Abi G. Aleyas

Functional Modulation of Dendritic Cells and Macrophages by Japanese Encephalitis Virus through MyD88 Adaptor Molecule-Dependent and -Independent Pathways

Dendritic cells (DCs) are potent initiators of T cell-mediated immunity that undergo maturation during viral infections. However, few reports describing the interactions of DCs with Japanese encephalitis virus (JEV), which remains the most frequent cause of acute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macrophages. JEV replicated its viral RNA in both cells with different efficiency, and JEV infection of macrophages followed the classical activation pathway of up-regulation of tested costimulatory molecules and proinflammatory cytokine production (IL-6, TNF-α, and IL-12). On the contrary, JEV-infected DCs failed to up-regulate costimulatory molecules such as CD40 and MHC class II. Of more interest, along with production of proinflammatory cytokines, DCs infected by JEV released antiinflammatory cytokine IL-10, which was not detected in macrophages. Moreover, signaling through MyD88 molecule, a pan-adaptor molecule of TLRs, and p38 MAPK in JEV-infected DCs was found to play a role in the production of cytokines and subversion of primary CD4+ and CD8+ T cell responses. We also found that IL-10 released from JEV-infected DCs led to a reduction in the priming of CD8+ T cells, but not CD4+ T cells. Taken together, our data suggest that JEV induces functional impairment of DCs through MyD88-dependent and -independent pathways, which subsequently leads to poor CD4+ and CD8+ T cell responses, resulting in boosting viral survival and dissemination in the body.

Cytokine GM-CSF Genetic Adjuvant Facilitates Prophylactic DNA Vaccine against Pseudorabies Virus through Enhanced Immune Responses

Granulocyte/macrophage colony‐stimulatory factor (GM‐CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen‐presenting cells (APCs) to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells (DCs). This study evaluated the utility of GM‐CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) in a murine model. The co‐injection of GM‐CSF DNA enhanced the levels of serum PrV‐specific IgG with a 1.5‐ to 2‐fold increase. Moreover, GM‐CSF co‐injection inhibited the production of IgG2a isotype. However, it enhanced production of an IgG1 isotype resulting in humoral responses biased to the Th2‐type against PrV antigen. In contrast, the co‐administration of GM‐CSF DNA enhanced the T cell‐mediated immunity biased to the Th1‐type, as judged by the significantly higher level of cytokine IL‐2 and IFN‐γ production but not IL‐4. When challenged with a lethal dose of PrV, the GM‐CSF co‐injection enhanced the resistance against a PrV infection. This suggests that co‐inoculation with a vector expressing GM‐CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen.

Multifront assault on antigen presentation by Japanese encephalitis virus subverts CD8+ T cell responses.

Japanese encephalitis virus (JEV) is a frequent cause of acute and epidemic viral encephalitis. However, there is little information describing the mechanisms by which JEV subverts immune responses that may predispose the host to secondary infections. In this study, we found that JEV induced the subversion of CD8+ T cell responses in a transient manner that was closely correlated with viral multiplication. Subsequently, analysis using a TCR-transgenic system revealed that CD8+ T cells purified from JEV-infected mice showed impaired responses, and that naive CD8+ T cells adoptively transferred into JEV-infected recipients showed less expanded responses. Furthermore, JEV altered the splenic dendritic cell (DC) subpopulation via preferential depletion of CD8α+CD11c+ DCs without changing the plasmacytoid DCs and induced a significant reduction in the surface expression of MHC class II and CD40, but not MHC class I, CD80, and CD86 molecules. Finally, JEV was shown to inhibit the presentation of MHC class I-restricted Ag in DCs, and this immune suppression was ameliorated via the activation of DCs by TLR agonists. Collectively, our data indicate that JEV precludes the functions of Ag-presenting machinery, such as depletion of CD8α+CD11c+ DCs and downregulation of MHC class I-restricted Ag presentation, thereby leading to immune subversion of CD8+ T cells.

Impaired cross-presentation of CD8α+ CD11c+ dendritic cells by Japanese encephalitis virus in a TLR2/MyD88 signal pathway-dependent manner.

Cross‐presentation is the pathway by which exogenous antigens are routed for presentation by MHC class I molecules leading to activation of antiviral CD8+ T‐cell responses. However, there is little information describing the modulation of cross‐presentation and the impact of pathogen‐derived signals associated with Japanese encephalitis virus (JEV), which is one of the most common causes of encephalitis in humans. In this study, we demonstrate that JEV infection could suppress in vivo cross‐presentation of soluble and cell‐associated antigens, thereby generating weak CD8+ T‐cell responses to exogenous antigens, as evaluated by CFSE dilution of adoptively transferred CD8+ T cells and in vivo CTL killing activity. Furthermore, CD8α+CD11c+ dendritic cells (DCs), which are known to be far more efficient at cross‐presenting soluble antigens, played a specific role in contributing to JEV‐mediated inhibition of the cross‐presentation of exogenous antigens through interference with effective antigen uptake. Finally, this study provides evidence that TLR2‐MyD88 and p38 MAPK signal pathway might be involved in JEV‐mediated inhibition of cross‐presentation of soluble and cell‐associated antigens. These observations suggest that the modulation of cross‐presentation of exogenous antigens through TLR signaling has important implications for antiviral immune responses against JEV infection and the development of effective vaccination strategies.

Genetic co-transfer of CCR7 ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus

The CC chemokine receptor 7 (CCR7) and cognate CCR7 ligands, CCL19 and CCL21, help establish microenvironments in lymphoid tissue that can facilitate encounters between naive T cells and mature dendritic cells (DCs). This study was conducted to determine if CCR7 ligands can augment the immunogenicity of a DNA vaccine that expresses glycoprotein B (gB) of the pseudorabies virus (PrV). The genetic co‐transfer of CCR7 ligands along with a PrV DNA vaccine increased the levels of serum PrV‐specific immunoglobulin (Ig) G by 2‐ to 2.5‐fold. In addition, the level of PrV‐specific IgG2a isotype was significantly enhanced by co‐injection of CCR7 ligand DNA, which indicates that CCR7 ligand biases the humoral immunity toward the Th1‐type pattern. The co‐injection of CCR7 ligand DNA consistently enhanced the level of Th1‐type cytokines (IL‐2 and IFN‐γ) produced by stimulated immune cells when compared with a group that was vaccinated with the PrV DNA vaccine. Also, the genetic co‐transfer of CCR7 ligand DNAs with PrV DNA vaccine provided prolonged survival against a virulent challenge by PrV. Moreover, the co‐administration of CCR7 ligand DNA increased the number of mature DCs into the secondary lymphoid tissues, which appeared to enhance the proliferation of PrV‐immune CD4+ T cells. Taken together, these findings indicate that CCR7 ligands are an attractive adjuvant for a PrV DNA vaccine that can offer protective immunity against the PrV.

Differential segregation of protective immunity by encoded antigen in DNA vaccine against pseudorabies virus

A murine model immunized with plasmid DNA vaccine expressing three glycoproteins pCIgB, pCIgC and pCIgD were used to examine the relative potency of major glycoproteins as well as the contribution of immunological parameters in providing protective immunity against the pseudorabies virus (PrV). Among the three glycoprotein‐encoded plasmid DNA vaccines, pCIgB produced the strongest response of PrV‐specific IgG in the sera. pCIgB and pCIgD also induced a contrast pattern of immunity that was biased to the Th1 and Th2 types, respectively. pCIgC showed the potent inducer of CD8+ T‐cell‐mediated CTL activity against PrV. In addition, a cocktail vaccination of all three glycoprotein‐encoded plasmid DNA vaccines induced the production of both cytokine types, Th1 and Th2 with levels that were the same as that of each immunogen. With regard to protective efficacy, pCIgB induced the most effective protection against a virulent virus challenge and a cocktail vaccination appeared to offer complete protection against a 5 LD 50 challenge, but not a 10 LD 50 one. pCIgD induced protection that was same as pCIgB, but pCIgC offered no effective protection. These results show the relative potency of the three glycoprotein‐encoded PrV DNA vaccines in inducing protective immunity against PrV infection. The results in this study support previous results showing the importance of Th1‐type CD4+ T cells and their antibodies in conferring protection.

Low‐dose antigen‐experienced CD4+ T cells display reduced clonal expansion but facilitate an effective memory pool in response to secondary exposure

The strength and duration of an antigenic signal at the time of initial stimulation were assumed to affect the development and response of effectors and memory cells to secondary stimulation with the same antigen. To test this assumption, we used T‐cell receptor (TCR)‐transgenic CD4+ T cells that were stimulated in vitro with various antigen doses. The primary effector CD4+ T cells generated in response to low‐dose antigen in vitro exhibited reduced clonal expansion upon secondary antigenic exposure after adoptive transfer to hosts. However, the magnitude of their contraction was much smaller than both those generated by high‐dose antigen stimulation and by naïve CD4+ T cells, resulting in higher numbers of antigen‐specific CD4+ T cells remaining until the memory stage. Moreover, secondary effectors and memory cells developed by secondary antigen exposure were not functionally impaired. In hosts given the low‐dose antigen‐experienced CD4+ T cells, we also observed accelerated recall responses upon injection of antigen‐bearing antigen‐presenting cells. These results suggest that primary TCR stimulation is important for developing optimal effectors during initial antigen exposure to confer long‐lasting memory CD4+ T cells in response to secondary exposure.

Polarization of protective immunity induced by replication-incompetent adenovirus expressing glycoproteins of pseudorabies virus

Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-γ and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.