By David Kilpatrick, PhD and Abbas Vafai, PhD

INTRODUCTION: The detection and differentiation of HSV-1 & -2 infections present challenges in a hospital point of care environment. There are many laboratory tests which can be performed, including serological assays, polymerase chain reactions, as well as virus neutralization and/or culture. However, only simple, quick serological assays, provide for rapid POC analysis. The close amino acid sequence homology between HSV-1 & -2, presents another challenge for type specificity. In this paper, Kalantari-Dehaghi et al.,1Kalantari-Dehaghi M, et al., 2012 J. Virol 86:4328-4339. Discovery of Potential Diagnostic and Vaccine Antigens in Herpes Simplex Virus 1 and 2 by Proteome-Wide Antibody Profiling. presented excellent data on the antigenic comparisons of both viruses.

DISCUSSION: The authors in this paper produced HSV-1 and -2 proteome microarrays by PCR in a T7 expression vector followed by an expression in E. coli-based in vitro transcription-translation system (IVTT) and probed them against a panel of patient sera using commercial gG-1 (HSV-1) and gG-2 (HSV-2) enzyme-linked immunosorbent assays. Reactive antigens for both HSV-1 and -2, as well as several which were cross-reactive between both virus types were identified. They found both membrane and non-membrane viral proteins were antigenic; however, the type-specific antigens were predominantly membrane proteins. The most commonly recognized antigens were structural. They derived P values by comparing seropositive and seronegative donors using Bayesian t-tests. Twenty-two HSV-1 antigens which were seroreactive with HSV-1 seropositive group were identified. Of those, glycoproteins G and E (US4 and US8) yielded the two highest HSV-1 type-specific signals. The HSV-1 seropositive group also recognized 9 HSV-2 antigens. They also identified 33 HSV-2 seroreactive antigens in the HSV-2 sero- positive group, and 19 of these also reacted to HSV-1 antigens. For HSV-2, the UL44/gC and US6/gD were the best type-specific antigens. While US4/gG has been used for several type-specific tests for HSV-1, their data suggests that UL44/gC might be a candidate antigen for the development of HSV-2 type-specific tests (97% specificity). They suggested that ideal antigens for serodiagnostic assays may be less dependent on membrane proteins. Others have suggested that the most important T cell epitopes in HSV may be integument or other noncapsid proteins2Carmack, MA et al 1996. J. Infect. Dis. 174:899-906. T cell recognition and cytokine production elicited by common and type-specific glycoproteins of herpes simplex virus type 1 and 2. 3Hosken N, et al. 2006. J. Virol. 80:5509-5515. Diversity of the cD8+ T-cell response to herpes simplex virus type 2 proteins among person with genital herpes. 4Koelle DM, et al. 1994. J. Infect. Dis. 169:956-961. Direct recovery of herpes simplex virus (HSV)-specific T lymphocytes clones from recurrent genital HSV-2 lesions. 5Koelle DM, et al., 1994. J. Virol. 68-2803-2810. Antigenic specificities of human CD4+ T-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions.. Tegument proteins, such as HSV-2 UL41 (and several others) have been identified as major targets for effector T cells 6Carmack, MA et al 1996. J. Infect. Dis. 174:899-906. T cell recognition and cytokine production elicited by common and type-specific glycoproteins of herpes simplex virus type 1 and 2. 7Hosken N, et al. 2006. J. Virol. 80:5509-5515. Diversity of the cD8+ T-cell response to herpes simplex virus type 2 proteins among person with genital herpes. 8Koelle DM, et al. 1994. J. Infect. Dis. 169:956-961. Direct recovery of herpes simplex virus (HSV)-specific T lymphocytes clones from recurrent genital HSV-2 lesions. 9Koelle DM, et al., 1994. J. Virol. 68-2803-2810. Antigenic specificities of human CD4+ T-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions.. Tegument protein from HSV-2 UL51 showed 88% specificity for HSV-2 (and 0% with HSV-1). Diagnostic assays detect strong antibody signals for US6/gD (from both HSV-1 and -2) and UL48 for HSV-2, these are both CD4 target antigens.

CONCLUSION: In this study, the authors found that sera from HSV-1 seropositive donors were specific for HSV-1 antigens while sera from HSV-2 seropositive donors were reactive against both HSV-1 and HSV-2 antigens. They suggest that this type-specific antibody profile may have been due to the expression system used and to the correct folding of the proteins created. However, they postulated that the asymmetry in antigen recognition between the two virus groups could also be due to the biological differences between HSV-1 and HSV-2 infections (route of entry or site of primary infection).

By David Kilpatrick, PhD and Abbas Vafai, PhD

INTRODUCTION: Various assays have been developed over the last thirty years for detecting Herpes virus antibodies in patient sera. The three most commonly used assays are Western blot assay (WBA), enzyme-linked immunosorbent assay (ELISA), and enzyme immnodot assays (EIA). Of these, the Western blot assay (WBA) is still considered the “gold standard” in the detection of antibodies to viral proteins. The sensitivity of this assay is primarily due to the detection of antibodies to more than one viral protein in the single assay. Read more

By David Kilpatrick, PhD and Abbas Vafai, PhD

INTRODUCTION: There are several commercial immunoassays for HSV on the market (Western blot, Dot blot or standard ELISA assays). Of these, the University of Washington Western blot assay (UW-WB) is considered the “gold standard” for assays to detect Herpes virus antibodies in patient sera1University of Washington Western blot. https://depts.washington.edu/. While this FDA approved assay is considered to be very accurate, a second confirmatory assay is often preferred as most patients are unaware of their genital Herpes status2Ashley-Morrow, Rhoda, and David Friedrich, 2003. The inaccuracy of Certain Commercial Enzyme Immunoassays in Diagnosing Genital Infections with Herpes Simplex Virus Types 1 and 2. Am J Clin Pathol, 120:839-844.. Read more

By David Kilpatrick, PhD and Abbas Vafai, PhD

INTRODUCTION: Finding antigens which can be used for human Herpes virus (HHV) assays can be challenging. Many technical concerns need to be addressed, including how to express sufficient quantities of each antigen, and once produced, how the immune system responds to each antigen. In this paper, Brenner et. al, cloned and expressed 16 HHV antigens to be used in a fluorescence capture bead assay. Read more