Journal of Infectious Diseases Advance Access published January 16, 2014 1
Mimicking herpes simplex virus 1 and herpes simplex virus 2 mucosal
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behavior in a well characterized human genital organ culture
Lennert Steukers1,*, Steven Weyers3, Xiaoyun Y. Yang1, Annelies P. Vandekerckhove1, Sarah Glorieux1, Maria Cornelissen4, Wim Van den Broeck2, Marleen Temmerman3
Laboratory of Virology
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Department of Morphology, Faculty of Veterinary Medicine, Ghent University,
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Salisburylaan 133, B-9820 Merelbeke, Belgium Department of Obstetrics and Gynaecology
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Department of Basic Medical Sciences, Ghent University Hospital, De Pintelaan 185, B-
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9000 Ghent, Belgium
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Corresponding author: Lennert Steukers, Salisburylaan 133, 9820 Merelbeke, Belgium,
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Phone: 00 32 9 264 73 75, Fax: 00 32 9 264 74 95, email:
[email protected]
© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e‐mail:
[email protected].
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and Hans J. Nauwynck1
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Abstract We developed and morphologically characterized a human genital mucosa explant model
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(endocervix and ectocervix/vagina) to mimic genital herpes infections caused by herpes simplex virus 1 (HSV-1) and 2 (HSV-2). Subsequent analysis of HSV entry receptor
expression throughout the menstrual cycle in genital tissues was performed and the evolution of HSV-1/-2 mucosal spread over time was assessed. Nectin-1 and -2 were found to be
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mainly to some connective tissue cells. Both HSV-1 and HSV-2 exhibited a plaquewise
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mucosal spread across the basement membrane and induced prominent epithelial syncytia.
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expressed in all tissues during the entire menstrual cycle. HVEM expression was limited
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Introduction Human genital herpes is worldwide one of the most important sexually transmitted infections
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(STI). The principal causative agent is herpes simplex virus type 2 (HSV-2); however, the
frequency of primary genital herpes infections caused by herpes simplex virus type 1 (HSV-1) is on the increase [1]. Additionally, genital herpes lesions promote the transmission of human immunodeficiency virus (HIV). HSV-2 has been shown to modulate the mucosal
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to a more prone susceptibility to HIV [1, 2]. No efficacious HSV-vaccine is currently on the
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market, and the likelihood of one coming to market soon is low [3].
Rodent models are considered as golden standard for genital herpes research [4, 5]. Although they play a robust role in elucidating aspects of primary genital mucosa infections, they
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clearly lack homology. Potential species-specific differences in cellular mediators of infection and invasion, impede extrapolation of results. For example, several entry mediators have been
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described for HSV, including nectins (nectin-1 and nectin-2), herpes virus entry mediator (HVEM) and specific sites in heparan sulfate. Although nectin homologues in mice resemble the human forms, mouse nectin-2 is not functional as entry receptor for HSV-1 and HSV-2 whereas human nectin-2 clearly mediates entry of HSV-2 and certain HSV-1 recombinants [6]. Interestingly, until now, information on either the presence and distribution of entry
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mediators or on its potential in vivo role within the genital tract is scarce. In this study we optimized a human genital mucosa explant model in a similar way as a
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previously optimized bovine genital mucosa explant model [7]. Next, we analyzed the expression patters of different herpes virus entry receptors including nectin-1, nectin-2 and HVEM in both endocervix and ectocervix/vagina throughout the menstrual cycle. Finally, we modeled the mucosal behavior in genital mucosa of both HSV-1 and HSV-2 by inoculating
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microenvironment including recruiting of submucosal dendritic cells, which might contribute
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genital organ cultures and compared the HSV-1 and HSV-2 replication characteristics within
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the same patients.
Patients and methods
Pieces of healthy genital mucosa were gathered from 15 women undergoing a routine
of progesterone/estradiol levels and HSV-specific neutralizing antibody titers (complementdependent seroneutralization test). The latter test does not allow discrimination between
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antibodies raised against HSV-1 and HSV-2. All persons provided written informed consent and the ethics committee of the Ghent University Hospital approved the study (EC/2010/152).
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(see Supplementary Table 1).
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Different patients of various stages of the menstrual cycle were included for all experiments
A more detailed description of the in vitro model, the applied techniques for evaluating tissue viability/morphology and the inoculation method, is provided in a Supplementary Methods section and is similar as described elsewhere [7].
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The strains used in this study for inoculation of human genital mucosa explants were HSV-1 strain F (ATCC VR-733) and HSV-2 strain MS (ATCC VR-540), cultured on Vero cells
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(ATCC CCL-81). HSV-inoculated explants were collected at 0h and 48h post inoculation (pi).
Evaluation of relative HSV entry receptor expression was performed on non-cultured, noninoculated tissues (time point 0h) of 10 different patients at various menstrual cycle stages by
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hysterectomy (Ghent University Hospital). In addition, serum was collected for determination
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means of immunofluorescence stainings. Cryosections of 10µm were fixed in methanol (20°C, 100%, 20 min). We used mouse monoclonal antibodies CK41, L14 and D-5 (Santa
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Cruz Biotechnology) directed against nectin-1 (1:50 in PBS), nectin-2 (1:20 in PBS) and HVEM (1:50 in PBS) respectively. After 1h incubation at 37°C, samples were thoroughly washed and incubated at 37°C for 1h with goat anti-mouse FITC®.
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fixed in methanol. In a first part, to visualize the basement membrane (BM) barrier, we used
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primary goat anti-collagen IV antibodies (Southern Biotech, 1:50 in PBS), secondary biotinylated rabbit anti-goat antibodies (Sigma, 1:100 in PBS) and tertiary streptavidin-Texas Red® antibodies (Molecular Probes, 1:50 in PBS). In a second part, HSV-1 or HSV-2
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antibodies were stained with primary mouse monoclonal antibodies against HSV-1 gD (1:100 in PBS, 10% NGS) or HSV-2 gB (1:50 in PBS, 10% NGS)(Santa Cruz Biotechnology)
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respectively, followed by secondary FITC®-labeled goat anti-mouse antibodies (Molecular Probes, 1:100 in PBS, 10% NGS). Next, measurements of average HSV-1/-2 plaque latitude and plaque penetration underneath the BM were performed at 0h and 48h pi.
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SPSS software (one way ANOVA, Post-hoc Bonferroni and Tukey’s HSD) was used to evaluate the variance. The results are given as means + standard deviation of different (≥3)
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independent experiments. Results with P values of ≤ 0.05 were considered significant.
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From all HSV-inoculated explants, 20 µm thick consecutive cryosections were made and
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Results Serum reproductive hormone levels and HSV-specific neutralizing antibody titer of the
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patients are given in Supplementary Table 1.
First, no significant changes were observed in the occurrence of apoptosis during the in vitro
Supplementary Figure 1). Second, light microscopical analysis showed endocervical mucosa to be lined by a simple columnar epithelium interspersed by mucus-producing cells.
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Ectocervical epithelium consisted of a nonkeratinized stratified squamous epithelium, similar as the vaginal epithelium. For all tissues, no significant changes in epithelial thickness could
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be observed because of in vitro culture (see Supplementary Figure 1). Analysis of the composition of the connective tissue revealed no significant differences in percentage nuclei or collagen due to cultivation. The lamina reticularis thickness, as part of the BM, could be
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maintained throughout tissue culture (see Supplementary Figure 1). Third, applying TEM, epithelial integrity and BM continuity (lamina densa) were found to be conserved at all time points for both endocervical and ectocervical tissues.
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Regardless of the phase of the menstrual cycle, expression pattern and localization of all HSV entry receptors was noticed to be consistent for both endocervix and ectocervix/vagina
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(Figure 1). In general, for all patients, both nectins were clearly more abundantly expressed in endocervix compared to ectocervix/vagina. At all times, the nectin-2 antibody staining was found to be more intense than the antibody staining for nectin-1 in the genital tract. In endocervical epithelium nectins were highly present on the luminal side of the cell, thus apicolateral. In ectocervical/vaginal epithelium, nectin-1 and -2 expression was most intense
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cultivation of endocervical and ectocervical mucosa up to 96h (end of the experiment) (see
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in the stratum spinosum and minimal in the stratum basale. Loss of nectin-1 and -2 expression was seen within the superficial layers of the ectocervix/vagina. Consequently, whenever
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epithelial thickness shrivels, nectin expression was located closer to the luminal side, as was observed in postmenopausal women and women in the luteal phase of the menstrual cycle. The majority of HVEM expressing cells was observed in the lamina propria and deeper
connective tissue. Scarcely distributed single cells resident within the epithelium were also
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HSV-1 and HSV-2 both replicated plaquewise in endocervical and ectocervical mucosa of all included patients (Figure 2). In ectocervical mucosa, HSV plaques were often localized at
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places of slight abrasion within the superficial layers. All HSV-induced ectocervical plaques were situated within the stratum spinosum and some extended to the stratum basale. Infection
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within the stratum superficialis was never observed. Interestingly, large multinucleated giant cells were observed in HSV epithelial plaques in the ectocervix. Starting from 48h pi, few (
