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. 2024 Jul 16:15:1438371.
doi: 10.3389/fimmu.2024.1438371. eCollection 2024.

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus

Chenhe Lu  1 , Haimin Li  1 , Wenjing Chen  1 , Hui Li  2 , Jiayu Ma  1 , Peng Peng  1 , Yan Yan  1 , Weiren Dong  1 , Yulan Jin  1 , Shiyue Pan  1 , Shaobin Shang  2 , Jinyan Gu  1 , Jiyong Zhou  1   3
Affiliations

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus

Chenhe Lu et al. Front Immunol. .

Erratum in

Abstract

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion.

Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus.

Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells.

Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

Keywords: IFN-γ; chimeric pseudorabies virus; circovirus; immunity; memory responses.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Generation of recombinant PRV with the Cap protein of PCV2. (A) The construction strategy of PRV-Cap. The PCV2 Cap gene was inserted into the gE extracellular region of parent PRV HD/c virus strain by homologous recombinant transfer vector, and then the fluorescent labeled EGFP gene was removed in vitro by the Cre-LoxP recombinant enzyme system to obtain the recombinant PRV with only the exogenous Cap gene. (B) IFA and (C) Western blotting assays of Cap-gE fusion protein expression in Vero cells inoculated with 1 MOI of PRV-Cap-EGFP, PRV-Cap, and PRV HD/c virus at 24 h post infection (D) Identification of inserting the Cap gene in Cap-EGFP and PRV-Cap by nucleic acid electrophoresis using gE-US7-9 primers.
Figure 2
Figure 2
Detection of expression and genetic stability of Cap-gE chimeric protein in PRV-Cap. (A) One-step growth curves and plaque detection of PRV-Cap and parent PRV HD/c virus (MOI=1) infected PK-15 cells. The infected cells were incubated at 37°C for 2 h, washed with DMEM three times, and cells were finally cultured with DMEM containing 2% FBS. Viral titers were measured at indicated time points post infection. The plaque formation unit (PFU) was detected by the limited dilution plaque assay and TCID50 was determined by Reed-Muench method. (B) The abundance of the Cap-gE fusion protein in the supernatant of PRV-Cap was detected by Western blotting compared with the original Cap protein of PCV2. (C) The virus particles purified by 20% Sorbitol Cushion ultra-centrifugation were examined by transmission electron microscopy with anti-Cap mAbs as described in Materials and methods. (D) The purified Cap virions were violently mixed with chloroform and ice bath for 20 min to remove the virus envelope for Western blotting detection. (E) The genome of PRV-Cap was extracted, and PCR detection and sequencing were performed with gE-US7-9 primers. (F) Western blotting detection of each generation of 108.0 TCID50/ml PRV-Cap supernatant. NS, p >0.05.
Figure 3
Figure 3
Immunogenicity of the PRV-Cap. (A) The mouse model of PRV-Cap vaccination. (B, C) The gB and Cap ELISA antibody titers at 7, 14, and 21 dpv. (D, E) Neutralization antibody titers against PRV or PCV2 at 21 dpv. (F, G) Survival rates of mice immunized post 3 weeks against PRV-DX (106.5 TCID50/ml, 0.1 ml) or PCV2 ZJ/c (107.0 TCID50/ml, 0.45ml) challenge. (H, I) The viral loads in the lungs and spleens of mice were measured by qPCR assay on 3rd day after PRV-DX challenge, and PRV genomic copies were calculated according to the CT value of the PRV-gB standard curve. (J, K) The viral loads in the lungs and spleens of mice were measured by qPCR assay on day 21 after PCV2 challenge, and PCV2 genomic copies were calculated according to the CT value of the PCV2-Rep standard curve. (L, M) Immunohistochemical analysis of the lungs, spleens and inguinal lymph nodes of PRV DX or PCV2 ZJ/c challenged mice. Magnification, 80×; Scale bars, 20 μm. Data are expressed as means ± standard errors of the means. NS, p >0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4
Figure 4
Dynamic changes of activation and expansion of different immune cell subsets after PRV-Cap immunization. (A, B) The expression of activation marker CD69 in CD4 T cells, CD8 T cells, γδ T cells, and B cells at 7 and 14 dpv. (C, D) The expression of expansion marker Ki67 in CD4 T cells, CD8 T cells, and B cells at 7 and 14 dpv. (E, F) The proportion and representative cytometric profiles of Tfh cells (CD4+PD-1+BCl-6+) and GC B cells (B220+GL7+FAS+) at 7 and 14 dpv. (G) The percentage and representative cytometric profiles of CD40-positive B cells (CD19+CD40+) at 7 and 14 dpv. (H) The expression and representative cytometric profiles of CD107a in NK cells and CTLs at 14 dpv. NS, p >0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5
Figure 5
PRV and PCV2 specific cytokine production profile from splenocytes of PRV-Cap vaccinated mice at 14 dpv. (A) The specific proliferation response of splenocytes was detected using the CCK8 method. Splenic lymphocyte suspension was stimulated with PRV-DX or PCV2 inactivated antigen (MOI=1) for 48 h, then antigen-specific proliferative response was detected at OD450 using CCK8. The stimulation index (SI) = (test group OD450 - blank control OD450)/(negative control OD450 - blank control OD450). (B) Representative images of IFN-γ ELISpot wells and mean spot size from the various vaccine groups. The secreting spots forming cells (SFC) were counted using the ELISpot method after 24 h stimulation with PRV-DX or PCV2 inactivated antigen. (C) The proportion of PRV or PCV2 specific CD4+ T cells that produced IFN-γ, TNF-α or IL-2 single cytokine, any two cytokines, and triple cytokines. (D–H) Percentage of CD4+ T cells that produced single, double, and triple representative TH1 cytokines after PRV-DX or PCV2 stimulation (MOI=1, 24h). (I–L) Percentage of CD8+ T cells that produced single or double cytokines after PRV-DX or PCV2 stimulation (MOI=1, 24h). Data expressed as mean ± sd from five mice per group. NS, p >0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 6
Figure 6
Percentage of memory T and memory B cells in splenocytes at 14 dpv. (A) The proportion and representative cytometric diagrams of CD4+ memory Th1 cells. (B, C) Statistical analysis and representative cytometry diagrams of Tcm and Tem in CD4+ or CD8+ T cells. (D) The proportion and representative cytometric diagrams of MBCs in B220 positive B cells. Data expressed as mean ± sd from five mice per group. NS, p >0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 7
Figure 7
Analysis of the memory response of PRV-Cap induced T cells. The booster immunization with PRV-DX (106.5 TCID50/ml, 0.1 ml) or PCV2 (107.0 TCID50/ml, 0.45ml) was performed in mice at 28 days after initial immunization with the PRV-Cap. (A–C) Percentage of CD4+ T cells stimulated by PRV-Cap (MOI=1, 24h) to produce single, double, and triple representative TH1 cytokines at 7, 14, 21, and 28 dpv and at 7 days post booster immunization with PRV-DX or PCV2. (D, E) Percentage of CD8+ T cells stimulated by PRV-Cap (MOI=1, 24h) to produced single or double cytokines at 7, 14, 21, and 28 dpv and 7 days post booster immunization with PRV-DX or PCV2. Data expressed as mean ± sd from five mice per group. NS, p >0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by National Key R&D program of China (2022YFD1800804), the Key Research and Development project of Zhejiang Province (2020C02011) and the Fundamental Research Funds for the Central Universities (2022-KYY-517101-0005).

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