Intravaginal Zinc Oxide Tetrapod Nanoparticles as Novel Immunoprotective Agents against Genital Herpes

Herpes simplex virus-2 is an enveloped dsDNA virus. Globally, it is one of the most common human pathogens. HSV-2 has been found to cause mucosal, ocular, and neurologic infections; however, it typically causes genital infections (genital herpes) manifesting as severe lesions in and around the genitalia, accompanied by inflammation of infected and nearby tissues (1, 2). In addition, disease recurrence and associated morbidity are very common, despite therapy (1). Genital herpes is thus a top-priority healthcare concern (1, 2). However, virtually all the attempts to develop an effective vaccine have failed and current HSV-2 antivirals are inefficient, essentially because of the development of drug resistance and failure to prevent recurrence (1, 3). Highly innovative strategies are needed to develop an effective prophylaxis against HSV-2 infection and associated diseases.

Rapid emergence of nanotechnology, and nanoparticle use in medicine, has led to numerous novel discoveries in the field of drug development (4). Using nanoparticles as an alternative to traditional therapies has unprecedented advantages such as enhanced efficacy, reduced side effects, and more targeted localization in disease sites (4–6). Previous studies suggested that uniquely synthesized zinc oxide (ZnO) nanoparticles suppress viral infection in vitro by binding to HSV-2 virions and rendering them ineffective for a productive infection (7).

In this article, we provide evidence for the in vivo efficacy of specially designed and synthesized ZnO tetrapod nanoparticles (ZOTEN) that prevent HSV-2 infection and disease. In addition, ZOTEN therapy creates a platform for viral Ag presentation, provides adjuvant benefits, and enhances cell-mediated and humoral anti–HSV-2 immunity. This work presents proof for a novel microbivac concept (8), which has the potential to prevent infection to sero-negative individuals. ZOTEN provides a platform for virus capture and presentation of neutralized virions to mucosal APCs for initiating and boosting adaptive immunity. Our study also demonstrates the feasibility that intravaginal route can be used for vaccine delivery.

Human vaginal epithelial (VK2/E6E7) cells were obtained from ATCC. VK2/E6E7 cells were passaged in keratinocyte serum-free medium (Life Technologies/BRL, Carlsbad, CA) supplemented with epidermal growth factor and bovine pituitary extract. African green monkey kidney (Vero) cells were provided by Dr. P.G. Spear (Northwestern University, Chicago, IL). Human cervical (HeLa) cells were provided by Dr. B.S. Prabhakar (University of Illinois at Chicago). Vero cells and HeLa cells were maintained in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS and 1% penicillin-streptomycin.

Two strains of HSV-2 were used: wild type HSV-2(333) and β-galactosidase–expressing HSV-2(333)gJ⁻ (9). High-titered stocks of the virus strains were propagated by infecting Vero cells at a low multiplicity of infection (MOI) of 0.01 and harvesting cells when cytopathic effect was 90–100%. Cell-associated virus particles were released by sonication. Both virus strains were titered on Vero cells and stored at −80°C.

Female BALB/c mice 6–8 wk of age were used for experiments. All animal studies were performed in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and also adhered to the guidelines of the World Medical Association’s Declaration of Helsinki. Seven days before infection, animals were injected with progesterone. Vaginal swabs were collected according to protocol mentioned earlier with slight modifications (10). In brief, vaginal swabs were collected by gently inserting presoaked calcium alginate fiber–tipped applicator swabs (Fisherbrand) into the vagina and gently brushing the side walls of the vagina three times. The swabs were then placed in 1 ml DMEM containing 5% (v/v) FBS, 1% gentamicin, and stored at −80°C until titered. Before titering, the swabs were thawed and vigorously vortexed for 30 s. Serial dilutions of the washes were used to generate the plaques on Vero cells.

Virus replication and spread were assessed by sacrificing mice as specified on days post viral inoculation. The vaginas were removed and split open longitudinally. Vaginal tissue was then fixed in 2% paraformaldehyde 0.02% Nonidet P-40 in PBS for 6 h. After fixation the organs were stained overnight with X-gal (1 mg/ml 6-bromo-4-chloro-3-indolyl-b-d-galactopyranisude, 3 mM potassium ferrous cyanide, 3 mM ferric cyanide, 2 mM magnesium chloride 0.1 mM EGTA, 0.01 sodium deoxycholate, and 0.02 Nonidet P-40). The staining of the vaginal tissue was scored on a scale from 0 to 5: 0 = no blue staining; 1 = 10–20% of surface covered in blue lesions; 2 = 30% of the surface covered in blue lesions; 3 = 40–50% of surface covered in blue lesions; 4 = 60–70% of surface covered in blue lesions; 5 = 80–100% covered in blue lesions. All examiners who have done scoring were masked regarding the treatment condition of each mouse.

Extracted vaginas were semiquantitatively assessed for inflammation. The inflammation of vaginal tissue was scored on a scale from 0 to 4: 0 = no inflammation; 1 = mild inflammation; 2 = moderate inflammation; 3 = severe inflammation and mild tissue damage; 4 = severe inflammation, severe tissue damage/necrosis of tissue. All examiners who have done scoring were masked regarding the treatment condition of each mouse.

Fourteen days postinfection, mice were sacrificed and dorsal root ganglion was removed for fixation, embedding in paraffin, and sectioning as described earlier (11). Sections were stained with H&E, and a semiquantitative scale was made to determine the level of inflammation within spinal cord, meninges, dorsal root ganglion, and nerve root.

BALB/c female mice were injected with 0.1 ml medroxyprogesterone acetate (Depo-Provera) at a concentration of 25 mg/ml to allow mice to get on the same menstrual cycle. Seven days after injection, mice were inoculated with HSV-2(333) [or HSV-2(333)gJ⁻] with or without ZOTEN. A viral dose of 5 × 10⁵ PFU was used for all infections. After challenge, mice were monitored for 21 d to evaluate clinical signs of disease and system tolerance of various different treatments. Also, infected mice were monitored for death or severe morbidity requiring euthanasia. The day of death or sacrifice was recorded for each animal.

The presence of Abs against HSV-2 in mouse sera was detected according to a protocol mentioned earlier with slight modifications (12). Virus neutralization was measured by evaluating the level of inhibition that sera had on the entry of β-galactosidase reporter virus HSV-2(333)gJ⁻. Serum was incubated with virus for 30 min before addition to cells. Two hours postinfection, virus and sera inoculum was removed, and infection continued for additional 4 h. O-nitrophenyl-β-d-galactopyranoside (ONPG) substrate solution was added to cells for 30 min to 1 h at 37°C. Tecan plate reader was used for the collection of OD values.

ZOTEN structures were synthesized by a recently developed flame transport synthesis (FTS) approach at Kiel University (13, 14). The preparation of ZOTEN starts from spherical zinc microparticles obtained commercially. Synthesis involves Zn particles, polyvinyl butyral (PVB) powder, and ethanol. A mixture is prepared and burned together in the furnace at 900°C. The flame (generated due to the burning of sacrificial polymer PVB) carries the Zn microparticles upward, where they are subsequently transformed into the vapor state in the form of individual Zn atoms, Zn dimers, Zn trimers, etc. The highly unstable atomic variants in the Zn vapor immediately start participating in nucleation and growth processes with the help of available native oxygen molecules in the environment. First, Zn and O combine to form a primary cluster and once the stable nucleus has been formed, further available Zn and O atoms contribute to conventional 1D spike growth. Homogeneous nucleation results in growth of tetrapod-type structures. PVB decomposes completely into CO₂ and O₂, and tetrapods growth occurs in normal ambient conditions. This highly efficient process gives a ZOTEN actual yield of 99.9%. Mishra et al. (14) show that this process creates uniform ZnO tetrapods (ZOTEN) by electron microscopy. The shape and size of the synthesized structures were investigated using scanning electron microscopy at 10-kV electron beam acceleration voltage and 18-μA filament (LaB6) current (14). The scanning electron microscopy results confirmed the tetrapod shape of the ZnO structures with arm diameters in the range of 200 nm to 1 μm and arm lengths in the range of 5 to 30 μm. Identical ZOTENs were used for all experiments demonstrated in this article.

After vaginal challenge with HSV-2(333), mice were observed for the development of clinical signs of disease for 21 d or until death/sacrifice. Mice were examined for the development of external lesions, hair loss, inflammation, and skin lesions around the vaginal opening, rectum, tail base, and hind legs. Mice were also monitored for the development of neurologic signs of disease such as fecal/urine retention, hind-limb paralysis, hunched posture, lethargy, dehydration, and ruffled fur. Any animal exhibiting severe morbidity was sacrificed. Each mouse was given an infection severity score from 0 to 5: 0 = no lesions; 1 = slight hair loss and inflammation; 2 = hair loss, inflammation, and small lesion <0.5 cm; 3 = significant hair loss, broken skin, scabbing, multiple pustules, bleeding; 4 = large lesions on vaginal opening, rectum, and hind leg, significant hair loss, broken skin >1 cm; 5 = severe scabbing and bleeding, multiple lesions, lesions on tail base, pustules, severe inflammation/bleeding. All examiners who have done scoring were masked regarding the treatment status of each mouse.

After BALB/c mice were injected with Depo-Provera, one of five treatment conditions was used to prime the immune system: 1) uninfected and untreated; 2) UV-inactivated virus alone; 3) ZOTEN and UV-inactivated virus; 4) alum and UV-inactivated virus; and 5) alum, ZOTEN, and UV-inactivated virus. Mice were sacrificed 21 d postinoculation. Spleens were removed and single-cell suspensions were made. Isolated splenocytes were treated with ACK lysis buffer for 5 min at room temperature to lyse RBCs. After incubation, equal numbers of cells were seeded in a round-bottom 96-well plate for 24 h. Cells were then challenged with HSV-2 for 2 d before being labeled with BrdU (Invitrogen). After BrdU labeling, stimulated splenocytes were stained with anti-mouse CD4-PE and anti-mouse CD8. FACS analysis was performed to determine the level of T cell proliferation. Results are shown as the percentage of lymphocytes that have incorporated BrdU into newly formed cells.

To measure the cytotoxicity of T lymphocytes against VK2/E6E7 target cells, we performed CTL killing assays as previously described (15, 16). In brief, target cells were labeled with Vybrant Dil Cell-Labeling Solution (Life Technologies) and then incubated with T lymphocytes at the indicated E:T ratios. After 4 h, cells were harvested; TO-PRO-3-iodide, a membrane-impermeable DNA stain, was added to each culture (0.5 μM final concentration), and cells were finally analyzed by flow cytometry. Background and maximum TO-PRO-3 iodide staining were obtained by incubating target cells with medium and detergent, respectively. The percentage specific lysis was calculated as [(% TO-PRO-3-iodide⁺PKH26⁺ cells in E:T cell coculture − % TO-PRO-3-iodide⁺PKH26⁺ cells in medium)/(% TO-PRO-3-iodide⁺PKH26⁺ cells in detergent − % TO-PRO-3-iodide⁺PKH26⁺ cells in medium] × 100%.

Dendritic cells were isolated according to a protocol mentioned earlier (17). Cells were isolated, counted, and cultured in glass-bottom dishes (MatTek Corporation, Ashland, MA). ZOTEN (0.1mg/ml) and 1 × 10⁶ PFU GFP HSV-2(333) were added to the dish and incubated for 1 h in 37°C before being imaged under the microscope (Zeiss Confocal 710 using a 63× oil objective lens).

The following statistical tests were used for various experiments. Appropriate statistical tests are mentioned in the figure legends for every respective experiment. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post hoc t test, Student t test, Mantel–Cox log-rank test, two-way ANOVA, and Kaplan–Meier survival analysis using GraphPad; *p ≤ 0.5, **p ≤ 0.01, ***p ≤ 0.001.

The Animal Care and Use Committee at the University of Illinois has approved all animal studies and the experimental protocols used in this study.

The ZnO tetrapod micronanoparticles used in this study were synthesized by the recently introduced FTS approach (14). In the FTS process, Zn microparticles and PVB powders are used as precursor material. Zn microparticles are mixed in PVB powder in the ratio of 1:2; then the mixture (in a ceramic crucible) is heated inside a muffle-type furnace up to 900°C for 30 min in normal air environment. During the synthesis process, sacrificial PVB polymer maintains the necessary separation between Zn microparticles, which are transported and simultaneously converted into nanomicroscale ZnO tetrapods by the generated flame inside the furnace. After this process ZnO tetrapods are harvested from the ceramic crucible (14, 18, 19). The microstructural evolutions of the synthesized ZOTEN were characterized using scanning electron microscope (20 keV Philips-FEI XL30 equipped with LaB₆ filament). ZOTEN consisted of spikelike structures, which meet at a central point (Fig. 1A). The process of ZOTEN formation is highly efficient and the actual yield is 99.9% (20).

To assess the virucidal potential of ZOTEN therapy in vivo, we used a mouse vaginal HSV-2 infection model (20). Female BALB/c mice were made susceptible to intravaginal infection by inducing the diestrus stage (uncornified vaginal epithelium) of the estrous cycle by injecting Depo-Provera (progesterone) (21). Mice were inoculated with HSV-2(333), in the presence and absence of ZOTEN, to allow the assessment of viral entry and spread within various organs and tissues. All mice were infected with 5 × 10⁵ PFUs. Mice were examined daily for clinical symptoms of infection such as hair loss, inflammation, and lesion development on the vagina, rectal opening, tail base, and legs. Although all infected mock-treated mice displayed external lesions by 5 d postchallenge, ZOTEN-treated mice showed significantly reduced lesion development, because <20% of ZOTEN-treated animals developed external lesions on day 14 postinfection and beyond (Fig. 1B). Thus, it appeared that ZOTEN decreases the rate and extent of lesion formation after HSV-2 infection.

Mock-treated mice began to lose hair and developed small lesions around the vaginal opening at 5 d postinfection, which continuously increased in number and size as infection progressed, eventually leading to the formation of pustules and bleeding at the base of the tail (Fig. 1C). In contrast, <20% of ZOTEN-treated mice showed slight inflammation at the vaginal opening with minor hair loss at day 5, crusting at the proximal region of the back leg by day 10 (Fig. 1D). The remaining (>80%) ZOTEN-treated mice were comparable with uninfected mice, displaying no hair loss or lesion development. To further assess the effect of ZOTEN on lesion development postinfection, we gave infected mice daily infection severity scores based on the progression of infection and related disease symptoms. Extent and severity of lesion development were significantly reduced by ZOTEN treatment (Fig. 1E). Together, these results confirmed that ZOTEN treatment as a microbicide significantly suppresses the acute symptoms of HSV-2 infection.

Having observed the strong ability of ZOTEN to suppress acute HSV-2 genital disease, we then sought to determine the effect of ZOTEN on viral loads in the infected mice vaginas. BALB/c mice infected with β-galactosidase–expressing HSV-2 gJ⁻ were sacrificed 5 d after challenge. The vaginas were extracted and stained with X-gal to assess the level of viral entry that occurred within the vaginal epithelium. Stained tissue sections were scored on a scale of 0 to 5, where 0 signifies no X-gal staining, whereas 5 signifies extensive X-gal staining on the epithelium. We found that ZOTEN treatment decreased X-gal staining, suggesting significantly lowered viral loads by the treatment (Fig. 2A). Average staining score of ZOTEN-treated mice was ∼2.1, contrary to a score of ∼4.4 for mock group (Fig. 2B). In addition, by measuring inflammation in extracted vaginas according to criteria mentioned in 2Materials and Methods, we found that ZOTEN-treated mice had vaginas with less inflammation and tissue damage (Fig. 2C), consistent with decreased viral loads in the tissues.

To further confirm the effect of ZOTEN treatment on virus levels in infected mice, we swabbed the mice vaginas for 5 consecutive days after intravaginal challenge, to measure the levels of virus within the vaginal lumen. Viral titers recovered from vaginal swabs revealed that ZOTEN-treated mice produced significantly lower viral titers at all points of infection tested (Fig. 2D). These results indicate the strong ability of ZOTEN to suppress HSV-2 vaginal infection in mice. Reduced redness, inflammation, hair loss, and lesion development, along with decreased viral replication levels by ZOTEN treatment, strongly support its potential as an intravaginal microbicide against HSV-2 infection.

A hallmark of the establishment of a chronic HSV-2 infection is virus dissemination within the body, leading to tissue inflammation (1). Because HSV-2 spreads predominantly to tissues of the peripheral nervous system and CNS (1, 2, 22), we assessed these tissues for signs of inflammation or tissue damage, suggestive of HSV-2 spread. Therefore, dorsal root ganglia and spinal cords of mock- or ZOTEN-treated mice were removed, fixed, sectioned, and stained with H&E, to determine inflammation levels (Fig. 3A), and scored on a semiquantitative scale according to criteria mentioned in 2Materials and Methods (Fig. 3B). Hence, ZOTEN-treated mice showed significantly less inflammation in the spinal cords than the mock-treated group, reflecting less viral dissemination into neuronal tissues.

Additional signs of HSV-2 infection spread include development of neurologic disease, weight loss, and finally, animal death (1, 2, 22). Consistent with decreased inflammation by ZOTEN treatment, reduced neurologic disease symptoms were also observed upon treatment (Fig. 3C, 3D). Furthermore, infection-induced weight loss was much less remarkable among ZOTEN-treated mice (Fig. 3E). To further confirm the impact of ZOTEN treatment on overall animal health and vitality, we assessed mortality rates of ZOTEN- or mock-treated mice. ZOTEN-treated mice had a significantly lower mortality rate than mock-treated mice, because 100% of the mice within the ZOTEN-treated group survived the 21-d period of evaluation. Conversely, 50% of the mock-treated mice died by day 10 postinfection, reaching 80% mortality by day 14 (Fig. 3F). Collectively, these data indicate the ability of ZOTEN to reduce chronic disease development associated with inflamed tissues, neurologic disease symptoms, weight loss, and mortality.

We have noticed that ZOTEN has a tendency to bind HSV-2 viral particles (7); thus, we reasoned whether this interaction may enhance viral Ag accessibility to, and engulfment by, APCs such as dendritic cells. Dendritic cells were isolated from spleens of mice and were infected with HSV-2 in the presence of ZOTEN (HSV-2–ZOTEN combination) for 2 h, and clearance of viral Ags by dendritic cells was monitored microscopically. We observed gradual removal of ZOTEN-bound viral particles (Fig. 4A), suggesting that ZOTEN may facilitate virus uptake by dendritic cells in the mucosal layer of the vaginal tract.

Adaptive immune responses (both cell mediated and humoral) are key lines of defense against viral infections, such as HSV-2 genital herpes (9, 22–26). Thus, having noticed the enhanced Ag uptake by APCs upon ZOTEN treatment, we sought to determine the adaptive immune response profile as a function of HSV-2 genital infection in vivo, in the presence or absence of ZOTEN treatment. First, we investigated whether ZOTEN can exert adjuvant properties to support development of adaptive immune responses to viral Ags. Using UV-inactivated HSV-2 virus as a source of viral Ags, we determined levels of infection-induced CD8 and CD4 T lymphocytes as a function of ZOTEN treatment. Mice were sacrificed and splenocytes were isolated 14 d postinoculation. We found that a combination of inactivated HSV-2 and ZOTEN increased T cell numbers to a greater extent than inactivated HSV-2 treatment alone (Fig. 4B). Also, the combination of alum and HSV-2 increased T cell proliferation just as much as the combination of HSV-2 and ZOTEN. These data suggest that ZOTEN can serve as an efficient adjuvant.

Having found that ZOTEN can act as an adjuvant, we further assessed the CTL cell memory responses to infection, an important adaptive immune response against viral infections (9, 24). Splenocytes from different groups of mice were assessed for developing a memory response to reinfection by HSV-2. Vaginal epithelial cells (VK2/E6E7) were infected with HSV-2(333) at an MOI of 0.1, stained with Vybrant dye, and incubated for 24 h before coculture with the splenocytes. The splenocytes were incubated with infected cells for 4 h and TO-PRO-3 dye was added to culture. Cytotoxic T cell–mediated VK2/E6E7 cell lysis creates a Vybrant–TO-PRO-3 double-positive population of cells. We found that splenocytes of mice infected and treated with ZOTEN and inactivated HSV-2 showed significantly higher cytotoxicity toward HSV-2–infected cells, compared with those from inactivated HSV-2 alone (Fig. 4C). ZOTEN-alum combination showed a higher enhanced cytotoxic T cell response, indicating a strong immunogenicity-enhancing property of ZOTEN, which may be used in combination with existing adjuvants to achieve synergism.

Because HSV-2 genital disease is characterized by a high recurrence rate, we wanted to further explore the abilities of ZOTEN to protect against reinfection. We have found that ZOTEN stimulates memory T cell–mediated immune responses (Fig. 4C) and wanted to assess humoral immunity in response to infection in vivo upon treatment with ZOTEN, especially considering that HSV-2 suppresses humoral immunity via various mechanisms to enable uninterrupted infection (27). Sera were evaluated for their ability to inhibit entry of HSV-2 and suppress infection of cells. We found that sera from ZOTEN-treated mice significantly reduced the ability of the virus to infect cells (Fig. 4D). Interestingly, despite the fact that ZOTEN adjuvant properties were comparable with alum (Fig. 4B), induction of virus-neutralizing Ab production in mice was a unique property of ZOTEN (Fig. 4D). Altogether, data suggest that ZOTEN enhances adaptive immune responses to HSV-2 infection, including memory response to reinfection, and to prevent genital disease recurrence (Fig. 4), in addition to its abilities to suppress acute (Fig. 1) and chronic (Fig. 2) disease.

The global widespread nature of HSV-2, its high recurrence rate, and the high susceptibility of the female reproductive tract to a plethora of sexually transmitted diseases including HSV-2 (28) highlight the need for new ways to prevent or control this debilitating infection. Previously failed vaccine studies against genital herpes in humans have focused mostly on i.m. immunization formulated in the oil-in-water emulsion with alum-based adjuvants. There is a lot of interest in examining alternate adjuvants and routes of immunization to elicit strong protective immunity to genital herpes to obtain higher efficacy of protection. The new vaccine designs or treatments need to aim to prevent disease transmission and spread to uninfected individuals. Current therapies provide little effect against disease, because of drug resistance, toxicities, and disease recurrence, and to date no vaccine is currently available for prevention or treatment of genital herpes (29). Thus, to our knowledge, this study reports the very first evidence for the novel microbivac concept (8), which is expected to guide future microbicide and vaccine development strategies against persistent infections. The microbivac concept uses a virus-trapping agent that provides immediate protection by trapping virus particles and then presents them for mucosal immunity development. ZOTEN prevents cell entry by inhibiting the interaction between virus and cellular receptors. HSV bound to ZOTEN are taken up by APCs in the mucosal surface and are presented to prime the immune system. Given its potential use as a microbicide, ZOTEN is expected to provide immunity boost every time the virions are trapped by it. This article also represents a report of a potential intravaginal vaccine, which we found to be effective in mice. Although most nanoparticles have been administered i.p., intranasally, or orally, we show that intravaginal inoculation can be used to provide microbial protection.

Demonstrating the virucidal potential of ZOTEN therapy in vivo, ZOTEN-treated mice showed significantly reduced lesion development, because <20% of ZOTEN-treated animals developed external lesions or detectable inflammation (Fig. 1). We found that ZOTEN treatment decreased viral loads and that ZOTEN-treated mice produced significantly lower viral titers at all points of infection tested (Fig. 2). Average staining score of ZOTEN-treated mice was ∼2.1, contrary to a score of ∼4.4 for mock group (Fig. 2B). We also found that ZOTEN-treated mice had less inflammation in the spinal cords than the mock-treated group, reflecting less viral dissemination into neuronal tissues, reduced neurologic disease symptoms, or weight loss (Fig. 3C, 3D). As an overall assessment of animal health and vitality, ZOTEN-treated mice had 100% survival rate compared with the 50–80% mortality rates seen with the mock-treated mice at days 10 and 17, respectively (Fig. 3F).

ZOTEN traps HSV-2 virions (7), but given its relatively large size, it cannot enter epithelial cells. We used high-resolution fluorescence microscopy to demonstrate its partial engulfment of virions by dendritic cells. Gradual removal of ZOTEN-bound GFP viral particles was observed via live-cell imaging (Fig. 4A), suggesting that ZOTEN may facilitate virus uptake by dendritic cells, which may facilitate a rapid clearance of trapped virions from the vaginal tract and process the virions for Ag presentation. Interestingly, a combination of inactivated HSV-2 and ZOTEN increased T cell numbers significantly to a greater extent than inactivated HSV-2 treatment alone (Fig. 4B). The initial differences in CD4 and CD8 responses (Fig. 4B) generated by the ZOTEN approach may appear relatively small, but they are statistically significant. It is also worth noting that the responses are expected to keep growing over time, especially given the unique advantages generated by the microbivac approach. ZOTEN is expected to be used repeatedly as a microbicide, and its virus-trapping abilities and adjuvant-like properties will boost adaptive immunity, including CD4/CD8 responses, every time HSV-2 virions are trapped by this microbicide. An additional advantage is that the immunity will develop against the whole virus as Ags. Although subunit vaccine studies in the past that used just adjuvanted HSV-2 gD were unable to elicit strong protective immunity against HSV-2 in clinical trials, the polyantigenic response has been alluded to be more beneficial in an HSV vaccine that uses a mutant virus by deleting gD to prevent cell entry and cell-to-cell spread (30–35).

The combination of alum and HSV-2 increased T cell proliferation just as much as the combination of HSV-2 and ZOTEN. These data suggest that ZOTEN can provide an additional benefit as an efficient adjuvant. We found that splenocytes of mice primed with ZOTEN showed significantly higher cytotoxicity toward HSV-2–infected cells, compared with those from control infected-untreated mice (Fig. 4C). ZOTEN-alum combination showed a higher enhanced cytotoxic T cell response, indicating a strong immunogenicity-enhancing property of ZOTEN, which may be used in combination with existing adjuvants to achieve synergism. We also found that sera from ZOTEN-treated mice significantly reduced the ability of the HSV to infect and enter cells, which was a unique property of ZOTEN (Fig. 4D). Another interesting observation was that HSV-2 serum entry inhibition was unique to ZOTEN. Alum enhances general endogenous CD4 T cell responses, Ab production, and CD8 T cell priming with the help of macrophages, mast cells, and eosinophils (36). The induction of cytotoxic response through generation of Abs in alum is nonspecific when compared with the ZOTEN+HSV-2 complex. According to Fig. 4B, alum and ZOTEN have both separately shown to increase CD4 and CD8 cell populations. However, in Fig. 4D, serum entry inhibition studies have shown that blocking entry of HSV-2 is specific to ZOTEN, but not alum, which suggests that Abs against HSV-2 are specifically produced in ZOTEN-treated mice. This could be attributed to the specific B cell response caused by priming the immune system against HSV-2 in HSV-2/ZOTEN-treated mice. Altogether, these data suggest that ZOTEN enhances adaptive immune responses to HSV-2 infection, including memory response to reinfection, suggesting the powerful properties of ZOTEN to prevent genital disease recurrence (Fig. 4), in addition to its abilities to suppress acute (Fig. 1) and chronic (Fig. 2) diseases.

In the animal studies presented in this study and other in vitro studies performed earlier, ZOTEN was chosen to develop the microbivac concept on the basis of multiple parameters. The chemical content, ZnO, is very well-characterized in cosmetic and clinical studies and well-tolerated during topical applications (37). Many beauty products contain ZnO in different formats (e.g., powder, liquid, or gel) and it has been tested in clinical trials for anti-HSV effects. However, for further development of ZOTEN for potential clinical usage as a microbivac against genital herpes, additional dose optimization studies and efficacy determination studies will be needed. Although mouse is generally a good model of HSV-2 genital infection, additional models such as rabbits or guinea pigs, which spontaneously reactivate the virus, need to be examined for microbicide and vaccine efficacy studies (38). Likewise, toxicology and pharmacokinetic studies and lead optimization studies need to be performed for future clinical applications. In the studies presented in this article, no significant adverse effects or toxicity was exhibited in the animal studies even at the highest dosage; however, long-term efficacy, off-target effects, and toxicology studies will be needed. Furthermore, recent advances in topical formulations need to be combined to even further reduce any side effects of ZOTEN. Design of more specific and even higher binding ZOTEN in the future could provide some advantages over the nonselective ZOTEN. Overall, to our knowledge, the described studies represent the very first inhibition of viral infection and protection from future infection by a microbivac. Thus, our studies represent an approach to antivirals with extensive potential derived from studies of viral entry inhibition and the promise of microbivac therapy.

In conclusion, this article provides evidence on the usefulness of ZOTEN as an effective virostatic platform, which also provides adjuvant benefits in combination with the captured virions for induction of protective immunity against genital herpes in mice. ZnO tetrapod structures could be applied to contraceptives as a means of drug delivery, which will allow the release of the drug in a localized and efficient manner. Given that ZOTEN works therapeutically in vitro (7), it is likely that existing symptoms can also be improved in infected mice because ZOTEN will inhibit the spread of newly produced HSV-2 by trapping them. Future studies investigating the direct therapeutic aspect of ZOTEN in vivo will be performed. In addition, future clinical studies are likely to provide more information on versatile use of ZOTEN to control persistent infections.

In the future, our results require further understanding of ZOTEN for induction of protective immunity in the female genital tract against genital herpes. Additional studies will be needed to elucidate the relative contribution of T cells and Ab response to the observed protection in mice. Moreover, studies using rabbits or guinea pig models of recurrent genital herpes are required to evaluate the potential of robust protective immunity to primary and recurrent genital herpes. A comparative harmonized analysis of different adjuvants, including those tested previously in human trials, is also required to pinpoint the relative usefulness of ZOTEN versus other adjuvants and for optimizing the immunization regimens for induction of protective immune responses to HSV-2.

Y.K.M. and R.A. thank the Deutsche Forschungsgemeinschaft Grant under the scheme AD/183/10-1.

The authors have no financial conflicts of interest.