Screening and identification of dominant B-cell epitopes of human cytomegalovirus UL138 and its clinical applications

Human cytomegalovirus (HCMV) belongs to the herpesvirus family of β-double-stranded linear DNA viruses. The virions are approximately 200–300 nm in diameter and encode approximately 250 open reading frames. HCMV is the most common and most harmful pathogen causing congenital and perinatal infection in newborns. The incidence of congenital HCMV infections in newborns is approximately 0.2–6.1% [1]. There are also asymptomatic infections, in which congenital infection of HCMV in 5–17% of asymptomatic children shows varying degrees of neurological sequelae after birth, including hearing impairment, retinal choroiditis, delayed mental development, nervous system stunting, epilepsy and dyskinesia [2]. Latent infection is an important biological feature of HCMV [3,4]. Due to changes in the physiological or pathological state of the body, latent HCMV can be repeatedly activated and enter the stage of proliferative infection, which can be seriously life-threatening to individuals with low immunity (such as tissue transplant recipients or patients with AIDS). Both latent-activated and primary infections of pregnant women can induce intrauterine infection with HCMV, and most of the mother-to-child transmission of HCMV is caused by latent virus activation. After HCMV establishes a latent infection, the viral genome persists, although infectious viral particles cannot be detected, and only a few latent related genes are detected. Under certain conditions, however, the virus can be activated from the latent state and undergoes transition to an active infectious state [5]. Therefore, detection of HCMV latent infection is of considerable significance for early diagnosis and prevention.

HCMV is detected by virus isolation, PP65 antigen detection, serum IgM, IgG antibody detection, and nucleic acid detection. Although the gold standard for HCMV diagnosis is HCMV isolation culture, HCMV proliferates very slowly in human fibroblast culture in vitro, with a replication cycle of 36–48 h. The gold standard method is time consuming, cumbersome to perform, and requires specialized equipment. Moreover, it cannot be used for early detection; even if the negative results of separation are not completely excluded from HCMV active infection, it is not suitable as a routine method for detecting HCMV infection. Fluorescence quantitative PCR is a new technology that has been introduced in recent years. The target genes reported in the literature for real-time PCR detection of HCMV infection include pp65-based UL54, UL83, UL123, US17 and UL75. But the vast majority of genes being difficult to accurately measure due to low expression, including LUNA, the UL133-UL138 region, US28, UL111A and others. Fluorescent PCR techniques are difficult to detect regions that are not in a replicative state. Furthermore, positive gene detection for using real-time PCR can only represent HCMV proliferative infection and is not sensitive to the detection of intracellular HCMV. Thus, it that cannot truly reflect the HCMV infection status in humans.

Previous studies have shown that the HCMV UL138 gene may play a role in HCMV latency, immune escape, virus propagation in the host, or pathogenesis [6]. Virus strain lacking the UL138 gene cannot establish a latent infection state, indicating the expression of this gene plays an important role in the latent infection mechanism of HCMV [6]. It is now clear that the HCMV UL138 transcript is a polycistronic structure, a complete membrane protein that localizes to the Golgi apparatus, leading to latent infection of HCMV through a novel mechanism [7]. Therefore, it is considered that the latent infection of HCMV can be determined by detecting the UL138 antigen.

On the surface of both T cells and B cells, there are specific antigen receptors that can recognize the corresponding epitopes. Peptide antigens based on B cell epitopes can be used for serological detection [8]. Accordingly, in the present study, we used IEDB to analyze resources online with linear epitope prediction results as the main reference and considering the prediction results of surface accessibility, β-turn, flexibility, hydrophilicity and antigenicity. On the basis of an analysis of latent infection-related proteins, we selected the HCMV UL138 gene and predicted the dominant epitopes of the encoded protein that are recognized by host B cells. Using the identified epitopes, we established a method for detecting HCMV latent infections with high specificity and sensitivity.

Materials & methods

Specimens & experimental animals

Thirty-three EDTA-anticoagulated venous blood samples were collected from children aged 1 year and younger at the Wenzhou Medical University Yuying Children's Hospital. Similarly, EDTA-anticoagulated venous blood samples were obtained from 140 gender-matched healthy individuals at the First Affiliated Hospital of Wenzhou Medical University. None of the individuals in the two groups were known to have hepatitis B, hepatitis C, HIV, syphilis, EBV or other infections. All procedures involving human subjects were approved by the First Affiliated Hospital of Wenzhou Medical University and the Ethics Committee of Yuying Children's Hospital of Wenzhou Medical University. All subjects or their guardians provided informed consent. Female BALB/c mice (6 to 8 weeks old) were purchased from the Experimental Animal Center of Wenzhou Medical University.

Main materials & reagents

The main materials and reagents used in the present study were as follows: A Roche Electrochemiluminescence Kit (Tiangen Biochemical Technology Co., Ltd, Beijing, China); 2× Taq PCR Mastermix (Tiangen Biochemical Technology Co., Ltd); Agarose Gel Recovery Kit (Tiangen Biochemical Technology Co., Ltd); Small plasmid kit (BIO-RAD, US); PVDF membrane (Sigma, US); IPTG (BIO-RAD, US); 30% polyacrylamide; Abcam mouse anti-His-tag antibody (Hangzhou Lianke biological Technology Co., Ltd); HRP-labeled goat anti-mouse IgG (H + L) and HRP-labeled goat anti-human IgG (H + L) (Biyuntian Biotechnology Institute); CMV IgG and CMV IgM (Roche Diagnostics GmbH); UL138 gene detection kit. Primers were synthesized by Shanghai Biotech, and the peptides were synthesized by Shanghai Liang Biotechnology Co., Ltd.

Prediction of the transmembrane region of pUL138 protein & B cell epitope screening

The pUL138 protein transmembrane region was predicted online using the protein tool DAS (http://www.sbc.su.se/∼miklos/DAS/). Online prediction of the pUL138 protein epitopes recognized by B cells was performed using IEDB analysis resources (http://tools.immuneepitope.org/tools/bcell/iedb_input), with linear epitope prediction as the main reference, taking into considering surface accessibility, β-turn prediction in terms of rotation angle, flexibility, hydrophilicity and antigenicity.

Construction & identification of the pET21a–d(+)/UL138 recombinant plasmid

On the basis of the results of DAS prediction, the gene sequence corresponding to the 29 amino acids in the transmembrane region was removed, and a primer pair was designed from the sequence of the UL138 gene cytoplasmic region. The upstream and downstream primer sequences were 5′-CGCGGATCCATGCACTGGCACGATACCTT-3′ and 5′-CCGCTCGAGTCATTAATGGTGATGGTGGT-3′, respectively. The amplified product was 465 bp in length. PCR amplification was carried out using the pGEX-4T-1/UL138 recombinant plasmid as the template. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The amplified product was identified by agarose gel electrophoresis, and a target band with the correct size was recovered. The recovered product was digested with the restriction endonucleases BamHI and XhoI and ligated into the pET21a–d(+) plasmid digested with the same enzymes. The ligated product was subsequently transferred into Escherichia coli DH5α competent cells. pET21a–d(+)/UL138 recombinant plasmids containing sequences in the correct orientation were isolated using a plasmid miniprep kit according to the manufacturer's instructions.

pUL138 full-length protein expression & purification

Fifty microliters of a glycerol stock of E. coli BL21(DE3) cells was used to inoculate 5 ml of LB liquid medium. Similarly, 50 μl of glycerol stocks of bacteria containing pET21a–d(+) and pET21a–d(+)/UL138 were used to inoculate 5 ml LB/ampicillin liquid medium, which was incubated overnight. The following day, 100 μl of the overnight culture was added to 5 ml of LB/ampicillin liquid culture and incubated for 4 h, and then induced by the addition of IPTG at a final concentration of 0.8 mmol/l, followed by shaking at 2500 r/min for 6 h. The culture was centrifuged; the resulting pellet was resuspended in 1/20 volume PBS and the suspension was placed in ice for 20 min. The cells were precipitated after sonication, and the inclusion bodies were treated as required. The cells were disrupted overnight in 2 ml of 8 M urea (pH 8.0) at 4°C. The resultant protein suspension in 8 M urea was added to a dialysis bag, which was then placed in a beaker containing an appropriate amount of column equilibration solution and dialyzed overnight at 4°C. The Ni-NTA particle-bound proteins were eluted by Ni-NTA particle affinity chromatography using a 250 mmol/l imidazole (pH 8.0) eluent, and the protein concentration in the column equilibrium solution was determined using a nucleic acid protein analyzer. After protein separation by 12% SDS-PAGE electrophoresis, the gel was soaked in Coomassie blue staining solution, stained on a shaker at 20°C for 2 h, and then destained. The separated proteins were transferred to a PVDF membrane for western blotting analysis; mouse anti-His-tag antibody, HCMV-infected serum, pUL138-immunized mouse serum and pre-immune serum were used as the primary antibodies. The PVDF membrane was immersed in horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-human IgG solution and incubated at 37°C. After 90 min, the membrane was washed five-times with TBST. The pUL138 protein was detected using ECL chromogenic agent, according to the manufacturer's instructions.

Preparation of immune serum

Eight-week-old female BALB/c mice were equally divided into blank group, control group, target protein group, and epitope groups of 1–5, with six rats allocated to each group. Fifty micrograms of purified pUL138 full-length protein and each of the five epitope polypeptides coupled with Keyhole limpet hemocyanin were mixed with an equal volume of adjuvant (first immunization with complete Freund's adjuvant, and second and third immunizations with incomplete Freund's adjuvant). Then, PBS was added to bring the volume to 300 μl and injected into mice by subcutaneous injection. The blank group received no treatment with any reagent or protein. The PBS group was subcutaneously injected with 300 μl of sterilized PBS solution for each immunization. Each immunization interval was 2 weeks. Blood was taken from the tail 1 day before immunization and from the eyelids at 14 days after the final immunization. After the serum was centrifuged, the supernatant was transferred to an Eppendorf tube and stored at -80°C for subsequent use.

Indirect ELISA test

To evaluate the ability of the synthesized epitope polypeptide to recognize positive sera, the synthesized epitope polypeptide was diluted to 20 μg/ml with a coating solution. Then, 100 μl of the synthesized epitope polypeptide was added to each well of a 96-well plate for coating at 37°C for 1 h, followed by the addition of Tris containing 5% skim milk and blocking in salt buffer for 1 h. In each well, BALB/c mouse serum (1:40 dilution) was immunized with full-length pUL138 protein and incubated at 37°C for 90 mins. After washing five-times with PBS, HRP-labeled goat anti-mouse IgG (1:1:1000 dilution) was added followed by incubation at 37°C for 90 mins. After washing five-times with PBS, TMB coloring solution was added and kept at 37°C in the dark for 15 min, after which the absorbance of each well was measured at 490 nm. The ratio of positive serum to negative serum OD was calculated, and the difference between groups was analyzed using SPSS15.0 software for analysis of variance.

The synthesized epitope polypeptide and pUL138 full-length protein were coated with the coating solution at a concentration of 20 μg/ml, and 100 μl was added to each well of a 96-well plate. The solution was coated at 37°C for 1 h and blocked in a Tris salt buffer containing 5% skim milk for 1 h. Sera from adults, normal healthy children, and mice (1:40 dilution) were added and incubated at 37°C for 90 min. The rest of the steps are the same as before. For each specimen, assays were performed in three wells, and the mean value of three readings per specimen was taken as the absorbance value of the specimen for ELISA detection.

The same serum samples were examined for HCMV infection using HCMV (IgG and IgM) kits and UL138 gene assay kit. The test results were recorded as required and analyzed using SPSS 15.0 statistical software. The x² test was used to compare the rates between groups, and McNemar's method was used for detection. p < 0.05 was considered statistically significant.

Western blotting analysis

To verify the immunogenicity of the synthesized epitope polypeptide, the full-length pUL138 protein was used as the antigen and the mouse serum immunized with the five dominant B cell epitopes was used as the primary antibody to detect whether it can recognize the full-length protein of pUL138. The gel was prepared according to the steps of WB, an equal amount of UL138 full-length protein was added to each well, electrophoresis, membrane transfer, and closure were performed, the primary antibody was incubated separately according to the band cuts, and the respective primary antibody was added to each sample for incubation, the first well was incubated with the UL138 full-length protein immune serum, the second well was incubated with the epitope 1 immune serum, and so on, the seventh well was incubated with the preimmune serum, and the second antibody was incubated, and the cuts were finally merged into a single membrane for exposure.

The main operation steps are shown in the flowchart (Supplementary Figure 1).

Results

Analysis of the secondary structure of pUL138 protein & screening for dominant B cell epitopes

Analysis of the transmembrane region of pUL138 protein revealed that the first 12 N-terminal amino acids constitute a signal peptide. Amino acids 15 to 30 were located in the transmembrane region and the remaining C-terminal amino acids were located in the intracellular regions (Figure 1A). Based on the online prediction of pUL138 protein epitopes recognized by B cells (Figure 1B), with linear epitope prediction as the main reference and taking into consideration surface accessibility, β-turn, flexibility, hydrophilicity and antigenicity (Table 1), we identified five potential candidate B cell epitopes. The predicted superior B cell epitopes are shown in Table 2.

Analysis of the immunogenicity of dominant pUL138 B cell epitopes

The mice were immunized with synthetic peptides of the five identified dominant B cell epitopes, and the specific antibody induced by itself was recognized by ELISA for each epitope and the full-length UL138 protein. The results showed that as the number of immunizations increased, the five dominant B cell epitopes induced specific antibodies in mice (Figure 2A). Using the full-length UL138 protein as an antigen, ELISA was performed to detect the antibody in the third immune serum of epitope peptide. The results showed that the antibody induced by epitope peptide can specifically recognize the full-length UL138 protein, and the recognition ability of 3 and 4 was slightly stronger than that of 1, 2 and 5 (Figure 2B). Western blotting analysis further confirmed that all five epitope peptide antibodies recognized the full-length protein of UL138; the epitope peptide 5 showed the weakest recognition ability (Figure 3A).

Antigenicity analysis of the dominant B epitopes of pUL138 protein

Using the synthetic polypeptides of the five dominant B cell epitopes as antigens coating the ELISA plate and the third mouse immune serum of pUL138 full-length protein as the primary antibody, the recognition of candidate B cell epitopes by pUL138-specific antibodies was determined. The results showed that candidate epitopes 1 and 2 were recognized by pUL138-specific immune sera compared with control sera, whereas binding of epitopes 3, 4 and 5 to the full-length UL138 protein immune sera was weak (Figure 3B). There were no significant differences in pre-immune serum. These results indicate that candidate epitopes 1 and 2 are the dominant B epitopes in full-length UL138 protein-induced immune response.

Evaluation of pUL138 & dominant B epitopes in the clinical diagnosis of HCMV infection (for this evaluation we used only epitopes 1 & 2)

We determined the detection conditions for recombinant polypeptides of pUL138 and the dominant B epitopes in HCMV latent infection. Untreated BALB/c mouse serum was used as a negative control. The peptides and full-length pUL138 protein were incubated with each of the five dominant B cell epitopes, and sera from adults, normal healthy children, and mice, with dilutions of 1:40, 1:160, 1:640, 1:2560, 1:10,240 and 1:40,960, were used for the primary antibodies. We found that the gradient of the full-length pUL138 protein and predominant B epitope-synthesized peptides in the serum dilution ratio between 1:40 and 1:160 was large; therefore, we selected 1:40 as the standard dilution for each serum. We found that 1, 2, 4 and 5 epitope peptides can distinguish children from adults (Figure 4A).

Detection of HCMV latent infection in human serum using full-length pMV138 protein & synthetic dominant B epitope peptides

Using ELISA, we detected five dominant B cell epitopes and pUL138 protein in the sera of adults, normal healthy children, and mice (Figure 4B). The cut-off value was calculated by taking the average value of the mouse serum as a reference. The positive rates of the five dominant B cell epitopes and the pUL138 protein were as follows: epitope 1: 99.50% of adults, 100.00% of normal children; epitope 2: adults 98.91%, normal children 100.00%; epitope 3: adults 100.00%, normal children 100.00%; epitope 4: adults 98.91%, normal children 100.00%; epitope 5: adults 100.00%, normal children 100.00%; pUL138 full length protein: adults 100.00%, normal children 100.00% (Figure 5).

The results obtained using the five dominant B cell epitopes and pUL138 protein for detection in human serum were compared with the results of HCMV (IgG and IgM) and UL138 gene assays (see Table 3). The results showed that for the full-length pUL138 protein and synthesized epitope peptides, the positive rate of serum detection was higher than that using other detection methods, and the detection results were significantly different (p < 0.05). In addition, the data showed that in serum samples that tested positive for IgM, the OD values of the synthetic epitope peptides were higher than the OD value of the IgM negative sample (p < 0.05).

Discussion

Currently there are studies suggesting that in China the prevalence of CMV infection fluctuates from 93.91 to 97.03%. PUL138 has been reported to play an important role in the establishment of latent HCMV infection [9,10]. In this study, we aimed to identify the dominant B cell-recognized epitopes of UL138 protein that are recognized by host B cells. We also established a method for detecting HCMV latent infections with high specificity and sensitivity using the identified epitopes.

Few serological assays are available to detect specific strains of CMV infection. A study developed a novel multiplexed ELISA based on three major genotypes encoding UL144, and three UL144 proteins produced by recombinant strains [11]. It determined the critical optical density for differentiating between UL144 antibody-positive and antibody-negative sera. The overall sensitivity, specificity, positive predictive value and negative predictive value of the multiple ELISA test were good in 303 sera. Li Baoqing et al. used the carrier of HPV16 L1 to express UL138 multi-B epitope chimeric VLPs and produced a better humoral immune response in mice [12]. CMV-IgG is secreted by B cells; thus, detection of serum CMV-IgG is a commonly used indicator for assessing the sensitization status of serum CMV-specific B cells [13,14]. Clinical studies have shown that CMV-specific memory B cells are abundantly expressed in all serum CMV-IgG-positive kidney transplant patients, whereas CMV-specific memory B cells are also found in 30% of serum CMV-IgG-negative kidney transplant patients [15]. Even in patients whose serum is negative for IgG antibodies, specific memory B cells in the body have been found to be immune to CMV virus. Accordingly, we speculate that B cells could be used as indicators of latent infection–activation status. On the basis of this, given that HCMV only infects humans [1], we selected female BALB/c mice for immunization and accordingly obtained the corresponding serum for the full-length pUL138 protein and synthetic peptides of the dominant B epitopes. Indirect ELISA was used to verify the immunogenicity and antigenicity of the epitope peptides. In the immunogenicity assay, the antibodies induced by the five epitope synthetic peptides were able to recognize the full-length protein of UL138, among which epitopes 3 and 4 had a stronger ability to do so, and WB verified once again the ability of the antibodies induced by the five epitope synthetic peptides to recognize the full-length protein of UL138, and in the antigenicity assay, epitopes 1 and 2 were more likely to be recognized by the specific immune sera of pUL138, which verified that the epitope synthetic peptides and the full-length protein of pUL138 can be applied to the detection of the clinical serum sample for the infection of HCMV infection.

Next, assay experiments of synthetic peptides of the dominant B epitope on serum samples from children, healthy adults and mice showed that the OD values of the untreated BALB/c mouse serum samples were significantly lower than those of the samples obtained from children and healthy adults. In both mice and healthy humans, the five candidate B cell epitope peptides were detected in close to 100% of serum samples. The positive rate of HCMV IgG in human serum samples was 90%, whereas that of IgM, UL138 DNA and UL138 cDNA was 10.4, 93.06, and 93.64%, respectively. There was a significant difference between the results obtained for HCMV (IgG and IgM) and UL138 gene detection (p < 0.01). We found that the clinically commonly used IgG could not detect the latent HCMV infection status of the population. Instead, we believe that the five B-cell dominant epitopes in the transmembrane region of the pUL138 protein and the full-length pUL138 protein may be able to detect not only the CMV infection status, but also the latent infection status, so the positive rate is close to 100%. 5 B-cell dominant epitopes in the transmembrane region of the pUL138 protein and the full-length pUL138 protein may be more sensitive for detecting the latent infection of HCMV. It can be assumed that dominant epitopes of B cells and the full-length pUL138 protein can more closely reflect the true latent infection status of the human body than can IgG, and the positive detection rate is significantly improved. The B-cell dominant epitopes are also a perfect substitute for the full-length pUL138 protein for detection. The detection rate using B cell epitopes was found to be considerably higher than that using either UL138 DNA or cDNA. It is suspected that the immune response of B cell epitope is early and the UL138 gene is preemptively expressed [7]. Nevertheless, establishing whether the quantitative detection of serum using UL138 B cell dominant epitopes is positively correlated with latent infection–activation will require further analysis. This experiment also has limitations, the five B-cell dominant epitopes in the transmembrane region of the pUL138 protein and the full-length pUL138 protein were detected to be close to 100%, which does not allow for differentiation between previously infected, currently infected, and latently infected populations, and its accuracy, which needs to be further verified. In addition, human CMV-negative serum specimens were not collected, so the control was mouse serum as a negative control, which needs to be improved subsequently.

In this study, we concluded that the B-cell dominant epitope in the transmembrane region of pUL138 protein can effectively detect CMV infection and population-based virus testing is more sensitive than other tests. The B-cell dominant epitope of the transmembrane region of pUL138 protein not only enables the simple, economic, and rapid diagnosis of active HCMV at an early stage, but also facilitates large-scale, standardized operation that would be readily accepted by primary hospitals. Therefore, we believe that the technique described herein has important clinical significance.

Conclusion

The five predicted dominant B cell epitopes enabled detection of human serum HCMV latent infection with high sensitivity and can therefore be used as a viable alternative to the traditional HCMV IgG detection method.