Heterogeneity and Breadth of Host Antibody Response to KSHV Infection Demonstrated by Systematic Analysis of the KSHV Proteome

<div><p>The Kaposi sarcoma associated herpesvirus (KSHV) genome encodes more than 85 open reading frames (ORFs). Serological evaluation of KSHV infection now generally relies on reactivity to just one latent and/or one lytic protein (commonly ORF73 and K8.1). Most of the other polypeptides encoded by the virus have unknown antigenic profiles. We have systematically expressed and purified products from 72 KSHV ORFs in recombinant systems and analyzed seroreactivity in US patients with KSHV-associated malignancies, and US blood donors (low KSHV seroprevalence population). We identified several KSHV proteins (ORF38, ORF61, ORF59 and K5) that elicited significant responses in individuals with KSHV-associated diseases. In these patients, patterns of reactivity were heterogeneous; however, HIV infection appeared to be associated with breadth and intensity of serological responses. Improved antigenic characterization of additional ORFs may increase the sensitivity of serologic assays, lead to more rapid progresses in understanding immune responses to KSHV, and allow for better comprehension of the natural history of KSHV infection. To this end, we have developed a bead-based multiplex assay detecting antibodies to six KSHV antigens.</p></div

Eigenvector converted blood volume (EV1) and oxygenation (EV2) images.

<p>Two adjacent KS lesions over time are shown in the images in arbitrary units of a patient with a confirmed pathological complete response. A digital photograph of the lesions is shown at baseline (week 0).</p

Multi-spectral instrument.

<p>Linearly polarized light is projected onto the skin and diffuse reflectance images are captured at the CCD camera after passing narrow band filters for wavelength selection.</p

Cross-sections through blood volume and blood oxygenation at baseline in a KS lesion.

<p>Cross-sections were chosen to be centered on the lesion, for both blood volume (a) and oxygenation (c). The EV1 and EV2 are depicted in arbitrary units as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083887#pone-0083887-g002" target="_blank">Fig. 2</a>. The average of 5 cross sections for blood volume (b) shows the typical increase and for oxygenation (d) shows a decrease inside the lesion area. A surrounding halo of increased oxygenation can be seen, especially to the immediate right of the lesion. The width of the lines in (b) and (d) reflect the range of +/− 1 standard deviation for the 11 cross sections at each point.</p

Eigenvector converted blood volume (EV1) and oxygenation (EV2) images.

<p>The KS lesion is shown over time in arbitrary units of a patient with progressive disease. A digital photograph of the lesion is shown at baseline (week 0).</p

Patient Characteristics and Response to Treatment.

<p>In patient 6, the target lesion flattened, but some other flat lesions became nodular and new lesions appeared, so the patient met the overall criteria for progressive disease.</p

Time courses of NESD for EV1 and EV2.

<p>(a) Time course for the NESD of blood volume (EV1) for 5 lesions that showed a clinical response (black) and two lesions that did not show a clinical response (grey). Patients whose lesion responded to treatment show a clear decrease in NESD of blood volume (EV1) over time, whereas those whose lesion progressed show an increase. (b) Time course for the NESD of blood oxygenation (EV2); again the 5 lesions that responded clinically are shown in black, and the 2 lesions that did not respond are shown in grey. The errobars shown correspond to the error on the estimate of the standard deviation. If the errorbar is not visible, the error is smaller than the marker size.</p

Heatmap showing seroreactivity of HAMB and RDP subjects to each antigen.

<p>A. Subjects with documented KSHV-associated diseases. B. Healthy blood donors. Subjects are shown in rows, antigens in columns. Color intensity is proportional to background-subtracted optical density (OD). HIV seropositivity is indicated in red; latent KSHV proteins are identified in green.</p

Bead-based assay.

<p><b>A</b>. Comparison of reactivity to individual antigens in RDP and HAMB subjects (Mann-Whitney test) <b>B</b>. Receiver operating characteristics. <b>C</b>. Assessment of signal specificity by antigen pre-adsorption. MFI, Median Fluorescence Intensity.</p

Characteristics of study participants.

#<p>For all subjects, the United States is the country of current domicile, but not necessarily the country of birth; African regions described using United Nations designations.</p><p>*Patients had Kaposi sarcoma and/or primary effusion lymphoma and/or multicentric Castleman's disease in remission (not symptomatic); some had multiple diagnoses.</p>†<p>CD4 counts at time of serologic evaluation available on 33 of 36 HIV-infected subjects.</p