Establishment and characterisation of a herpes simplex virus type 2 murine challenge model for the evaluation of a novel microbicide
Kgoe, Tumelo Olebogeng
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Introduction: Herpes simplex virus type 2 (HSV-2) has been a global epidemic for many years. This disease is prevalent in Europe, North America, and Africa. HSV-2 is one of the most common sexually transmitted infections (STIs) in low-income countries with high incident rates of HIV/AIDS. With more than 20 million cases reported annually, it has become an issue of great concern. As an incurable STI, preventative alternatives are preferred over the development of treatments. There is a need to develop preventative strategies to effectively reduce HSV infection. Animal models are the preferred biological systems for the testing of safety and efficacy of the newly developed products, including vaccines and novel microbicides. In our study, we developed and characterised a murine model which can be applied in the evaluation of Bi-SP, a novel bismuth (III) complex which has previously been shown to display anti-microbial activity in vitro against HSV-2 and other STI causing agents. Methods: For model establishment, six to eight-week old fasted male (n = 8) and medroxyprogesterone pretreated female (n = 8) BALB/c mice were administered 10 μl of sterile phosphate buffered saline (PBS) rectally and vaginally respectively. Ten minutes after PBS administration, the mice were infected with 16.6 μl of HSV-2 strain G at a viral titer of 1 x 106 plaque forming units. The mice were monitored for infection twice a day for the duration of the study. Vaginal and rectal swab samples were collected from all animals 2 days post infection, immediately before euthanasia and after euthanasia. The swab samples were assayed for viable virus by virus plaque assay and CD4+ and CD8+ analysis using flow cytometry. Rectal and vaginal tissue samples from male and female mice respectively were collected from euthanised animals for histology. The tissue samples were fixed in formalin and were processed through a series of dehydration and elucidation steps and embedded in histoplast wax. The tissues were transversely sectioned with a microtome at 5 – 7 μm sections. Sectioned bands were mounted on glass microscopic slides and were stained with hematoxylin & eosin. The stained tissues were evaluated for epithelial cell disruption and evidence of inflammation under a compound microscope. Safety assessment of Bi-SP was conducted in male (n = 5) and female BALB/c mice (n = 5) at 5 μg/animal. Administration of the novel microbicide was done rectally and vaginally using a 2 – 20 μl pipette. Rectal and vaginal tissue samples were collected 2 hours after Bi-SP administration for histopathology. In determining the efficacy of the novel microbicide, female mice were administered Bi-SP at 5 μg/animal in PBS (n = 10), 5 μg/animal in a hypo-osmotic 2.7% HEC gel (n = 10) and 2.7% HEC gel only as control (n = 10), vaginally. The male mice were administered Bi-SP at 5 μg/animal (n = 20) and 10 μg/animal (n = 10) in PBS, rectally. Ten minutes after microbicide administration, the mice were infected with HSV-2 strain G at a titer of 1 x 106 PFU. The male (n = 1) and female (n = 1) control mice were administered with sterile PBS. The experiments were conducted in a biosafety level (BSL)-3 laboratory and the mice were monitored for infection twice a day over a period of 19 days. During the course of the study, the animals were monitored and clinical scores were assigned. Once the animals reached the humane endpoint clinical score, they were euthanised. The rectal and vaginal swab samples were collected 2 days post-infection and immediately before euthanasia. For virus plaque assay, only swabs collected 2 days post infection were used while CD4+ and CD8+ analysis was done using swabs from 2 days post infection and immediately before euthanasia. Vaginal and rectal tissues were excised from the female and male mice respectively, after euthanasia. The tissues were sliced open longitudinally and stored in formalin for histological analysis. Results: The survival rate of male and female BALB/c mice infected with HSV-2 was reduced to 0%, 9 days post infection. All the animals reached severe clinical symptoms between days seven and nine post infection. The symptoms included severe inflammation perianally and perivaginally in male and female mice respectively. Severe swelling of the bladder and constipation in the digestive tract were observed. Virus plaque assays confirmed the presence of viable virus in the collected swab samples. Histological evaluation revealed elevated levels of inflammatory infiltrate in the submucosa and severe disruption on the mucosa, more specifically the glands and the glandular crypts in rectal tissues collected from male mice. A thinned squamous epithelium and an infiltration of inflammatory cells was observed in vaginal tissue collected from female mice, which was indicative of tissue damage. The presence of the virus from swab samples and the effect HSV-2 had on the vaginal and rectal mucosa is confirmation of the susceptibility of BALB/c mice to HSV-2 strain G. Bi-SP at 5 μg/animal did not cause tissue damage. The microbicide demonstrated significant activity (p<0.05) against HSV-2, subsequently increasing the survival rates from 0% in the PBS administered groups of both male and female mice, to 10% in female mice administered with Bi-SP at 5 μg/animal in PBS and 60% in female mice administered with Bi-SP at 5 μg/animal in 2.7% HEC gel. Female mice administered with 2.7% HEC gel only also had a survival rate of 60%. The survival rate in male mice increased to 40% in mice administered with 5 μg/animal and 30% in mice administered with 10 μg/animal in PBS. The presence of viable virus in swab samples was confirmed using plaque assay and CD4+ and CD8+ cells were detected in vaginal swabs but not in rectal swabs. Conclusion: Both female and male BALB/c mice were highly susceptible to HSV-2, whether infected vaginally or rectally, making them a model for use in microbicide safety and efficacy studies. The susceptibility of BALB/c mice allowed for the study on the effects of the novel microbicide Bi-SP, on HSV-2 in vaginal and rectal tissues. Bi-SP was proven to be safe to use at 5 μg/animal as it did not cause any tissue damage in the vaginal and rectal mucosa. The microbicide improved the survival of HSV-2 infected mice, an indication of the antiviral effects against HSV-2 Therefore, we can conclude that BALB/c mice were highly susceptible to HSV-2 strain G at 1 x 106 pfu and are a suitable model for rectal and vaginal HSV-2 challenge studies. Although Bi-SP displayed significant antiviral activity against HSV-2, the microbicide will be need to be optimised to yield better survival rates than it has for the current study.
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