Esther Tarrab

Nanoparticle Adjuvant Sensing by TLR7 Enhances CD8+ T Cell–Mediated Protection from Listeria Monocytogenes Infection

Developing new adjuvants and vaccination strategies is of paramount importance to successfully fight against many life-threatening infectious diseases and cancer. Very few adjuvants are currently authorized for human use, and these mainly stimulate a humoral response. However, specific Abs are not sufficient to confer protection against persisting infections or cancer. Therefore, development of adjuvants and immunomodulators able to enhance cell-mediated immune responses represents a major medical need. We recently showed that papaya mosaic virus nanoparticles (PapMV), self-assembled from the coat protein of a plant virus and a noncoding ssRNA molecule, are highly immunogenic in mice. PapMV can be used either as a vaccine delivery platform, through fusion of various epitopes to the coat protein or as adjuvant to enhance humoral immune responses against coadministered Ags or vaccines. However, the mechanisms that confer these immunomodulatory properties to PapMV and its ability to enhance T cell vaccines remain unknown. Using immunization studies in mice, we demonstrate in this paper that PapMV represents a novel TLR7 agonist with strong immunostimulatory properties. More importantly, pretreatment with PapMV significantly improves effector and memory CD8+ T cell responses generated through dendritic cell vaccination increasing protection against a Listeria monocytogenes challenge.

Potentiating Cancer Immunotherapy Using Papaya Mosaic Virus-Derived Nanoparticles

The recent development of novel immunotherapies is revolutionizing cancer treatment. These include, for example, immune checkpoint blockade, immunomodulation, or therapeutic vaccination. Although effective on their own, combining multiple approaches will most likely be required in order to achieve the maximal therapeutic benefit. In this regard, the papaya mosaic virus nanoparticle (PapMV) has shown tremendous potential as (i) an immunostimulatory molecule, (ii) an adjuvant, and (iii) a vaccine platform through its intrinsic capacity to activate the innate immune response in an IFN-α-dependent manner. Here, we demonstrate that intratumor administration of PapMV significantly slows down melanoma progression and prolongs survival. This correlates with enhanced chemokine and pro-inflammatory-cytokine production in the tumor and increased immune-cell infiltration. Proportions of total and tumor-specific CD8(+) T cells dramatically increase following PapMV treatment whereas those of myeloid-derived suppressor cells (MDSC) concomitantly decrease. Moreover, systemic PapMV administration prevents metastatic tumor-implantation in the lungs. Importantly, PapMV also synergistically improves the therapeutic benefit of dendritic cell (DC)-based vaccination and PD-1 blockade by potentiating antitumor immune responses. This study illustrates the immunostimulatory potential of a plant virus-derived nanoparticle for cancer therapy either alone or in conjunction with other promising immunotherapies in clinical development.

Transient Complement Inhibition Promotes a Tumor-Specific Immune Response through the Implication of Natural Killer Cells

Using cobra venom factor to induce transient C3 molecule exhaustion in a mouse model of melanoma, Janelle and colleagues show that the antitumor immune response is NK dependent, uncovering a link between complement proteins and NK cells in tumor development. Although the role of the complement system in cancer development has been studied, its involvement in the development of an antitumoral immune response remains poorly understood. Using cobra venom factor (CVF) to inhibit the complement cascade via C3 molecule exhaustion in immunocompetent mice bearing B16gp33 melanoma tumors, we show that transient inhibition of the complement system allowed for the development of a more robust gp33-specific antitumoral CD8+ T-cell response. This immune response proved to be natural killer (NK) dependent, suggesting an interaction of complement proteins with this cellular subset leading to T lymphocyte activation and enhanced cytotoxic T-cell activity against tumor cells. This study demonstrates for the first time the implication of the complement system in the development of NK-mediated cytotoxic T-cell–dependent antitumoral immune responses. The complement pathway could therefore be a potent therapeutic target to improve NK-dependent antitumoral immune responses in patients with cancer. Cancer Immunol Res; 2(3); 200–6. ©2013 AACR.

Complement Component 3 Regulates IFN-α Production by Plasmacytoid Dendritic Cells following TLR7 Activation by a Plant Virus–like Nanoparticle

The increasing use of plant viruses for the development of new vaccines and immunotherapy approaches poses questions regarding the mechanism by which the mammalian immune system recognizes these viruses. For example, although natural Abs (NA) and complement are key components of the innate immune system involved in the opsonization, phagocytosis, and destruction of microorganisms infecting mammals, their implication in plant virus recognition and immunogenicity is not well defined. In this study, we address the involvement of NA and the complement system in the activation of innate immunity through engagement of TLR7 with papaya mosaic virus (PapMV)-like nanoparticles. We demonstrate that NA, although binding to PapMV, are not involved in its recognition by the immune system. On the other hand, C3 strongly binds to PapMV nanoparticles and its depletion significantly reduces PapMV’s interaction with immune cells. Unexpectedly, however, we observed increased immune cell activation following administration of PapMV to complement-depleted mice. TLR7 activation by PapMV in the absence of C3 induced higher IFN-α production, resulting in superior immune cell activation and increased immunotherapeutic properties. In conclusion, in this study we established the involvement of the complement system in the recognition and the phagocytosis of PapMV nanoparticles and identified an unsuspected role for C3 in regulating the production of IFN-α following TLR7 activation.

Expression of Viral Antigen by the Liver Leads to Chronic Infection Through the Generation of Regulatory T Cells

Background & Aims The constant exposure of the liver to food and bacterial antigens through the mesenteric circulation requires it to maintain tolerance while preserving the ability to mount an effective immune response against pathogens. We investigated the contribution of the liver’s tolerogenic nature on the establishment of chronic viral infections. Methods TTR-NP mice, which express the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) specifically in hepatocytes under control of a modified transthyretin (TTR) promoter, were infected with the Armstrong (Arm) or WE acute strains of LCMV. Results The infection persisted for at least 147 days in TTR-NP mice. Expression of NP by the liver induced a strong peripheral tolerance against NP that was mediated by interleukin-10-secreting CD4+ regulatory T cells, leading to high PD-1 (programmed death-1) expression and reduced effector function of virus-specific T cells. Despite an active immune response against LCMV, peripheral tolerance against a single viral protein was sufficient to induce T-cell exhaustion and chronic LCMV Armstrong (Arm) or WE infection by limiting the antiviral T-cell response in an otherwise immunocompetent host. Regulatory T-cell depletion of chronically infected TTR-NP mice led to functional restoration of LCMV-specific CD4+ and CD8+ T cell responses and viral clearance. Conclusions Expression of a viral antigen by hepatocytes can induce a state of peripheral tolerance mediated by regulatory T cells that can lead to the establishment of a chronic viral infection. Strategies targeting regulatory T cells in patients chronically infected with hepatotropic viruses could represent a promising approach to restore functional antiviral immunity and clear infection.

Altered immunodominance hierarchies of CD8+ T cells in the spleen after infection at different sites is contingent on high virus inoculum.

Activated epitope-specific CD8+ T cells after virus infection can be organized into hierarchies (immunodominance), based on their ability to focus the response on few viral determinants. The mechanisms responsible for immunodominance can be multifactorial, with CD8+ T cells precursor frequencies recently highlighted as a key regulator. Employing the LCMV infection model, we demonstrate that the hierarchies were altered when comparing different sites of infection but only at high viral doses. These findings have significant implications when investigating immunity to viruses with different replication abilities that may override the influence of T cell precursor frequencies.

The outcome of cross‐priming during virus infection is not directly linked to the ability of the antigen to be cross‐presented

The initiation of CD8+ T cell (CTL) immune responses can occur via cross‐priming. Recent data suggested a relationship between cross‐presentation and immunodominance of epitope‐specific T cells. To test this association, we evaluated the efficacy of cross‐presentation for several virus epitopes in vitro and examined if this can be extrapolated in vivo. Employing lymphocytic choriomeningitis virus (LCMV), we demonstrate that the cross‐presentation and cross‐priming of LCMV antigens were dominated by NP396, but not NP205 when analyzing the LCMV‐NP. Although with LCMV‐GP, cross‐presentation was dominated by GP276, and cross‐priming was dominated by GP33. Importantly, although NP396 was significantly more efficient than GP33 in cross‐presentation, cross‐priming of their specific CTL was comparable. In a subsequent virus challenge after cross‐priming, GP33‐specific CTL dominated the response. Accordingly, based on our data, the ability of viral epitopes to be cross‐presented in vitro does not entirely reflect what would occur in cross‐priming. Thus, weak cross‐presenting antigens may still cross‐prime an efficient CTL response depending on other in vivo elements such as the naïve T‐cell precursor frequencies.

Type I Interferon Impairs Specific Antibody Responses Early during Establishment of LCMV Infection

Elicitation of type I interferon (IFN-I) has been shown to both enhance and impair cell-mediated immune responses in acute and persistent viral infections, respectively. Here, we show that, in addition to its effect on T cells, IFN-I drives impairment of specific antibody responses through interaction with B cells in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. This impairment was limited to the T cell-dependent B cell response and was associated with disruption of B cell follicles, development of hypergammaglobulinemia (HGG), and expansion of the T follicular helper cell population. Antigen-specific antibody responses were restored by ablation of IFN-I signaling through antibody-mediated IFN-I receptor blockade and B cell-specific IFN-I receptor knockout. Importantly, IFN-I receptor deficiency in B cells also accelerated the development of LCMV neutralizing antibodies and alleviated HGG. These results provide a potential therapeutic target toward efficient treatment measures that limit immunopathology in persistent viral infections.

Dok-1 and Dok-2 Regulate the Formation of Memory CD8+ T Cells.

Diverse signals received by CD8+ T cells are integrated to achieve the required magnitude of cell expansion and the appropriate balance of effector/memory CD8+ T cell generation. Notably, the strength and nature of TCR signaling influence the differentiation and functional capacity of effector and memory CD8+ T cells. Dok-1 and Dok-2, the two members of the Dok family expressed in T cells, negatively regulate TCR signaling in vitro. However, the role of Dok proteins in modulating T cell function in vivo has not yet studied. We studied the function of Dok-1 and Dok-2 proteins in the regulation of the CD8+ T cell response to vaccinia virus infection. Comparison of responses to vaccinia virus expressing OVA peptide SIINFEKL by wild-type and Dok-1/2−/− CD8+ OT-I cells showed that the absence of Dok-1 and Dok-2 slightly reduced the magnitude of virus-specific effector CD8+ T cell expansion. This was not due to reduced proliferation or enhanced apoptosis of effector CD8+ T cells. Dok-1/2–deficient effector CD8+ T cells showed increased cell surface TCR expression following virus infection in vivo and increased expression of granzyme B and TNF upon stimulation with peptide Ag ex vivo. Finally, Dok-1/2–deficient effector CD8+ T had a severe defect in survival that resulted in impaired generation of memory CD8+ T cells. These results reveal the critical involvement of Dok-1 and Dok-2 in a negative-feedback loop that prevents overactivation of CD8+ T cells and promotes memory formation.

Efficacy of a Virus-Like Nanoparticle As Treatment for a Chronic Viral Infection Is Hindered by IRAK1 Regulation and Antibody Interference

Although vaccination has been an effective way of preventing infections ever since the eighteenth century, the generation of therapeutic vaccines and immunotherapies is still a work in progress. A number of challenges impede the development of these therapeutic approaches such as safety issues related to the administration of whole pathogens whether attenuated or inactivated. One safe alternative to classical vaccination methods gaining recognition is the use of nanoparticles, whether synthetic or naturally derived. We have recently demonstrated that the papaya mosaic virus (PapMV)-like nanoparticle can be used as a prophylactic vaccine against various viral and bacterial infections through the induction of protective humoral and cellular immune responses. Moreover, PapMV is also very efficient when used as an immune adjuvant in an immunotherapeutic setting at slowing down the growth of aggressive mouse melanoma tumors in a type I interferon (IFN-I)-dependent manner. In the present study, we were interested in exploiting the capacity of PapMV of inducing robust IFN-I production as treatment for the chronic viral infection model lymphocytic choriomeningitis virus (LCMV) clone 13 (Cl13). Treatment of LCMV Cl13-infected mice with two systemic administrations of PapMV was ineffective, as shown by the lack of changes in viral titers and immune response to LCMV following treatment. Moreover, IFN-α production following PapMV administration was almost completely abolished in LCMV-infected mice. To better isolate the mechanisms at play, we determined the influence of a pretreatment with PapMV on secondary PapMV administration, therefore eliminating potential variables emanating from the infection. Pretreatment with PapMV led to the same outcome as an LCMV infection in that IFN-α production following secondary PapMV immunization was abrogated for up to 50 days while immune activation was also dramatically impaired. We showed that two distinct and overlapping mechanisms were responsible for this outcome. While short-term inhibition was partially the result of interleukin-1 receptor-associated kinase 1 degradation, a crucial component of the toll-like receptor 7 signaling pathway, long-term inhibition was mainly due to interference by PapMV-specific antibodies. Thus, we identified a possible pitfall in the use of virus-like particles for the systemic treatment of chronic viral infections and discuss mitigating alternatives to circumvent these potential problems.