This is the first study to examine the soluble mucosal immune environment during symptomatic genital herpes outbreaks and following ACV treatment. The findings suggest that the ex vivo
anti-HSV activity of CVL is increased at the time of an outbreak compared to HSV-seronegative controls, although the difference did not reach statistical significance. The anti-HSV activity measured likely underestimates the local inhibitory activity, as CVL represents an ~50-fold dilution of genital secretions 23,24
. Possibly, the increased activity observed in cases reflects a local host response to prevent virus spreading to the upper genital tract. Indeed, although virus may be detected in the cervix by PCR, HSV DNA is more frequently isolated from the lower genital tract, and cervical disease is uncommon 16,25
. In this study, only five of 10 participants had detectable CVL HSV DNA when presenting with external genital lesions.
The anti-HSV activity fell in cases during ACV treatment (Day 7) to levels comparable to that observed in HSV-seronegative controls, and then rebounded on Day 14. Concentrations of pro-inflammatory mediators followed a similar temporal pattern (). To further explore the rebound in HSV inhibitory activity on Day 14, we analyzed data from asymptomatic, HSV-2 seropositive subjects at a single time point (baseline) who were not being treated with antivirals (n=16). These women had participated in IRB-approved microbicide safety studies. The CVL anti-HSV activity in this asymptomatic cohort was similar to that observed on Day 14 in the cases (median 47% vs. 69%, respectively; p=0.38) and higher than that observed in HSV-2-seronegative controls (47% vs. 25%, respectively; p=0.08). This suggests that the rebound observed on Day 14 may reflect a return to the steady state in HSV-seropositive women, independent of recent lesions. The higher anti-HSV activity in the cases and asymptomatic HSV-2 seropositive women may represent a mucosal response to subclinical viral shedding, which was likely suppressed by ACV treatment. Larger studies with more frequent sampling in symptomatic and asymptomatic women are needed to determine whether there is an association between subclinical shedding and CVL inhibitory activity.
The anti-HSV activity correlated with several pro-inflammatory, antimicrobial proteins and IgG, which is consistent with prior studies 10,11,27
. Whether these mediators directly contribute to the CVL anti-HSV activity or reflect a biomarker of other unidentified antiviral factors will require fractionation and depletion studies. Possibly, HSV-specific antibodies, which were not evaluated previously reflecting the lack of established assays, contribute to the CVL inhibitory activity in cases. Efforts to specifically deplete CVL of immunoglobulin with Protein A were unsuccessful, as other proteins including HNP1-3 were also lost. In ongoing studies, we are adapting a commercial ELISA (GenWay Biotech, San Diego, CA) designed to measure HSV-specific IgG in blood to measure antibodies in CVL. HSV-specific IgG was detected from three of 10 cases (Day 0 samples), three of 14 asymptomatic HSV-2 seropositive subjects, and none of the controls. There was no significant difference in anti-HSV activity when comparing subjects with detectable to those without detectable CVL HSV-specific IgG (p=0.67), suggesting that HSV-specific IgG contributes little to CVL inhibitory activity. Larger studies including measurements of mucosal HSV-specific IgG and IgA are needed.
Paradoxically, we observed that, compared to controls, CVL from cases were more likely to exhibit HIV inhibitory activity, although HSV recurrences have been associated with increased HIV shedding and an increased risk of HIV infection 4,28,29
. Perhaps, the inflammatory response to HSV overcomes the modest protective effect of CVL against HIV. The inflammatory response may recruit T cells to the dermal-epidermal junction, which could serve as HIV targets if the virus traverses the keratinized skin barrier, perhaps weakened by HSV-induced disruption of the epithelial barrier.
Protease inhibitors may also provide antiviral and anti-inflammatory activity. SLPI was present at significantly lower concentrations in CVL from cases compared to controls on Day 0. This observation is consistent with the in vitro
finding that HSV immediate early proteins down-regulate SLPI expression in epithelial cells and keratinocytes 30
. Whether the concentrations of other antiproteases were altered in CVL and how this contributes to CVL anti-HIV activity, and ultimately, to the risk of HIV infection, requires additional studies. A proteomic study found that antiproteases, including serpins and cystatins, were present at higher concentrations in CVL from HIV-highly exposed seronegative women compared to uninfected controls and correlated positively and significantly with pro-inflammatory cytokines 31
. The authors speculated that antiproteases might control inflammatory responses and provide a more potent antiviral mucosal environment, although they did not measure anti-HIV activity. In this study, HBD-2 correlated positively and significantly with CVL anti-HIV activity, but only among cases. A recent study also found that HBD-2 correlated with CVL anti-HIV activity of CVL, but only among HIV-infected women, not among healthy controls 12
Conversely, while CVL anti-HSV and anti-HIV activity was increased in cases, the bactericidal activity against E. coli
was reduced. These findings indicate that different molecules contribute to the activity against different pathogens. Moreover, the reduction in E. coli
bactericidal activity during an outbreak is consistent with the epidemiological link between BV and HSV 32,33
. Vaginal fluid from women with BV exhibited reduced inhibitory activity against E. coli
, which was partially restored after metronidazole treatment 34
. A substantial percentage of women with microbiota changes consistent with BV are asymptomatic by Amsel clinical criteria 35,36
and we did not assess vaginal microbiota populations in this study. We speculate that changes in vaginal bacteria including an increases in E. coli
colonization, loss of protective lactobacilli and/or an increase in anaerobic species may have characterized the microenvironment in the cases 35,37
In summary, despite several limitations, including small sample size, lack of parallel samples from asymptomatic HSV-2 seropositive women, infrequent sampling for silent HSV reactivation, and lack of specimens just prior to lesion appearance (when HSV replication may be highest), the findings obtained indicate that genital outbreaks are associated with increases in pro-inflammatory mediators and greater CVL anti-HSV activity compared to HSV seronegative controls. We speculate that these mucosal responses may limit HSV spread and prevent viral ascension into the upper genital tract, but may also paradoxically increase HIV acquisition risk. While the concentrations of inflammatory proteins and anti-HSV activity fell during treatment, they returned to pretreatment levels following ACV cessation. The rebound to a more inflammatory mucosal environment may contribute to the clinical observation that HSV-2 seropositivity is a risk factor for HIV acquisition, independent of overt herpetic lesions 6,38-44
. These findings underscore the need to further investigate subclinical shedding and its impact on mucosal immunity and HIV risk and to optimize strategies to suppress HSV reactivation.