Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity

Mature dendritic cells (DC) are the most potent antigen-presenting cells within the entire immune system. Interference with the function of these cells therefore constitutes a very powerful mechanism for viruses to escape immune responses. Several members of the Herpesviridae family have provided examples of such escape strategies, including interference with antigen presentation and production of homologous cytokines. In this study we investigated the infection of mature DC with herpes simplex virus type 1 (HSV-1) and the way in which infection alters the phenotype and function of mature DC. Interestingly, the T-cell-stimulatory capacity of these DC was strongly impaired. Furthermore, we demonstrated that HSV-1 leads to the specific degradation of CD83, a cell surface molecule which is specifically upregulated during DC maturation. These data indicate that HSV-1 has developed yet another novel mechanism to escape immune responses.     

Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3^−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.     

HLA-G*0105N null allele encodes functional HLA-G isoforms

Expression of the nonclassical HLA class I antigen, HLA-G, is associated with immune tolerance in view of its role in maintaining the fetus in utero, allowing tumor escape, and favoring graft acceptance. Expressed on invasive trophoblast cells, HLA-G molecules bind inhibitory receptors on maternal T lymphocytes and NK cells, thereby blocking their cytolytic activities and protecting the fetus from maternal immune system attack. The HLA-G gene consists of 15 alleles, including a null allele, HLA-G*0105N. HLA-G*0105N presents a single base deletion, preventing translation of both membrane-bound (HLA-G1) and full-length soluble isoforms (HLA-G5) as well as of the spliced HLA-G4 isoform. The identification of healthy subjects homozygous for this HLA-G null allele suggests that the HLA-G*0105N allele may generate other HLA-G isoforms, such as membrane-bound HLA-G2 and -G3 and the soluble HLA-G6 and -G7 proteins, which may substitute for HLA-G1 and -G5, thus assuming the immune tolerogeneic function of HLA-G. To investigate this point, we cloned genomic HLA-G*0105N DNA and transfected it into an HLA-class I-positive human cell line. The results obtained indicated that HLA-G proteins were indeed present in HLA-G*0105N-transfected cells and were able to protect against NK cell lysis. These findings emphasize the role of the other HLA-G isoforms as immune tolerogeneic molecules that may also contribute to maternal tolerance of the semiallogenic fetus as well as tumor escape and other types of allogeneic tissue acceptance.     

Immunization with replication-defective mutants of herpes simplex virus type 1: sites of immune intervention in pathogenesis of challenge virus infection.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) were used as a new means to immunize mice against HSV-1-mediated ocular infection and disease. The effects of the induced immune responses on pathogenesis of acute and latent infection by challenge virus were investigated after corneal inoculation of immunized mice with virulent HSV-1. A single subcutaneous injection of replication-defective mutant virus protected mice against development of encephalitis and keratitis. Replication of the challenge virus at the initial site of infection was lower in mice immunized with attenuated, wild-type parental virus (KOS1.1) or replication-defective mutant virus than in mice immunized with uninfected cell extract or UV-inactivated wild-type virus. Significantly, latent infection in the trigeminal ganglia was reduced in mice given one immunization with replication-defective mutant virus and was completely prevented by two immunizations. Acute replication in the trigeminal ganglia was also prevented in mice immunized twice with wild-type or mutant virus. The level of protection against infection and disease generated by immunization with replication-defective mutant viruses was comparable to that of infectious wild-type virus in all cases. In addition, T-cell proliferative and neutralizing antibody responses following immunization and corneal challenge were of similar strength in mice immunized with replication-defective mutant viruses or with wild-type virus. Thus, protein expression by forms of HSV-1 capable of only partially completing the replication cycle can induce an immune response in mice that efficiently decreases primary replication of virulent challenge virus, interferes with acute and latent infection of the nervous system, and inhibits the development of both keratitis and systemic neurologic disease.     

Herpes B Virus,Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8⁺ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions.     

Rabies virus expressing dendritic cell-activating molecules enhances the innate and adaptive immune response to vaccination

Our previous studies indicated that recruitment and/or activation of dendritic cells (DCs) is important in enhancing the protective immune responses against rabies virus (RABV) (L. Zhao, H. Toriumi, H. Wang, Y. Kuang, X. Guo, K. Morimoto, and Z. F. Fu, J. Virol. 84:9642-9648). To address the importance of DC activation for RABV vaccine efficacy, the genes for several DC recruitment and/or activation molecules, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein 1α (MIP-1α), were individually cloned into RABV. The ability of these recombinant viruses to activate DCs was determined in vitro and in vivo. Infection of mouse bone marrow-derived DCs with each of the recombinant viruses resulted in DC activation, as shown by increased surface expression of CD11c and CD86 as well as an increased level of alpha interferon (IFN-α) production compared to levels observed after infection with the parent virus. Intramuscular infection of mice with each of the viruses recruited and/or activated more DCs and B cells in the periphery than infection with the parent virus, leading to the production of higher levels of virus-neutralizing antibodies. Furthermore, a single immunization with recombinant RABV expressing GM-CSF or MDC protected significantly more mice against intracerebral challenge with virulent RABV than did immunization with the parental virus. Yet, these viruses did not show more virulence than the parent virus, since direct intracerebral inoculation with each virus at up to 1 × 10(7) fluorescent focus units each did not induce any overt clinic symptom, such as abnormal behavior, or any neurological signs. Together, these data indicate that recombinant RABVs expressing these molecules activate/recruit DCs and enhance protective immune responses.     

Neuronal Interferon Signaling Is Required for Protection against Herpes Simplex Virus Replication and Pathogenesis

Interferon (IFN) responses are critical for controlling herpes simplex virus 1 (HSV-1). The importance of neuronal IFN signaling in controlling acute and latent HSV-1 infection remains unclear. Compartmentalized neuron cultures revealed that mature sensory neurons respond to IFNβ at both the axon and cell body through distinct mechanisms, resulting in control of HSV-1. Mice specifically lacking neural IFN signaling succumbed rapidly to HSV-1 corneal infection, demonstrating that IFN responses of the immune system and non-neuronal tissues are insufficient to confer survival following virus challenge. Furthermore, neurovirulence was restored to an HSV strain lacking the IFN-modulating gene, γ34.5, despite its expected attenuation in peripheral tissues. These studies define a crucial role for neuronal IFN signaling for protection against HSV-1 pathogenesis and replication, and they provide a novel framework to enhance our understanding of the interface between host innate immunity and neurotropic pathogens.     

Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2＇-deoxycytidine

The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-AC） and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5＇ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components （APM） as well as β2 microglobulin （β2-m） expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.     

Induction of murine p30 by superinfecting herpesviruses.

The interaction of endogenous type C viruses with superinfecting herpes simplex virus type 2 (HSV-2) was investigated in two murine cell lines. Replication of HSV-2 was suboptimal in random-bred Swiss/3T3A cells and, in initial experiments, infection with a low virus-to-cell ratio resulted in carrier cultures with enhanced murine leukemia virus (MuLV) p30 expression. Immunofluorescence tests with Swiss/3T3A cells productively infected with HSV-2 also showed HSV-associated cytoplasmic antigens and enhanced MuLV p30 expression when compared with uninfected controls. Inactivation of HSV-2 with UV light did not abolish this reaction, although the number of cells expressing p30 was reduced. HSV-2 replicated more efficiently in a line of NIH Swiss cells (N c1 A c1 10). These cells are not readily inducible for type C expression by conventional methods; however, untreated and UV-inactivated HSV-2 induced both HSV-2-associated antigens and MuLV p30 in these cells. Although the Birch strain of human cytomegalovirus induced MuLV p30, neither mouse cytomegalovirus nor vesicular stomatitis virus induced MuLV p30 in either cell line.     

A functional role of HLA-G expression in human gliomas: an alternative strategy of immune escape

HLA-G is a nonclassical MHC molecule with highly limited tissue distribution that has been attributed chiefly immune regulatory functions. Glioblastoma is paradigmatic for the capability of human cancers to paralyze the immune system. To delineate the potential role of HLA-G in glioblastoma immunobiology, expression patterns and functional relevance of this MHC class Ib molecule were investigated in glioma cells and brain tissues. HLA-G mRNA expression was detected in six of 12 glioma cell lines in the absence of IFN-gamma and in 10 of 12 cell lines in the presence of IFN-gamma. HLA-G protein was detected in four of 12 cell lines in the absence of IFN-gamma and in eight of 12 cell lines in the presence of IFN-gamma. Immunohistochemical analysis of human brain tumors revealed expression of HLA-G in four of five tissue samples. Functional studies on the role of HLA-G in glioma cells were conducted with alloreactive PBMCs, NK cells, and T cell subpopulations. Expression of membrane-bound HLA-G1 and soluble HLA-G5 inhibited alloreactive and Ag-specific immune responses. Gene transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells (U87MG) rendered cells highly resistant to direct alloreactive lysis, inhibited the alloproliferative response, and prevented efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G were directed against CD8 and CD4 T cells, but appeared to be NK cell independent. Interestingly, few HLA-G-positive cells within a population of HLA-G-negative tumor cells exerted significant immune inhibitory effects. We conclude that the aberrant expression of HLA-G may contribute to immune escape in human glioblastoma.     

Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome

Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner strictly dependent on viral gene expression. Soluble CD83 enzyme-linked immunosorbent assays, together with Western blot analysis, demonstrated that CD83 rapidly disappears from the cell surface after contact with HSV-1 by a mechanism that involves protein degradation rather than shedding of CD83 from the cell surface into the medium. Infection experiments with an ICP0 deletion mutant demonstrated an important role for this viral immediate-early protein during CD83 degradation, since this particular mutant strain leads to strongly reduced CD83 degradation. This hypothesis was further strengthened by cotransfection of plasmids expressing CD83 and ICP0 into 293T cells, which led to significantly reduced accumulation of CD83. In strong contrast, transfection of plasmids expressing CD83 and a mutant ICP0 defective in its RING finger-mediated E3 ubiquitin ligase function did not reduce CD83 expression. Inhibition of the proteasome, the cellular protein degradation machinery, almost completely restored CD83 surface expression during HSV-1 infection, indicating that proteasome-mediated degradation and HSV-1 ICP0 play crucial roles in this novel viral immune escape mechanism.     

Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. The levels of virus-directed lysis varied widely from target to target, and we found that differences in virus-directed lytic efficiency could be attributed both to the characteristics of HSV-1 replication in the different targets and to the subgroup of natural effector cells which mediated lysis. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, we used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). We also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. Using complement-mediated elimination of Qa-5-positive or asialo-GM1-positive NK cells to distinguish NK from NC activity, we found that NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. In addition, we showed that both NK and NC cytotoxicities contributed to the lysis against the HSV-1-infected fibroblastoid line, M50, but the infected PU51R cells were killed by only NK effectors. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the 51Cr-release assay in the presence of anti-interferon serum. Because NC activity was not augmented by interferon, virus-enhanced NC lysis of M50-HSV and WEHI-HSV was not due to this nonspecific mechanism. Together, our data show that HSV-1 infection of NK/NC targets induces increased cytotoxicity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.     

表达新冠病毒S基因重组狂犬病病毒的构建及其免疫原性

【背景】新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)在全球流行已近3年,除对人类造成了巨大伤害,也影响了人类的伴侣动物。人的COVID-19疫苗已在全球应用,但动物用的新冠病毒疫苗却鲜有报道。【目的】研制兽用新冠病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)和狂犬病病毒(rabies virus,RABV)的二联苗。【方法】将合成的SARS-CoV-2 S基因和S1基因分别克隆至RABV弱毒疫苗株rHEP-Flury基因组G与L基因间,并将2个重组质粒分别与辅助质粒共转染至BHK-21细胞中,拯救重组病毒rHEP-nCOV-S和rHEP-nCOV-S1。通过RT-PCR、Western blotting和荧光抗体染色,验证重组病毒、确证S和S1蛋白在RABV中成功表达。再将重组病毒接种NA细胞及成年小白鼠,测定病毒的体外生长特性、重组病毒的致病性及免疫原性。【结果】免疫荧光结果显示,转染7d后细胞上清均出现了绿色免疫荧光,表明已成功拯救嵌合SARS-CoV-2S和S1基因的重组病毒RABV rHEP-nCOV-S和rHEP-nCOV-S1,并且rHEP-nCOV-S1的增殖和扩散能力强于亲本株rHEP-Flury,但rHEP-nCOV-S与亲本株无显著差异。Western blotting结果显示,在目的位置处均出现72kDa和144kDa特异性条带,表明S和S1蛋白在重组RABV中高效表达。重组病毒免疫6周KM小鼠后,小鼠的体重变化与亲本RABV基本一致,重组病毒诱导小鼠产生狂犬中和抗体。【结论】本研究拯救出了嵌合SARS-CoV-2 S/S1基因的重组RABV,为动物COVID-19载体疫苗的研发奠定了基础。     

An inactivated recombinant rabies virus displaying the Zika virus prM-E induces protective immunity against both pathogens

The global spread of Zika virus (ZIKV), which caused a pandemic associated with Congenital Zika Syndrome and neuropathology in newborns and adults, prompted the pursuit of a safe and effective vaccine. Here, three kinds of recombinant rabies virus (RABV) encoding the prM-E protein of ZIKV were constructed: ZI-D (prM-E), ZI-E (transmembrane domain (TM) of prM-E replaced with RABV G) and ZI-F (signal peptide and TM domain of prM-E replaced with the region of RABV G). When the TM of prM-E was replaced with the region of RABV G (termed ZI-E), it promoted ZIKV E protein localization on the cell membrane and assembly on recombinant viruses. In addition, the change in the signal peptide with RABV G (termed ZI-F) was not conducive to foreign protein expression. The immunogenicity of recombinant viruses mixed with a complex adjuvant of ISA 201 VG and poly(I:C) was tested in BALB/c mice. After immunization with ZI-E, the anti-ZIKV IgG antibody lasted for at least 10 weeks. The titers of neutralizing antibodies (NAbs) against ZIKV and RABV at week 6 were all greater than the protective titers. Moreover, ZI-E stimulated the proliferation of splenic lymphocytes and promoted the secretion of cytokines. It also promoted the production of central memory T cells (TCMs) among CD4+/CD8+ T cells and stimulated B cell activation and maturation. These results indicate that ZI-E could induce ZIKV-specific humoral and cellular immune responses, which have the potential to be developed into a promising vaccine for protection against both ZIKV and RABV infections.