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. 2024 Feb 21;20(2):e1012023.
doi: 10.1371/journal.ppat.1012023. eCollection 2024 Feb.

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

Sydney J Bennett  1   2 , Dicle Yalcin  2 , Sara R Privatt  1   2 , Owen Ngalamika  3 , Salum J Lidenge  4   5 , John T West  2 , Charles Wood  1   2
Affiliations

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

Sydney J Bennett et al. PLoS Pathog. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Overview of the antibody responses against the KSHV proteome.
(A) The antibody repertoire of 59 KS patients and 22 ASY individuals was determined using a phage library expressing 56-mer peptides scanning the entire KSHV proteome. Quantification of the Ab response is represented as breadth (i.e., sum of reactive peptides) and magnitude (i.e., MLXP: -log10(p)). Created with BioRender.com. (B) and (C) The log10-scaled average magnitude of the antibody responses to KSHV proteins for each individual is represented as a heatmap. See also S1 Fig. Abbreviations: Kaposi Sarcoma (KS), asymptomatic (ASY), phage immunoprecipitation and sequencing (PhIP-Seq), endemic KS (EnKS), epidemic KS (EpKS), human immunodeficiency virus 1 (HIV).
Fig 2
Fig 2. The breadth and magnitude of the antibody response against KSHV.
(A) The breadth and (B) magnitude of the antibody response against KSHV was compared between KS and ASY. Significant comparisons were determined using Mann-Whitney statistical tests. In the violin plots, each data point represents an individual, the dashed lines represent the median, and the dotted lines represent the quartiles. (C) Pearson correlation and linear regression of breadth and magnitude, where the solid line represents the goodness-of-ft, and the dashed lines represent the 95% confidence interval. See also S2 and S6 Figs. Abbreviations: Kaposi Sarcoma (KS), asymptomatic (ASY), ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.
Fig 3
Fig 3. The magnitude of response against functional groups of KSHV proteins.
Each KSHV protein was categorized into a functional group, and the magnitude of the antibody response to each functional group as well as the individual protein was compared. Significant comparisons were determined using the Friedman test with Dunn’s multiple comparisons post hoc tests in (A). The functional groups were compared between the ASY and KS cohorts. In the volcano plots, each data point represents an individual, the middle line represents the median, and the outer lines represent the quartiles. (B) Means with standard errors of the mean (SEM) are shown. See also S3 and S4 Figs. Abbreviations: Kaposi Sarcoma (KS), asymptomatic (ASY), ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.
Fig 4
Fig 4. Epitope map of immunodominant proteins–ORF65 (capsid) and K8.1 (glycoprotein).
For (A) ORF65 and (B) K8.1, the known secondary structure and motifs are annotated across the proteins, followed by the multiple sequence alignment (MSA) of reference (GK18, JSC-1) and input library (E5LBZ2, Unspliced, K8.1A, K8.1B) sequences. Gray represents 100% conservation among the sequences, while mismatched residues are colored by their physiochemical properties as defined by the Zappo color scheme (green: hydrophilic, salmon: aliphatic/aromatic, orange: aromatic, fuchsia: conformationally special, yellow: cysteine only, red: negatively charged, blue: positively charged). Further, each peptide targeted by at least one individual was also aligned and colored by the percent reactivity (brown). To the right, the scaled average magnitudes (blue) are shown as a heatmap. Finally, the percent reactivity (sage) and average magnitude (black lines) of the amino acid-level responses are shown for KS and ASY. See also S5 Fig. Abbreviations: Kaposi Sarcoma (KS), asymptomatic (ASY), transmembrane (TM), s: small (A/C/D/G/N/P/S/T/V), u: tiny (A/G/S).
Fig 5
Fig 5. The effect of disease and HIV on the breadth and magnitude of the antibody response to KSHV proteins.
(A) The breadth and (B) magnitude of the antibody response to each KSHV protein were compared between KS and ASY. Means with standard errors of the mean (SEM) are shown. Additionally, the HIV status of the participants was considered when comparing the (C) breadth and (D) magnitude where each data point in the volcano plot represents a KSHV protein. Significant comparisons were determined using Mann-Whitney tests, and the underlines denote comparisons that remained significant after multiple hypothesis corrections using the two-stage step-up method (Benjamini, Krieger, and Yekutieli; FDR = 10%). See also S6 Fig. Abbreviations: Kaposi Sarcoma (KS), asymptomatic (ASY), human immunodeficiency virus 1 (HIV), endemic KS (EnKS), epidemic KS (EpKS), ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.
Fig 6
Fig 6. The effect of HIV on the overall antibody response to KSHV in KS and ASY.
(A) The breadth and (B) magnitude of the antibody response to KSHV were compared between EnKS, EpKS, HIV- ASY, and HIV+ ASY. Significant comparisons were determined using Kruskal-Wallis with Dunn’s multiple comparisons post hoc statistical tests. In the violin plots, each data point represents an individual, the dashed lines represent the median, and the dotted lines represent the quartiles. Abbreviations: Kaposi Sarcoma (KS), asymptomatic (ASY), human immunodeficiency virus 1 (HIV), endemic KS (EnKS), epidemic KS (EpKS), ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.
Fig 7
Fig 7. Top discriminative peptides predictive of KS/ASY status.
Discriminative peptides from the top-performing model (Model 5) are presented as a superimposed bar plot, where each bar represents the percent reactivity to the given peptide per group. Significant statistical differences in percent reactivities between KS and ASY were determined using Z-test for proportions. See also S7, S8, S9, and S10 Figs. Abbreviations: Kaposi Sarcoma (KS), asymptomatic (ASY), ***p<0.001, **p<0.01, *p<0.05.
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