The KMB17 cell line (IMB, CAMS, Yunnan, China) and the African green monkey kidney Vero cell line (ATCC, Manassas, VA, United States) were maintained in high glucose Dulbecco’s modified Eagle’s medium (Corning, NY, United States) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, United States). The culture medium was changed to DMEM supplemented with 2% fetal bovine serum after viral infection. The pathogenic HSV-1 strain 8F (Yu et al., 2010) and the HSV1 mutant M3 were used in our experiments. The mutant M3 was originated from HSV-1 strain 8F, in which a 30-bp (225–254) sequence of the ul7 gene, a 59-bp (375–433) sequence of the ul41 gene, and a 138-bp (937–1074) sequence of the LAT gene were deleted sequentially using the CRISPR/Cas9 method. The mutants were identified by PCR and sequencing of the PCR products, and the mutated clones were acquired through plaque screening of the harvest in Vero cells. The viruses were titered on Vero cells.

Four-week-old female BALB/c mice weighing 10–13 g (Vital River, Beijing, China) were purchased and housed in a specific pathogen-free facility of the Institute of Medical Biology. The mice were maintained under a 12-h light/dark cycle (lights on at 08:00 h) at 22 ± 1°C. The animals were housed individually and allowed free access to food and water. All efforts were made to minimize suffering or discomfort to the animals. Prior to the experiments, the animals were routinely acclimated (>1 week) to laboratory conditions to reduce potential stress effects during the experiments. The animal experiments were designed based upon the principles expressed in the “Guide for the Care and Use of Laboratory Animals” and “Guidance for Experimental Animal Welfare and Ethical Treatment.” The experimental protocols were reviewed and approved by the Yunnan Provincial Experimental Animal Management Association (approval number: SCXK [Dian] 2011-0005) and the Experimental Animal Ethics Committee of the Institute (Approval number: YIKESHENGLUNZI [2016] 54).

For the immunization experiment, the BALB/c mice were narcotized under 2% isoflurane inhalation and immunized via an intramuscular injection of 10³, 10⁴ or 10⁵ plaque-forming units (PFUs) of the HSV-1 mutant M3 or phosphate-buffered saline (PBS, sterile, pH 7.4) as a control. Splenic lymphocytes were isolated 1 and 2 months post-immunization for an ELISpot assay. Additionally, blood samples from the mice were tested for neutralizing antibodies on days 28, 42, and 56 after the primary injection.

After determining the optimal immunization dose, the BALB/c mice were immunized via an intramuscular injection of 10⁴ (PFUs) of the HSV-1 mutant M3 or PBS (sterile, pH 7.4) as a control. At 1 and 2 months post-immunization, the immunized and PBS-immunized mice were challenged with HSV1 8F, McKrae, and 17+ (2 × 10⁴ PFU/50 μL/mouse) via the respiratory tract. After the viral challenge, the weights of the mice were measured every 2 days. The survival rate was assessed over a 10-day period. The splenic lymphocytes were isolated 6 days post-viral challenge for an ELISpot assay. Tissues were obtained at 3, 6, and 9 days post-viral challenge and subjected to an assessment of mouse organ pathology.

Virus titration was analyzed by performing a micro-titration assay according to a standard protocol. For the titration of various mouse organs, viral stocks were ground and homogenized as much as possible, and the homogenate supernatants were then serially diluted 10 times and added to 96-well plates coated with Vero cells. The plates were incubated at 37°C in 5% CO₂ and scored for the presence of cytopathic effect (CPE) 7 days post-infection. All virus-related experiments were performed in a large room under BSL-2 conditions.

A neutralization assay was performed in accordance with standard protocols. Briefly, a mixture of diluted serum (1:4, 1:8, 1:16, 1:32, and 1:64) and virus at a titer of 100 times the 50% cell culture infectious doses (CCID₅₀)/100 μL were incubated at 37°C for 2 h. The mixture was then added to Vero cells in 96-well plates and incubated at 37°C. The CPE of the virus was observed after 1 week.

BALB/c mice were sacrificed after anesthetization with ether, and the spleens were removed aseptically into Hank’s Balanced Salt Solution (Corning, NY, United States). A single-cell suspension was prepared through the gentle dispersion of the cells. Red blood cells were removed using 5 mL of mouse lymphocyte separation solution (Solarbio, Beijing, China) per mouse spleen for 30 min. The cells were washed and suspended in RPMI 1640 medium (Corning, NY, United States) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin.

Splenic lymphocytes were isolated as described above. A mouse IFN-γ ELISpot Kit (MABTECH Inc., Cincinnati, OH, United States) was used according to the manufacturer’s protocol. Briefly, the plate was conditioned and seeded with splenic lymphocytes prior to addition of the stimulant (the two 95% purity peptides: gB498-505: SSIEFARL and ICP6822-829: QTFDFGRL), which mainly targeted the CD8⁺ T lymphocytes (Salvucci et al., 1995; Wallace et al., 1999; St Leger et al., 2011). The cells were then incubated at 37°C for 24 h. After incubation, the cells were removed, and the plate was developed. The colored spots were counted using an automated ELISpot reader (CTL, Cleveland, OH, United States), with spot-forming cells (SFCs) representing HSV1 M3-specific IFN-γ-producing T cells.

The mouse organs were fixed in 10% formalin and embedded in paraffin into tissue blocks. Approximately two slides per organ were stained with hematoxylin and eosin (H&E) to assess the morphology. For the immunohistochemical examinations, the specimens were immersed in 0.3% hydrogen peroxide in PBS for 10 min to block the intrinsic peroxidase activity. After rinsing with distilled water, the specimens were heated at 90°C for 30 min for antigen retrieval. The specimens were blocked with 5% bovine serum albumin (BSA) in PBS at 37°C for 15 min. The specimens were then incubated with a rabbit polyclonal anti-HSV-1 antibody (Abcam, Cambridge, United Kingdom) at 4°C for 12 h. The sections were incubated with poly-HRP anti-rabbit IgG (ZSGB-BIO, Beijing, China) at 37°C for 30 min after rinsing with PBS. Enzyme immunohistochemistry was performed using a standard avidin-biotin-peroxidase method with the 3,3′-diaminobenzidine (DAB) substrate (Tiangen, Beijing, China) according to the manufacturer’s recommended protocol. After rinsing with water for 6 min, the sections were stained with hematoxylin to assess the cell nuclei. For each staining condition, a slide with rabbit IgG (Abcam, Cambridge, United Kingdom) was stained and examined in parallel as a control.

An in situ hybridization assay was performed using the Enhanced Sensitive ISH Detection Kit I (POD) (Boster, Wuhan, China) per the manufacturer’s recommendations. Briefly, frozen sections were fixed with 4% paraformaldehyde for 20 min at room temperature. The sections were immersed in 0.6% hydrogen peroxide in methanol to block the intrinsic peroxidase activity. After three rinses with distilled water, pepsin digestion was performed, followed by treatment in pre-hybrid solution for 3–4 h. Twenty microliters of the LAT probe (5′-CATAGAGAGCCAGGCACAAAAACAC-Dig-3′, 1 μg/mL hybrid solution) was pipetted onto each sample and left overnight in a humidity chamber. The sections were consecutively treated with sealing liquid for 30 min, mouse-anti-DIG for 60 min, SABC solution for 20 min, anti-mouse-HRP polymer for 20 min, and diaminobenzidine chromogen (DAB) for 10 min. After rinsing with water, the sections were stained with hematoxylin to assess the cell nuclei. LAT positivity was evaluated in the entire cell. Any brown dot-like signal from the cell was considered positive for LAT expression. Cells with no such signal were considered negative for LAT.

Trigeminal ganglions were removed from the sacrificed mice 9 days post wild-type virus challenge. The tissues were cut into smaller sections and placed on monolayers of Vero cells. CPE was monitored daily for 7 days. If no CPE was detected, the cultures were continuously blind passaged for three generations and the virus titrations of the third generation were determined by the standard protocol on Vero cells. Meanwhile, DNA was extracted from the culture mediums for PCR testing of HSV1. The genomic regions of the RS1, ul48, and ul44 genes were amplified by PCR using specific primers. The specific primer sets used are listed in Table 1.

The results of the various assays, which were performed in triplicate, are expressed as mean values with standard deviations. SPSS software was used for statistical analyses. The weights of the infected mice were evaluated using repeated measures. A survival analysis was performed to analyze the survival rate of infected mice. Differences of virus titers between two groups were evaluated using an independent sample t-test. A p-value of p < 0.05 was considered significant.

Based on the observation that M3 had an attenuated phenotype in mice, our experiments utilized high, medium and low M3 doses (10⁵, 10⁴, and 10³ PFU/mouse) for immunization via intramuscular injection into the mice. Each dose group contained 10 mice, which were used for immunological detection at two time points (1 and 2 months post-immunization; Figure 1A). All mice inoculated with any dose of the mutant strain developed a specific neutralizing antibody response in a dose-dependent manner (Figure 1B). An ELISpot response against the HSV1 gB and ICP6 antigens was observed in T cells that specifically secreted IFN-γ (Figure 1C). These immunological indicators were up-regulated in the immunized mice, indicating that M3 can induce specific immunity against HSV1. Also, in our previous work, we evaluated the immune protective efficiency of three doses. The results showed that 10³ PFU/mouse was ineffective to induce favorable immune response, whereas the mice immunized with 10⁴ PFU/mouse and 10⁵ PFU/mouse doses showed a similar immune protective efficiency (data not shown). Therefore, we prefer a lower dose of 10⁴ PFU/mouse to obtain the same immune protective effect with a higher safety. Hereafter, 120 mice were immunized with M3 (with a dose of 10⁴ PFU/mouse) via intramuscular injection, and 60 were used for the viral challenge at each time point. In parallel, an equal number of mice were challenged with the wild-type strains 8F, McKrae, and 17+ at the same dose as positive controls (Figure 2A). The immune response, including neutralizing antibodies and a specific ELISpot response against gB and ICP6 antigen peptides, was assessed 1 and 2 months after one injection of this virus followed by challenge with the HSV1 wild-type strains 8F, McKrae, and 17+ (2 × 10⁴ PFU/mouse) via the nasal mucosa (Figure 2A).

Clinical observations of the mice immunized with M3 did not reveal any visible symptoms, which suggested an attenuated phenotype of M3, similar to the findings of our previous work. Interestingly, we did not find any clinical changes in the immunized mice at 1 or 2 months after challenge with the three wild-type strains. The weight measurements of all the mice indicated that the immunized mice maintained their increasing weight trend over 10 days post-infection. In contrast, a decreasing trend was observed in the unimmunized mice over the same period (Figures 2B,C). Moreover, all of the animals in the immunized and 8F challenged (P-8F) groups survived at 1 month post-immunization, whereas three of eight mice (37.5%) in the McKrae challenge (P-McKrae) group and one of eight mice (12.5%) in the 17+ challenge (P-17+) group died (Figure 2D). All animals in the P-McKrae and P-17+ groups became ill, with most of the mice exhibiting symptoms of severe disease, including back arching and inverted hair, whereas six of the eight animals in the P-8F group developed back arching and inverted hair, and no animals in the immunization groups displayed these complications, at 1 month post-immunization (Figure 2E). In addition, at 2 months post-immunization, two of eight mice (25%) in the McKrae challenge (P-McKrae) group and one of eight mice (12.5%) in the 17+ challenge (P-17+) group died, whereas all of the animals in the other immunization groups survived (Figure 2F). All animals in the P-McKrae developed back arching and inverted hair, whereas five of seven animals in the P-17+ group and two of eight animals in the P-8F group developed back arching and inverted hair, and no animals in the immunization groups displayed these complications (Figure 2G).

The pathological assessment of the major organs of all mice post-viral challenge was an important evaluation of the immunological protective effect. The assessment suggested a trend in which the pathological changes showed the presence of fewer inflammatory lesions in the mice immunized with the M3 strain than in the unimmunized mice (Table 2). The observations focused on the cerebrum indicated an obvious inflammatory lesion and infiltration of inflammatory cells in the cerebrum in the unimmunized animals challenged with all three wild viral strains since day 3 post-viral challenges (Figure 3A). These lesions appeared to extend to more areas with more inflammatory cell aggregation around vesicular tissues, whereas the degeneration of some neurons associated with congestion in the tissues were presented on day 6 (Figure 3A). Moreover, inflammatory cell infiltration and bleeding in the meninx were observed in most of the mice. Conversely, the mice immunized with the M3 strain showed slight inflammatory cell aggregation and a few areas presenting slight congestion in the local area of the meninx (Figure 3A). These pathological changes presented a distinct trend at various time points in the immunized and unimmunized mice. We performed an immunohistochemical detection using a specific antibody against HSV1 on the same sections obtained from all mice. In cerebral sections from the unimmunized animals, many dispersed HSV1 antigenic-positive signals were observed in the areas with aggregated inflammatory cells (Figure 3B). In contrast, few antigen-positive signals were observed in the local tissues of the cerebrum, where inflammatory cells aggregated in the immunized mice (Figure 3B). These results indicated that the immunity induced in the mice immunized with M3 might be capable of controlling the pathological lesions induced by infection with the HSV1 wild-type strain.

The above comparative observations of the pathological changes induced by infection with the three wild viral strains in the immunized and unimmunized mice suggested that the severity of the pathological lesions was controlled in the immunized mice. To verify this observation, we evaluated the dynamic variations in the viral titration in the major organs of the immunized and unimmunized mice at 3, 6, and 9 days post-viral challenge. The viral titration for the CNS tissues, including various parts of the brain and spinal cord from the immunized mice, suggested a lower trend than those detected in the organs from the unimmunized mice (Figures 4A–D). However, no virus was traced in the other organs, not only in the immunized mice but also in the unimmunized mice. These data suggest that the immunity induced by M3, which can limit the pathological damage of nervous tissues and other tissues caused by the wild viral strain challenges, might be due to the inhibition of the proliferation of challenged virus in immunized individuals. Previous studies suggested that specific CD8+ cells play an important role in controlling herpes virus infection via a cytotoxic effect against infected host cells (Macleod et al., 2014). Therefore, our work examined specific T cell responses against the viral gB and ICP6 antigens using an ELISpot assay to assess IFN-γ specificity in all mice on day 6 post-viral challenge. We observed an increased specific ELISpot response in the immunized mice relative to that in the unimmunized mice (Figure 4E). This finding supports our prediction discussed above.

Reported studies of HSV1 infection in mice indicated that the virus could enter the trigeminal ganglion of mice for further possible latent infection while it induced acute infection in CNS tissues (Curanovic and Enquist, 2009). However, some works also suggested no positive data supporting the autonomic reactivation of latent HSV1 in the trigeminal ganglion of mice, even when the expression of LAT RNA was traced in this tissue (Webre et al., 2012). In this study, after 1 and 2 months of immunization with the M3 strain, the mice were challenged with three wild strains: McKrae, 17⁺, and 8F. During the observation period for acute infection, the trigeminal nerve samples obtained from all mice at days 3, 6, and 9 post-viral challenges were used for titration of the virus in this tissue followed by the identification of viral genomic DNA using PCR reaction; simultaneously, an in situ hybridization assay was performed to probe the LAT RNA expression to verify the viral transcription in the neurons. The results indicated significantly lower viral loads in the trigeminal ganglions of the mice immunized with the M3 strain than that in the unimmunized mice (Figures 5A,B). These results suggest that the immunization of M3 provided a mechanism to control the virus entering the trigeminal neurons. The identification using PCR with primers against the RS1, ul48, and ul44 genes of HSV1 was supportive to this suggestion (Figure 5C). As further evidence, the in situ hybridization detection showed a significantly lower LAT RNA level in the trigeminal nerves of the immunized mice than that in the unimmunized mice at days 6 and 9 (Figure 5D). Subsequently, homogenized trigeminal tissues were co-cultured with Vero cells to investigate the viral reactivation to obtain further evidence to support this immunity. The results indicated that the Vero cells co-cultured with trigeminal ganglion tissue from the control mice challenged by a wild-type strain, particularly P-17+, showed typical CPE (Figure 6A, Upper); and no CPE was observed in the cells in immunized mice after viral challenge, even after three passages (Figure 6A, Bottom). In addition, PCR using primers targeting the RS1, ul48, and ul44 genes of HSV1 supported these results (Figure 6B). These data support the conclusion that M3 immunization in mice establishes an effective clinical immune protection capable of defeating various wild strain challenges.