Adenoviral delivery of soluble ovine OX40L or CD70 costimulatory molecules improves adaptive immune responses to a model antigen in sheep.

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L- CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.

The Interplay between Bluetongue Virus Infections and Adaptive Immunity.

Viral infections have long provided a platform to understand the workings of immunity. For instance, great strides towards defining basic immunology concepts, such as MHC restriction of antigen presentation or T-cell memory development and maintenance, have been achieved thanks to the study of lymphocytic choriomeningitis virus (LCMV) infections. These studies have also shaped our understanding of antiviral immunity, and in particular T-cell responses. In the present review, we discuss how bluetongue virus (BTV), an economically important arbovirus from the Reoviridae family that affects ruminants, affects adaptive immunity in the natural hosts. During the initial stages of infection, BTV triggers leucopenia in the hosts. The host then mounts an adaptive immune response that controls the disease. In this work, we discuss how BTV triggers CD8+ T-cell expansion and neutralizing antibody responses, yet in some individuals viremia remains detectable after these adaptive immune mechanisms are active. We present some unpublished data showing that BTV infection also affects other T cell populations such as CD4+ T-cells or γδ T-cells, as well as B-cell numbers in the periphery. This review also discusses how BTV evades these adaptive immune mechanisms so that it can be transmitted back to the arthropod host. Understanding the interaction of BTV with immunity could ultimately define the correlates of protection with immune mechanisms that would improve our knowledge of ruminant immunology.

Inhibition of the IFN Response by Bluetongue Virus: The Story So Far.

Bluetongue virus (BTV) is the prototypical orbivirus that belongs to the Reoviridae family. BTV infection produces a disease in ruminants, particularly in sheep, that results in economic losses through reduced productivity. BTV is transmitted by the bite of Culicoides spp. midges and is nowadays distributed globally throughout subtropical and even temperate regions. As most viruses, BTV is susceptible to the IFN response, the first line of defense employed by the immune system to combat viral infections. In turn, BTV has evolved strategies to counter the IFN response and promote its replication. The present review we will revise the works describing how BTV interferes with the IFN response.

Vaccination With Recombinant Adenoviruses Expressing the Bluetongue Virus Subunits VP7 and VP2 Provides Protection Against Heterologous Virus Challenge.

Bluetongue virus (BTV) is the causative agent of a disease that affects domestic and wild ruminants and leads to critical economic losses. BTV is an arbovirus from the Reoviridae family that is typically transmitted by the bite of infected Culicoides midges. BTV possesses multiple serotypes (up to 28 have been described), and immunity to one serotype offers little cross-protection to other serotypes. The design of vaccines that provide protection across multiple serotypes is therefore highly desirable to control this disease. We previously reported that a recombinant replication-defective human adenovirus serotype 5 (Ad5) that expresses the VP7 inner core protein of BTV serotype 8 (Ad5VP7-8) induced T-cell responses and provided protection. In the present work, we evaluated as BTV vaccine the combination of Ad5VP7-8 with another recombinant Ad5 that expresses the outer core protein VP2 from BTV-1 (Ad5VP2-1). The combination of Ad5VP2-1 and Ad5VP7-8 protected against homologous BTV challenge (BTV-1 and BTV-8) and partially against heterologous BTV-4 in a murine model. Cross-reactive anti-BTV immunoglobulin G (IgG) were detected in immunized animals, but no significant titers of neutralizing antibodies were elicited. The Ad5VP7-8 immunization induced T-cell responses that recognized all three serotypes tested in this study and primed cytotoxic T lymphocytes specific for VP7. This study further confirms that targeting antigenic determinant shared by several BTV serotypes using cellular immunity could help develop multiserotype BTV vaccines.

Hemagglutinin protein of Peste des Petits Ruminants virus (PPRV) activates the innate immune response via Toll-like receptor 2 signaling.

The toll-like receptor (TLR) family comprises both cell-surface and intracellular receptors that recognize different types of pathogen-associated molecular patterns (PAMPs) leading to the production of pro-inflammatory cytokines and subsequent development of adaptive immunity. TLR2 is a cell-surface receptor initially thought to act as a bacterial sentinel but also shown to recognize a number of viral glycoproteins. In this study, we sought to characterize the role of TLR2 in the activation of the immune response by peste des petits ruminants virus (PPRV), a morbillivirus of the Paramixoviridae family that causes an acute, highly contagious disease in goats and sheep. Using human embryonic kidney (HEK) 293 cells stably expressing human (h)TLR2 but lacking any other TLR, we found that PPRV induces IL-8 production in a dose-dependent manner. That activation is only observed in cells expressing hTLR2 and is greatly reduced when the receptor is blocked by pretreatment with specific antibody. We identified hemagglutinin (H) as the viral protein responsible of TLR2 activation by performing the same assays with purified recombinant mammalian-expressed H protein. Exogenous addition of recombinant H protein to cell culture induces high levels of interleukin (IL)-8 only in TLR2-expressing cells. Moreover, H engagement on TLR2 in the monocytic cell line THP-1 activates extracellular-signal-regulated kinase (ERK) signaling. Stimulation of primary ovine dendritic cells with either inactivated PPRV or purified recombinant H protein results in transcription of pro-inflammatory cytokines and the secretion of the Th1-polarizing cytokine IL-12. The role of these host immune mechanisms in the control of PPR is discussed.

Virus-induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade.

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response, and viral pathogens have evolved multiple mechanisms to antagonize this pathway and to facilitate infection. Bluetongue virus (BTV), an orbivirus of the Reoviridae family, is transmitted by midges to ruminants and causes a disease that produces important economic losses and restriction to animal trade and is of compulsory notification to the World Organization for Animal Health (OIE). Here, we show that BTV interferes with IFN-I and IFN-II responses in two ways, by blocking STAT1 phosphorylation and by degrading STAT2. BTV-NS3 protein, which is involved in virion egress, interacts with STAT2, and induces its degradation by an autophagy-dependent mechanism. This STAT2 degradative process requires the recruitment of an E3-Ub-ligase to NS3 as well as NS3 K63 polyubiquitination. Taken together, our study identifies a new mechanism by which a virus degrades STAT2 for IFN signaling blockade, highlighting the diversity of mechanisms employed by viruses to subvert the IFN response.

Peste des Petits Ruminants Virus Fusion and Hemagglutinin Proteins Trigger Antibody-Dependent Cell-Mediated Cytotoxicity in Infected Cells.

The adaptive immune system utilizes multiple effector mechanisms to clear viral infections. Among those antibody-dependent cell-mediated cytotoxicity (ADCC) can help recognize and clear virus-infected cells. In the present work we evaluated ADCC contribution to immunity in two economically important viral diseases that affect ruminants: bluetongue and peste des petits ruminants. Immune sera obtained from sheep experimentally infected with bluetongue virus (BTV) serotype 8 or peste des petits ruminant virus (PPRV) IC'89 were used for this study. PPRV immune sera could bind to the surface of PPRV-infected ovine B cells while BTV immune sera was unable to bind to the surface of BTV-infected sheep cells but could recognize intracellular BTV antigens. BTV and PPRV immune serum ADCC potency was established using an ovine autologous cytotoxicity assay that employed an NK cell-enriched fraction as effector cells and a virus-infected B cell-enriched fraction as target cells. In this system, immune sera triggered ADCC against PPRV-infected cells, but not against BTV-infected cells. PPRV immune sera could recognize PPRV fusion and hemagglutinin proteins on the surface of transfected cells, and enhanced lysis of these cells in ADCC assays. This indicated that these viral antigens are natural ADCC targets during PPRV infection. The present work describes a novel effector immune mechanism against PPRV in the natural host that could contribute to virus clearance highlighting the importance of studying protective immune mechanisms to improve current vaccines by invoking all effector arms of immunity.

Vaccination with recombinant adenovirus expressing peste des petits ruminants virus-F or -H proteins elicits T cell responses to epitopes that arises during PPRV infection.

Peste des petits ruminants virus (PPRV) causes an economically important disease that limits productivity in small domestic ruminants and often affects the livestock of the poorest populations in developing countries. Animals that survive PPRV develop strong cellular and humoral responses, which are probably necessary for protection. Vaccination should thus aim at mimicking these natural responses. Immunization strategies against this morbillivirus using recombinant adenoviruses expressing PPRV-F or -H proteins can protect PPRV-challenged animals and permit differentiation of infected from vaccinated animals. Little is known of the T cell repertoire these recombinant vaccines induce. In the present work, we identified several CD4+ and CD8+ T cell epitopes in sheep infected with PPRV. We also show that recombinant adenovirus vaccination induced T cell responses to the same epitopes, and led to memory T cell differentiation. T cells primed by these recombinant adenovirus vaccines expanded after PPRV challenge and probably contributed to protection. These data validate the use of recombinant adenovirus expressing PPRV genes as DIVA strategies to control this highly contagious disease.

IL-10: A Multifunctional Cytokine in Viral Infections.

The anti-inflammatory master regulator IL-10 is critical to protect the host from tissue damage during acute phases of immune responses. This regulatory mechanism, central to T cell homeostasis, can be hijacked by viruses to evade immunity. IL-10 can be produced by virtually all immune cells, and it can also modulate the function of these cells. Understanding the effects of this multifunctional cytokine is therefore a complex task. In the present review we discuss the factors driving IL-10 production and the cellular sources of the cytokine during antiviral immune responses. We particularly focus on the IL-10 regulatory mechanisms that impact antiviral immune responses and how viruses can use this central regulatory pathway to evade immunity and establish chronic/latent infections.

Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses.

Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.