Our data shows that estradiol improves vaccine-elicited protection against genital herpes infection and disease and results in an enhanced antibody response. Others have previously examined the impact of estradiol on vaccine efficacy against genital herpes in mouse models using a thymidine kinase deficient virus as an attenuated vaccine [8
]. The results presented here are consistent with and extend those studies. If estradiol was going to modulate susceptiblity to and/or protection against HSV-2 infection, as we and they hypothesized, then the potential existed that it could be modulating susceptibility to the vaccine virus as well as or instead of the pathogenic virus challenge. We avoided this potential problem by using a subunit vaccine, so that replication of the immunogen is not a factor. Further, this vaccine candidate showed efficacy in initial phase III clinical trials, lending additional translational relevance. Another major concern we had was in the dosing schedule of the estradiol. In establishing whether estradiol could enhance vaccine-elicited protection, we wanted to be certain that an estradiol-thickened vaginal epithelium did not confound our ability to detect alterations in vaccine efficacy by leaving the mice physiologically impermeable to virus, especially since the magnitude of this effect is much greater in mice; at no time in the menstrual cycle is a woman’s vagina impermeable to HSV-2 infection. We confirmed that our results were not contaminated by any physiological impact of estradiol by directly imaging it, rather than trying to gauge or time the estrous cycles of the mice or serologically quantify the estradiol. The imaging studies presented here enabled us to visually verify that our administration of a sustained-release estradiol into the mouse model posed no physiological barrier to viral infection. Because we are able to demonstrate in that the vaginal epithelial thickness had returned to pre-estradiol values (or to progesterone-regulated values in the case of intact mice), it is then supportable to accept that the differences observed in the subsequent studies (incidence of infection, disease, circulating antibody, etc.) represented an estradiol-mediated impact on the immune system. Secondarily, the imaging studies confirmed the bioavailability and metabolic potency of the estradiol valerate preparation and dosage used.
It was also interesting to note, from the imaging studies, that the ovariectomized mouse vaginal epithelium, before estradiol treatment () and after estradiol treatment has lapsed (), is thinner than the progesterone-treated epithelium of an ovary-intact mouse (), which explains why ovariectomized are exquisitely sensitive to HSV-2 infection and are thus an ideal model to examine the impact of estradiol on vaccine efficacy. The gD/AS04 vaccine alone was able to reduce the incidence of infection by 20%, a trend towards protection against infection but falling short of statistical significance (). It should be noted that, in this regard, the ovariectomized mouse model parallels the clinical observations using the same vaccine candidate with which additional expanded phase III clinical trials are currently underway [4
]. In ovariectomized mice treated with estradiol valerate one week prior to the first vaccination, an additional gain of 20% vaccine efficacy was observed in comparison to vaccination alone, significantly reducing the incidence of infection by almost 40% compared to naïve controls (P<0.01) (). This is an exciting observation. Sterilizing immunity has not been widely believed to be a feasible goal of an HSV-2 vaccination in clinical application. It is anticipated that an HSV-2 vaccine brought to market would provide immunity sufficient to prevent disease and might reduce the burden of latent virus in the ganglia, which could in turn reduce the shedding into the genital shedding and transmission; modeling indicates that, in this way, a vaccine that provides nonsterilizing immunity can still significantly reduce the incidence of HSV-2 infection [24
]. However, adding estradiol to the vaccination regimen in ovariectomized mice directly enhanced protection against infection as well as enhancing the protection against disease. Vaccination alone significantly reduced the incidence of clinical disease overall by 34% (P<0.01) and among infected mice by almost 22% (P<0.01) (). The estradiol-treated groups had reduced incidence of disease by an additional 27.5% overall compared to vaccination (61.7% compared to naïve). Among infected mice, estradiol treated mice had an additional 22% reduction in incidence of disease compared to vaccination alone (not significant) and a 44 % reduction in incidence of disease compared to naïve controls (P<0.01). Thus the rigorous ovariectomized mouse model shows that estradiol treatment not only boosts vaccine efficacy to provide enhanced protection against disease but it also boosts it up to a threshold adequate to prevent infection.
Key effector and regulatory cells of the immune system express estrogen receptors and a mechanism involving an interfacing of the endocrine and reproductive systems with the immune system is not a novel idea [26
]. It has long been understood that the immune system of the female reproductive tract must be modulated to not attack and reject a fetus that is 50% foreign. In contrast, the idea that estradiol could be systemically modulating the formation of adaptive immune memory responses is much newer and is supported by our data [8
]. While the gD/AS04 vaccine is designed to stimulate both Th1
responses, our HSV-2-specific IgG ELISA data and neutralization assay suggest estradiol-treated mice produce additional HSV-2 gD-specific antibody. This explains, at least in part, why the estradiol-treated vaccinated mice had a better outcome than their vaccine-only counterparts.
The ovary-intact studies were consistent with the principles demonstrated in the ovariectomized mice and the literature. A complicating variable in intact studies is endogenous estrogen. This is particularly true when the studies are designed to test the modulation of vaccine-elicited immunity. In non-hormone studies this is controlled for by the administration of progesterone prior to viral challenge, which synchronizes and halts, at least temporarily, the estrous cycles of the mice. However, this is too late for the purposes of our studies because the endogenous estrogen may or may not have been significantly contributing the sum estradiol levels in the mouse. Moreover, such contributions, if significant, would have come at unsynchronized times in the development of the memory immune response to the vaccine. Also, progesterone is known to play an antagonistic role to estradiol in many endocrine pathways; some have suggested it does so in immune ones as well and this is an area of ongoing investigation [11
]. Therefore, we thought it important to examine and report the impact of estradiol on vaccine efficacy in intact animals because it provides an additional perspective on the translational relevance of our data (noting the ovariectomized mice performed more like the human vaccine trials than the intact mice) and because it then enables us to put this data in the context of all the other published HSV-2 mouse model data. Also noteworthy, there was a reduction in the incidence of disease across all viral challenge titres in the estradiol-treated groups compared to the groups receiving vaccination alone (data not shown), again suggesting an enhancement of efficacy against the background of already-robust protection, as is more fully explored in the ovariectomized studies.
While there are still additional questions to be explored regarding the impact of estradiol on infection and immunity, the studies described here present an important demonstration that estradiol’s effects go far beyond physiology and can shape the host response to vaccination. The conclusions drawn from these studies have implications that need to be explored using other animal models, other candidate vaccines and other infectious organisms so that, once the mechanism of action of estradiol on the host-response is clearly understood, it may be exploited in a wide range of scenarios to elicit optimal protection from otherwise suboptimal vaccines.