Discovery of Potential Diagnostic and Vaccine Antigens in Herpes Simplex Virus 1 and 2 by Proteome-Wide Antibody Profiling

Abstract

c Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1-or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1-and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli. Herpes simplex virus 1 (HSV-1) and HSV-2 cause significant human morbidity. HSV-2 is the causative agent of most recurrent genital herpes lesions and is sexually transmitted. Infections are often asymptomatic, and most infected individuals are unaware of the infection, yet HSV-2 is associated with an increased risk of HIV acquisition (33) and an increased risk during pregnancy of spontaneous abortion, premature birth, and perinatal herpes (12, 13). Unawareness of HSV-2 infection is also a major contributing factor to transmission to uninfected partners (64,65). In contrast, HSV-1 is usually transmitted during childhood and is found to be associated predominantly with orolabial infections (cold sores). Also, HSV-1 infection of the eye (ocular herpes) is the most common cause of infectious corneal blindness in industrialized countries. Both HSV-1 and HSV-2 establish lifelong latent infections within the dorsal root and trigeminal ganglia and are characterized by periodic reactivation and virus shedding from mucocutaneous epithelium. Owing to the different natural histories and outcomes of HSV-1 and -2 infections, accurate diagnosis of the HSV type is important for patient management and prognosis and controlling potential transmission. For example, knowing the specific HSV type can help the patient take appropriate precautions to prevent transmission of the disease to others. In particular, the identification of unrecognized HSV-2 infection can be used to carefully monitor virus shedding during pregnancy and minimize the risk of perinatal infection.Laboratory tests for HSV infection include virus culture, virus neutralization, PCR, and serological tests. Virus culture is considered the "gold standard" in the early stages of a primary infection. However, it is less sensitive during the healing stage of infection or during recurrent infections. Culture testi...

Functional Characterization of Ocular-Derived Human Alphaherpesvirus Cross-Reactive CD4 T Cells

“…These responses are of potential interest for vaccine development to confer cross-protection but may also be involved in immunopathogenesis. Whereas various aHHV cross-reactive B cell target Ags have been identified (52)(53)(54), T cell immunity directed to multiple aHHV is restricted to HSV-1 and HSV-2 (29,55,56). The current study reports three main findings.…”

Immunodominant “Asymptomatic” Herpes Simplex Virus 1 and 2 Protein Antigens Identified by Probing Whole-ORFome Microarrays with Serum Antibodies from Seropositive Asymptomatic versus Symptomatic Individuals

“…The construction of HSV-1 and HSV-2 proteome microarrays was described in detail in a previous report (39); proteome microarrays were fabricated, as described previously (19,20,54), by the PCR amplification of coding sequences in genomic DNA, followed by the insertion of amplicons into a T7 expression vector by homologous recombination and expression in coupled in vitro transcriptions-translations (IVTT) prior to printing onto microarrays. Gene sequences for PCR primer design were obtained from the NCBI (accession no.…”

“…Details on the potential use of the identified antigens for diagnostic purposes at a point of care (POC) were discussed in a previous report (39).…”

“…The construction of HSV-1 and HSV-2 proteome microarrays was described in detail in a previous report (39). Briefly, HSV-1 strain 17 template DNA was supplied as 5 overlapping genomic fragments cloned into cosmids (17).…”

“…A serum sample was defined as being seropositive for a particular antigen if the signal was ϾC plus 2 SD (where C is the average signal of 16 control spots of IVTT reaction mixtures lacking template DNA). Reactive antigens were ranked by the normalized signal intensity and by Benjamini-Hochberg-corrected P values after comparing the seropositive and seronegative donors by using Bayesian t tests, as we described previously (39). The intensity of antibody responses, specific to the products of the HSV-1 and HSV-2 ORFomes, was determined for each individual and ranked as follows: strong responses, with an average signal intensity (ASI) of Ͼ20,000; medium responses, with an ASI of between Ͼ10,000 and Ͻ20,000; low responses, with an ASI of between Ͼ5,000 and Ͻ10,000; and no responses, with an ASI of Ͻ5,000.…”