Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity

Direct vaccination with messenger RNA (mRNA) molecules encoding tumor-associated antigens is a novel and promising approach in cancer immunotherapy. The main advantage of using mRNA for vaccination is that the same molecule not only provides an antigen source for adaptive immunity, but can simultaneously bind to pattern recognition receptors, thus stimulating innate immunity. However, achieving both features remains challenging, as the complexation of mRNA required for immune-stimulating activity may inhibit its translatability. In this study, we present a new and more effective vaccine design: a two-component mRNA-based tumor vaccine that supports both: antigen expression and immune stimulation, mediated by Toll like receptor 7 (TLR7). The two-component mRNA vaccines, containing free and protamine-complexed mRNA, induce balanced adaptive immune responses providing humoral as well as T cell mediated immunity. This balanced immune response is based on the induction of antigen-specific CD4(+) T helper cells and cytotoxic CD8(+) T cells. Once activated, these CD4(+) and CD8(+) T cells secrete a wide set of cytokines, which drive a TH1 response. Immunization with the two-component vaccines induces sustained memory responses, mediated by antigen-specific memory T cells. Moreover, treatment of mice with the two-component mRNA vaccine mediates a strong antitumor response against OVA-expressing tumor cells, not only in a prophylactic but also in a therapeutic setting. In conclusion, two-component mRNA vaccines with self-adjuvanting activity induce balanced adaptive immune responses and mediate sustained antitumor activity.     

Protective T cell immunity in mice following protein-TLR7/8 agonist-conjugate immunization requires aggregation, type I IFN, and multiple DC subsets

The success of a non-live vaccine requires improved formulation and adjuvant selection to generate robust T cell immunity following immunization. Here, using protein linked to a TLR7/8 agonist (conjugate vaccine), we investigated the functional properties of vaccine formulation, the cytokines, and the DC subsets required to induce protective multifunctional T cell immunity in vivo. The conjugate vaccine required aggregation of the protein to elicit potent Th1 CD4+ and CD8+ T cell responses. Remarkably, the conjugate vaccine, through aggregation of the protein and activation of TLR7 in vivo, led to an influx of migratory DCs to the LN and increased antigen uptake by several resident and migratory DC subsets, with the latter effect strongly influenced by vaccine-induced type I IFN. Ex vivo migratory CD8-DEC205+CD103-CD326- langerin-negative dermal DCs were as potent in cross-presenting antigen to naive CD8+ T cells as CD11c+CD8+ DCs. Moreover, these cells also influenced Th1 CD4+ T cell priming. In summary, we propose a model in which broad-based T cell-mediated responses upon vaccination can be maximized by codelivery of aggregated protein and TLR7/8 agonist, which together promote optimal antigen acquisition and presentation by multiple DC subsets in the context of critical proinflammatory cytokines.     

Transient Pretreatment With Glucocorticoid Ablates Innate Toxicity of Systemically Delivered Adenoviral Vectors Without Reducing Efficacy

More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.     

Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations

Innate immunity to viral infection involves induction of the type I IFN response; however, dysfunctional regulation of this pathway leads to inappropriate inflammation. Here, we evaluated a nonconsanguineous family of mixed European descent, with 4 members affected by systemic inflammatory and autoimmune conditions, including lupus, with variable clinical expression. We identified a germline dominant gain-of-function mutation in TMEM173, which encodes stimulator of type I IFN gene (STING), in the affected individuals. STING is a key signaling molecule in cytosolic DNA-sensing pathways, and STING activation normally requires dimerization, which is induced by 2′3′ cyclic GMP-AMP (cGAMP) produced by the cGAMP synthase in response to cytosolic DNA. Structural modeling supported constitutive activation of the mutant STING protein based on stabilized dimerization. In agreement with the model predictions, we found that the STING mutant spontaneously localizes in the Golgi of patient fibroblasts and is constitutively active in the absence of exogenous 2′3′-cGAMP in vitro. Accordingly, we observed elevated serum IFN activity and a type I IFN signature in peripheral blood from affected family members. These findings highlight the key role of STING in activating both the innate and adaptive immune responses and implicate aberrant STING activation in features of human lupus.     

Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10-20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)gamma production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNgamma, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.     

CD4(+) T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease

Aspergillus fumigatus is a model fungal pathogen and a common cause of infection in individuals with the primary immunodeficiency chronic granulomatous disease (CGD). Although primarily considered a deficiency of innate immunity, CGD is also linked to dysfunctional T cell reactivity. Both CD4(+) and CD8(+) T cells mediate vaccine-induced protection from experimental aspergillosis, but the molecular mechanisms leading to the generation of protective immunity and whether these mechanisms are dysregulated in individuals with CGD have not been determined. Here, we show that activation of either T cell subset in a mouse model of CGD is contingent upon the nature of the fungal vaccine, the involvement of distinct innate receptor signaling pathways, and the mode of antigen routing and presentation in DCs. Aspergillus conidia activated CD8(+) T cells upon sorting to the Rab14(+) endosomal compartment required for alternative MHC class I presentation. Cross-priming of CD8(+) T cells failed to occur in mice with CGD due to defective DC endosomal alkalinization and autophagy. However, long-lasting antifungal protection and disease control were successfully achieved upon vaccination with purified fungal antigens that activated CD4(+) T cells through the endosome/lysosome pathway. Our study thus indicates that distinct intracellular pathways are exploited for the priming of CD4(+) and CD8(+) T cells to A. fumigatus and suggests that CD4(+) T cell vaccination may be able to overcome defective antifungal CD8(+) T cell memory in individuals with CGD.     

Viral vectors for inducing CD8+ T cell responses

CD8(+) T cells (T(CD8+)) can mediate protective immunity to intracellular pathogens and tumours. Viruses generate strong T(CD8+) responses and, therefore, represent attractive vectors for generating vaccines aimed at producing T(CD8+)-mediated protective immunity. This review will examine the immunological properties of viruses that make them good candidates as vaccine vectors, as well as the manipulations of both vector and antigen that may be required to produce an effective vaccine. The areas addressed include virus infection of dendritic cells in vivo, stimulation of the innate immune response via intracellular and extracellular pattern recognition receptors, the effect of antigenic form on the pathways of antigen presentation and the requirement for elimination of viral genes that target various aspects of the innate and adaptive immune response.     

The TNFR family members OX40 and CD27 link viral virulence to protective T cell vaccines in mice

Induction of CD8+ T cell immunity is a key characteristic of an effective vaccine. For safety reasons, human vaccination strategies largely use attenuated nonreplicating or weakly replicating poxvirus-based vectors, but these often elicit poor CD8+ T cell immunity and might not result in optimal protection. Recent studies have suggested that virulence is directly linked to immunogenicity, but the molecular mechanisms underlying optimal CD8+ T cell responses remain to be defined. Here, using natural and recombinant vaccinia virus (VACV) strains, we have shown in mice that VACV strains of differing virulence induce distinct levels of T cell memory because of the differential use of TNF receptor (TNFR) family costimulatory receptors. With strongly replicating (i.e., virulent) VACV, the TNFR family costimulatory receptors OX40 (also known as CD134) and CD27 were engaged and promoted the generation of high numbers of memory CD8+ T cells, which protected against a lethal virus challenge in the absence of other mechanisms, including antibody and help from CD4+ T cells. In contrast, weakly replicating (i.e., low-virulence) VACV strains were poor at eliciting protective CD8+ T cell memory, as only the Ig family costimulatory receptor CD28 was engaged, and not OX40 or CD27. Our results suggest that the virulence of a virus dictates costimulatory receptor usage to determine the level of protective CD8+ T cell immunity.     

固有免疫和适应性免疫与慢性丙肝的研究进展

固有免疫应答是抵抗病毒入侵的第一道防线,启动后产生一系列的细胞因子如干扰素等发挥作用,6～8周后,适应性免疫应答才被检测到。经过抗原刺激后,活化、增殖的效应细胞如CD4+和CD8+T细胞、调节性T细胞和自然杀伤细胞,产生效应分子如抗体和细胞因子而发挥作用。虽然有固有免疫和适应性免疫应答机制,大多数丙型肝炎病毒感染者仍不能清除病毒,会发展为慢性持续性感染。该文就固有免疫和适应性免疫在慢性丙型肝炎发病机制中的作用进行综述。     

The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice

Recombinant adeno-associated viruses (AAVs) have been used widely for in vivo gene therapy. However, adaptive immune responses to AAV have posed a significant hurdle in clinical application of AAV vectors. Recent advances have suggested a crucial role for innate immunity in shaping adaptive immune responses. How AAV activates innate immunity, and thereby promotes AAV-targeted adaptive immune responses, remains unknown. Here we show that AAV activates mouse plasmacytoid DCs (pDCs) via TLR9 to produce type I IFNs. In vivo, the TLR9-MyD88 pathway was crucial to the activation of CD8⁺ T cell responses to both the transgene product and the AAV capsid, leading to loss of transgene expression and the generation of transgene product–specific and AAV-neutralizing antibodies. We further demonstrate that TLR9-dependent activation of adaptive immunity targeting AAV was mediated by type I IFNs and that human pDCs could be activated in vitro to induce type I IFN production via TLR9. These results reveal an essential role for the TLR9-MyD88–type I IFN pathway in induction of adaptive immune responses to AAV and suggest that strategies that interfere with this pathway may improve the outcome of AAV-mediated gene therapy in humans.     

Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.     

CD47 blockade enhances the efficacy of intratumoral STING-targeting therapy by activating phagocytes

Activation of STING signaling plays an important role in anti-tumor immunity, and we previously reported the anti-tumor effects of STING through accumulation of M1-like macrophages in tumor tissue treated with a STING agonist. However, myeloid cells express SIRPα, an inhibitory receptor for phagocytosis, and its receptor, CD47, is overexpressed in various cancer types. Based on our findings that breast cancer patients with highly expressed CD47 have poor survival, we evaluated the therapeutic efficacy and underlying mechanisms of combination therapy with the STING ligand cGAMP and an antagonistic anti-CD47 mAb using E0771 mouse breast cancer cells. Anti-CD47 mAb monotherapy did not suppress tumor growth in our setting, whereas cGAMP and anti-CD47 mAb combination therapy inhibited tumor growth. The combination therapy enhanced phagocytosis of tumor cells and induced systemic anti-tumor immune responses, which rely on STING and type I IFN signaling. Taken together, our findings indicate that coadministration of cGAMP and an antagonistic anti-CD47 mAb may be promising for effective cancer immunotherapy.     

Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ib-specific T cells in host defense

The major histocompatibility complex (MHC) class Ib molecule H2-M3 primes the rapid expansion of CD8+ T cells by presenting N-formylated bacterial peptides. However, the significance of H2-M3-restricted T cells in host defense against bacteria is unclear. We generated H2-M3-deficient mice to investigate the role of H2-M3 in immunity against Listeria monocytogenes (LM), a model intracellular bacterial pathogen. H2-M3-deficient mice are impaired in early bacterial clearance during primary infection, with diminished LM-specific CD8+ T cell responses and compromised innate immune functions. Although H2-M3-restricted CD8+ T cells constitute a significant proportion of the anti-listerial CD8+ T cell repertoire, the kinetics and magnitude of MHC class Ia-restricted T cell responses are not altered in H2-M3-deficient mice. The fact that MHC class Ia-restricted responses cannot compensate for the H2-M3-mediated immunity suggests a nonredundant role of H2-M3 in the protective immunity against LM. Thus, the early H2-M3-restricted response temporally bridges the gap between innate and adaptive immune responses, subsequently affecting the function of both branches of the immune system.     

Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta

Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-alpha/beta in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8alpha+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-alpha/beta regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8alpha+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-alpha/beta, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.     

Engineering cross-presentation in vivo

Apoptotic bodies deliver antigens (Ags) to the cross-presentation pathways of dendritic cells (DCs), where their presentation has been associated with both the maintenance of tolerance as well as the induction of protective immunity. The manner in which apoptotic bodies are generated, their abundance in relation to local DCs, and the milieu in which they are generated appear to be the major factors determining whether apoptotic bodies will induce CD8+ T cell activation or anergy. These observations have been extended to the field of vaccination, where the engineered apoptosis of Ag-bearing/loaded cells in vivo has been used to prime strong CD8+ T cell immunity. This review will examine Ag capture and cross-presentation by DCs, with particular emphasis on the manipulation of apoptotic bodies in vivo for the purpose of vaccination.     

Engineering cross-presentation in vivo

Apoptotic bodies deliver antigens (Ags) to the cross-presentation pathways of dendritic cells (DCs), where their presentation has been associated with both the maintenance of tolerance as well as the induction of protective immunity. The manner in which apoptotic bodies are generated, their abundance in relation to local DCs, and the milieu in which they are generated appear to be the major factors determining whether apoptotic bodies will induce CD8(+) T cell activation or anergy. These observations have been extended to the field of vaccination, where the engineered apoptosis of Ag-bearing/loaded cells in vivo has been used to prime strong CD8(+) T cell immunity. This review will examine Ag capture and cross-presentation by DCs, with particular emphasis on the manipulation of apoptotic bodies in vivo for the purpose of vaccination.