Guinea pig genital herpes model: evaluation of vaccines and antiviral drugs
小鼠模型,中洪博元,动物模型
z-bo
2020-07-23
622
Type 1 (HSV-1) and type 2 (HSV-2) herpes simplex virus (HSV) infections are the most common human infections. The herpes simplex virus usually causes self-limiting oral and genital bullous ulcers that often recur. However, HSV can cause more serious diseases and reduced immune function in newborns, and is a common cause of encephalitis. In addition, HSV-2 infection is an important risk factor for HIV infection. Preventive and therapeutic vaccines are needed because the initial HSV infection causes an acute primary disease, which can then persist through latent neuroinfection and reactivate to produce a recurrent disease or virus. For acute and recurrent diseases and recurrent viral shedding, improved antiviral drugs are needed. The ultimate goal of HSV preventive vaccines is to completely prevent infection, but it is difficult to achieve this goal. Therefore, a vaccine that limits the initial amount of HSV replication and prevents or minimizes the clinical severity of acute diseases is a reasonable goal. The therapeutic vaccine goal for patients infected with HSV can not only reduce the frequency and severity of recurrent disease, but also prevent or reduce recurrent shedding during symptomatic and asymptomatic relapse.
Mouse model: The most commonly used models for studying HSV are inbred and inbred mouse models. Models include the brain, mouth, face, cornea, nose, skin, newborns and genitals. Rats are relatively cheap. This will help understand the molecular biology of acute and persistent infections and reactivation events. The main advantage of the mouse model is that a variety of reagents can be used to study immune and molecular biological responses. In addition, knockout and knock-in strains can be used to selectively evaluate gene activity and function. The consequences of genital infection are affected by the age of the mouse, the inbred line used, the amount of inoculation and the HSV line used. When using them, these variables must be considered. HSV-2 genital infections often have fatal consequences, which limits the effectiveness of these studies. In addition, vaginal infections in mice require the use of methoxyprogesterone to synchronize the estrus cycle, which leads to increased susceptibility and decreased immune response to the genital HSV-2 vaccine. After vaginal inoculation, the animal suffers from genital disease with erythema and swelling, but compared to guinea pigs, discrete lesions are usually not visible and more difficult to score. Since most animals die from infection after infection, it is not possible to assess potential infection, recurrent disease or shedding of recurrent virus, but thymidine kinase deficiency (TK-) virus is non-lethal mice. Note that you can use it in the model. In short, the mouse model is the most valuable tool to determine the local and systemic immune response to infection, identify possible protective responses, and is an early screening tool for cheap drugs and vaccines.
Rat model: The lethal genital cotton rat model has also been developed. Four days after taking medroxyprogesterone, cotton rats can infect HSV-1 and HSV-2 in the vagina. This model is being used to evaluate gD vaccines used in clinical trials. Although the two doses of vaccines were immunogenic, they failed to prevent HSV-2 disease or reduce genital HSV-2 replication. However, compared with HSV-2 genital diseases, this vaccine can significantly reduce vaginal HSV-1 titer and increase resistance to HSV-1. Since these findings are consistent with large clinical trials, the model is of interest. However, it should be noted that the research conducted in cotton rats used only two doses of the vaccine, while the clinical trials used three doses of the vaccine.
Non-human primate model: Another model for testing antiviral agents, vaccines and fungicides is the monkey vaginal HSV-2 infection model. This model has the unique ability to study the interaction between HSV-2 infection and HIV. Similar to the mouse genital HSV model, the animals were pretreated with methoxesterone 4-5 weeks before the vaginal injection of high-dose HSV-2. Animals were subsequently infected, but the incidence of lesions appeared to be lower in this model. In one study, only 10% of HSV-2 macaques had cervical inflammation and/or lesions. However, during the 2-year follow-up, approximately 50% of the animals had detectable HSV-2 excretion and recorded infections. Another study found similar findings. In the first 7 days after vaccination, infectious virus and HSV-2 DNA were continuously detected in genital secretions, but there were clinical symptoms of infection (pathological changes or systemic effects). HSV-2 DNA was detected in genital swabs at 42 and 56 days after HSV-2 vaccination, and persistent and spontaneous activation of the latent virus was also recorded. As seen in humans, HSV-2 infection also increases the frequency of vaginal SIV-RT infections.
Guinea pig model: HSV-2 genital model description: The main advantage of the HSV-2 genital guinea pig model is that infected animals will develop acute self-limited diseases that are easy to quantify. There are spontaneous relapses that are easy to detect. Ultraviolet rays can also cause recurrent lesions. In addition, the infected animals showed repeated shedding, with or without damage. An important factor in guinea pig infection is to mimic human disease. Another advantage is that animals can assess the presence and number of acute and persistent neurological infections, which is different from what humans can assess.
Operation: Use a pre-moistened calcium alginate cotton swab to open Hartley inbred female guinea pigs that usually weigh 200-300 grams, open the vaginal obturator membrane, and then drop 0.1 ml of HSV-2 directly into the vagina to do it. No pre-treatment of progesterone is required. Next, score the lesions caused by the virus on the skin of the external genitalia. The most common scoring criteria are 0, no disease, 1+, redness or swelling, 2+, some small vesicles, 3+, some large vesicles and 4+, some large infiltrations. I have ulcers. In addition, the above-mentioned scores can be assigned to animals with moderately severe injuries, that is, the scores are between 0.5, 1.5, 2.5, or 3.5. The skin is sparsely covered with fine hairs around the perineum and is considered to be the skin of the external genitalia. The severity usually peaks 5 to 7 days after infection and then gradually decreases.
Obtain vaginal swab samples at a specific time after infection to quantify the effect of treatment on vaginal virus replication. If possible, samples can also be taken from clinically normal external genital skin or external genital skin lesions. It is usually swabbed every day or every other day from 1-10 days, because acute viral shedding usually ends after 8-10 days. Take a sample with a pre-moistened calcium alginate swab and place the swab into a test tube containing 100 ml of penicillin, 50 mg of streptomycin, 50 mg of gentamicin, and 2.5 mg of amphotericin B. I will. And keep frozen. The viral load of the swab sample can be titrated by plaque assay or qPCR. After the acute disease is resolved, that is, after the vulvar lesion has healed, evaluate recurrent lesions and viral recurrence. The lesions healed in days 10-14, so the assessment of recurrent disease is usually started on the 15th day. The animals are checked daily for recurrence. Vaginal swabs are usually taken from the skin of the vagina and genitals between the 15th day and the 63rd to 70th day to check whether the virus has fallen off the skin of the vagina and genitals. Repeated shedding with or without lesions was observed. The amount of shedding can be quantified by quantitative PCR to quantify HSV DNA or culture to detect infectious virus. At the end of the experiment, the animals can be sacrificed, and the frequency and quantification of the potential viral load in the DRG and spinal cord can be determined. The incubation period of the virus can be determined by co-culturing the minced pieces to determine whether there is any reactivated virus. Alternatively, the amount of potential HSV DNA can be determined by qPCR quantification. When the virus titer of DRG is the highest, the therapeutic effect on the initial DRG infection can be measured by killing the animal about 3 to 8 days after infection. Efficacy: Guinea pigs are the only small animals that can be used to evaluate preventive and therapeutic vaccines and treat acute and recurrent diseases. In addition, as mentioned above, the model allows evaluation of several key virological and clinical endpoints of these two methods. The impact of preventive vaccines or treatment of acute diseases on early vaginal replication will be assessed by swab sampling 7-10 days after infection. The severity of the acute illness was also assessed. Intervention can prevent the replication of the virus, at least reduce the titer of the virus recovered from the vagina, or prevent the development of lesions.
After recovering from an acute illness, you can evaluate the animal for recurrent disease, which is usually a single blister or red disease. It can be measured as a cumulative score in different time periods. Since the incidence of lesions gradually decreases over time, the normal observation period is 7 to 8 weeks after recovery from the acute phase, that is, 15 to 64 days or 71 days. Similarly, the effect on virus recurrence can be quantified by collecting vaginal swabs during observation of recurrent disease in animals. Like humans, dropouts can occur with or without genital disease. We believe this is an important endpoint for evaluating vaccines and other interventions, because repeated shedding is the most important source of infection. Effective vaccines or early antiviral interventions will limit the spread and replication of the virus in the DRG during the initial infection. It is estimated that this reduction will affect subsequent recurrent diseases and recurrent virus transmission.. Therapeutic vaccines for recurrent diseases Or the evaluation of interventions is similar. After recovering from an acute illness, the animals are usually vaccinated 2-3 times at specified intervals. Antiviral drugs given at relapse may affect the recurrence of the disease or the shedding of the virus. Therefore, the procedure is similar to the procedure described above for assessing recurrent disease and recurrent virus shedding. Although there are many clinical and virological endpoints, the model of recurrent HSV disease in guinea pigs is limited by the number of relapses and the decrease in the number of relapse dropouts over time. Conclusion: Among many animal models of HSV genital diseases, guinea pigs are the most similar to human diseases. They have many advantages compared with other small animal models in the evaluation of drugs, biological agents and vaccines. .. However, this model has some limitations, which may lead to poor predictions. It is not yet possible to clarify the endpoint that best predicts the success of human trials, but only vaccines that can at least prevent acute disease, reduce subsequent relapses, and evaluate potential viral load can be evaluated. Has been suggested. Only therapeutic vaccines that can reduce disease recurrence and repeat virus shedding can be developed.
