Cellular and biochemical differences between two attenuated poxvirus vaccine candidates (MVA and NYVAC) and role of the C7L gene

The poxvirus strains NYVAC and MVA are two candidate vectors for the development of vaccines against a broad spectrum of diseases. Although these attenuated virus strains have proven to be safe in animals and humans, little is known about their comparative behavior in vitro. In contrast with MVA, NYVAC infection triggers greater cytopathic effect in a range of permissive and nonpermissive cell lines. The yields of NYVAC cell-associated virus in permissive cells (BHK-21) were slightly reduced compared with those of MVA infection. During the course of infection in HeLa cells, there is a translational block induced by NYVAC late in infection, which correlated with a marked increase in phosphorylation levels of the initiation factor eIF-2alpha. In contrast to MVA, the synthesis of certain late viral proteins was only blocked in NYVAC-infected HeLa cells. Electron microscopy (EM) analysis revealed that morphogenesis of NYVAC in HeLa cells was blocked at the stage of formation of immature viral forms. Phase-contrast microscopy, EM, flow cytometry, and rRNA analyses demonstrated that contrary to MVA, NYVAC infection induces potent apoptosis, a phenomenon dependent on activation of caspases and RNase L. Apoptosis induced by NYVAC was prevented when the virus gene C7L was placed back into the NYVAC genome, recovering the ability of NYVAC to replicate in HeLa cells and maintaining the attenuated phenotype in mice. Overall, our findings demonstrate distinct behavior between NYVAC and MVA strains in cultured cells, as well as a new role for the C7L viral gene as an inhibitor of apoptosis in NYVAC infection.     

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.     

The Canarypox Virus Vector ALVAC Induces Distinct Cytokine Responses Compared to the Vaccinia Virus-Based Vectors MVA and NYVAC in Rhesus Monkeys

Despite the growing use of poxvirus vectors as vaccine candidates for multiple pathogens and cancers, their innate stimulatory properties remain poorly characterized. Here we show that the canarypox virus-based vector ALVAC induced distinct systemic proinflammatory and antiviral cytokine and chemokine levels following the vaccination of rhesus monkeys compared to the vaccinia virus-based vectors MVA and NYVAC. These data suggest that there are substantial biological differences among leading poxvirus vaccine vectors that may influence resultant adaptive immune responses following vaccination.     

Expression of defective virus and cytokine genes in murine AIDS.

A syndrome characterized by severe immunodeficiency and lymphoproliferation develops in susceptible strains of mice infected with a mixture of murine leukemia viruses (MuLVs) designated LP-BM5 MuLV. The etiologic agent in this mixture has been shown to be a replication-defective virus (BM5d) with a 4.8-kb genome that required replication-competent helper viruses, primarily ecotropic (BM5e), for cell-to-cell spread in the host. In the present study, we studied the expression of BM5d and BM5e in tissues of infected mice at various times after inoculation in relation to the expression of cytokine genes that may contribute to the pathogenesis of this disorder. Northern (RNA) analysis of total RNA showed that BM5d was expressed at significant levels in lymphoid tissues within 1 week of infection and that the levels of expression increased with time after inoculation. By 16 weeks postinfection, BM5d was expressed in all tissues examined. Expression of BM5e was relatively more restricted to lymphoid tissues and was detected at lower levels than expression of BM5d at early times after infection, but this virus was expressed in all tissues by 16 weeks. Infection with the virus mixture was associated with constitutive expression of tumor necrosis factor in all tissues examined and of interleukin-1 (IL-1) in lymphoid tissues within 1 week of infection, and at later times with widespread expression of these cytokines and gamma interferon. Also, the levels of interferon regulatory factor 1 mRNA were significantly increased in all infected tissues during the infection. In contrast, expression of IL-3, IL-4, IL-5, and IL-6 was not detectable by Northern analysis of the respective mRNAs in any infected tissue at early or late times postinfection.     

Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity

Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity.     

IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo

Interferons (IFN) exert antiviral, immunomodulatory and cytostatic activities. IFN-alpha/beta (type I IFN) and IFN-lambda (type III IFN) bind distinct receptors, but regulate similar sets of genes and exhibit strikingly similar biological activities. We analyzed to what extent the IFN-alpha/beta and IFN-lambda systems overlap in vivo in terms of expression and response. We observed a certain degree of tissue specificity in the production of IFN-lambda. In the brain, IFN-alpha/beta was readily produced after infection with various RNA viruses, whereas expression of IFN-lambda was low in this organ. In the liver, virus infection induced the expression of both IFN-alpha/beta and IFN-lambda genes. Plasmid electrotransfer-mediated in vivo expression of individual IFN genes allowed the tissue and cell specificities of the responses to systemic IFN-alpha/beta and IFN-lambda to be compared. The response to IFN-lambda correlated with expression of the alpha subunit of the IFN-lambda receptor (IL-28R alpha). The IFN-lambda response was prominent in the stomach, intestine and lungs, but very low in the central nervous system and spleen. At the cellular level, the response to IFN-lambda in kidney and brain was restricted to epithelial cells. In contrast, the response to IFN-alpha/beta was observed in various cell types in these organs, and was most prominent in endothelial cells. Thus, the IFN-lambda system probably evolved to specifically protect epithelia. IFN-lambda might contribute to the prevention of viral invasion through skin and mucosal surfaces.     

Improved NYVAC-based vaccine vectors

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.     

Interplay between modified vaccinia virus Ankara and dendritic cells: phenotypic and functional maturation of bystander dendritic cells

Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus strain, currently under evaluation as a vaccine vector in various clinical settings. It has been reported that human dendritic cells (DCs) mature after infection with MVA, but reports on the functionality of DCs have so far been controversial. In this work, we studied the phenotype and functionality of MVA-infected DCs. As previously reported, we found that human monocyte-derived DCs upregulated CD86 and HLA-DR in response to MVA infection. Moreover, infected DCs produced a broad array of chemokines and cytokines and were able to activate and induce gamma interferon (IFN-γ) production both in CD4(+) and in CD8(+) allogeneic T cells and in specific autologous peripheral blood lymphocytes (PBLs). Analysis of DC maturation following infection with a recombinant green fluorescent protein (GFP)-expressing MVA revealed that upregulation of CD86 expression was mainly observed in GFP(neg) (bystander) cells. While GFP(pos) (infected) DCs produced tumor necrosis factor alpha (TNF-α), they were unable to produce CXCL10 and were less efficient at inducing IFN-γ production in CEF-specific autologous PBLs. Maturation of bystander DCs could be achieved by incubation with supernatant from infected cultures or with apoptotic infected cells. Type I IFNs were partially responsible for the induction of CXCL10 on bystander DCs. Our findings demonstrate for the first time that, in MVA-infected DC cultures, the leading role with respect to functionality and maturation characteristics is achieved by the bystander DCs.     

The combination of a chemokine,cytokine and TCR-based T cell stimulus for effective gene therapy of cancer

Cytotoxic T cells can recognize and kill tumor cells that present peptides derived from tumor-associated antigens (TAA) on their surface when associated with major histocompatibility complex (MHC) class I molecules. However, immune responses to tumor-associated antigens are often suppressed by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T cell anergy by the direct injection of vectors carrying genes encoding one of a variety of cytokines. Polyclonal stimulation of T cells, preferably via the TCR complex, results in a cascade of cytokines associated with T cell activation and thus may be better able to overcome T cell anergy. We have previously reported the use of the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, antibodies specific for the CD3epsilon chain (KT3). When injected into growing tumors, these constructs induce the activation of immune effector cells and result in rejection of the tumor. A variety of recombinant adenovirus (Ad) vectors expressing immunostimulatory and/or immunoattractant molecules have now been produced. With this collection of viruses, we have carried out in vivo analyses of combinations of vectors in tumor therapy experiments. For example, we have tested, in murine tumor models, the combination of MVA-KT3 with Ad expressing recently identified cytokines [for example interleukin-12 (IL-12), IL-18] as well as chemokines (e.g. RANTES, MIP1beta). One combination, MVA-KT3/Ad-IL-12/Ad-MIP1beta causes rejection of 100% of growing RENCA tumors. Much attention has been focused on cancer gene therapy using gene transfer of single agents. These data show that antigenic stimulation via the MHCI/TCR-CD3+cytokine+chemokine combination may provide a new and promising approach to cancer gene therapy which is more likely to bypass tumor immunosuppression mechanisms.     

Replication of modified vaccinia virus Ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene E3L

Highly attenuated modified vaccinia virus Ankara (MVA) serves as a candidate vaccine to immunize against infectious diseases and cancer. MVA was randomly obtained by serial growth in cultures of chicken embryo fibroblasts (CEF), resulting in the loss of substantial genomic information including many genes regulating virus-host interactions. The vaccinia virus interferon (IFN) resistance gene E3L is among the few conserved open reading frames encoding viral immune defense proteins. To investigate the relevance of E3L in the MVA life cycle, we generated the deletion mutant MVA-DeltaE3L. Surprisingly, we found that MVA-DeltaE3L had lost the ability to grow in CEF, which is the first finding of a vaccinia virus host range phenotype in this otherwise highly permissive cell culture. Reinsertion of E3L led to the generation of revertant virus MVA-E3rev and rescued productive replication in CEF. Nonproductive infection of CEF with MVA-DeltaE3L allowed viral DNA replication to occur but resulted in an abrupt inhibition of viral protein synthesis at late times. Under these nonpermissive conditions, CEF underwent apoptosis starting as early as 6 h after infection, as shown by DNA fragmentation, Hoechst staining, and caspase activation. Moreover, we detected high levels of active chicken alpha/beta IFN (IFN-alpha/beta) in supernatants of MVA-DeltaE3L-infected CEF, while moderate IFN quantities were found after MVA or MVA-E3rev infection and no IFN activity was present upon infection with wild-type vaccinia viruses. Interestingly, pretreatment of CEF with similar amounts of recombinant chicken IFN-alpha inhibited growth of vaccinia viruses, including MVA. We conclude that efficient propagation of MVA in CEF, the tissue culture system used for production of MVA-based vaccines, essentially requires conserved E3L gene function as an inhibitor of apoptosis and/or IFN induction.     

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10⁸ PFU) or low-dose (1 × 10⁷ PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.