigenicity studies and may prove useful to correlate KSHV humoral immunity with pathogenesis. Author summary The incidence of Kaposi sarcoma (KS) coincides with high prevalence of Kaposi sarcoma-associated herpesvirus (KSHV) infection, particularly in sub-Saharan Africa, Peru, and the Mediterranean. KSHV infection typically occurs during childhood and enters latency typified by the expression of latency-associated nuclear antigen (LANA). Despite recognition that LANA is highly antigenic, no previous study has defined the targeted epitopes. Our study presents the first high-resolution epitope map of LANA in KS patients and asymptomatic KSHV-infected individuals. High-throughput phage display, VirScan, was leveraged to elucidate epitopes across the LANA protein and compare them between KS and asymptomatic groups. A 24-amino acid peptide was found to be highly antigenic in both groups, regardless of co-infection with human immunodeficiency virus (HIV). In KS patients, HIV co-infection was associated with a lower response to this epitope, but HIV co-infection had little to no effect in the absence of cancer. Further studies of the identified epitope will determine whether the epitope has therapeutic, diagnostic, or viral decoy potential. The application of our novel findings will be instrumental when applied to the entire KSHV proteome in designing effective vaccines for KSHV infection and therapeutics to prevent progression to KS.

Automated summary

This study presents the first high-resolution epitope map of the latency-associated nuclear antigen (LANA) in Kaposi sarcoma (KS) patients and asymptomatic KSHV-infected individuals using high-throughput phage display (VirScan). The results show that a 24-amino acid peptide on LANA is highly antigenic in both groups, regardless of co-infection with human immunodeficiency virus (HIV). HIV co-infection is associated with a lower response to this epitope in KS patients, but has little to no effect in the absence of cancer. Further studies on this identified epitope may have therapeutic, diagnostic, or viral decoy potential. The application of these findings could be important for designing effective vaccines and therapeutics for KSHV infection and prevention of progression to KS.