Different Adjuvants Induce Common Innate Pathways That Are Associated with Enhanced Adaptive Responses against a Model Antigen in Humans.

To elucidate the role of innate responses in vaccine immunogenicity, we compared early responses to hepatitis B virus (HBV) surface antigen (HBsAg) combined with different Adjuvant Systems (AS) in healthy HBV-naïve adults, and included these parameters in multi-parametric models of adaptive responses. A total of 291 participants aged 18-45 years were randomized 1:1:1:1:1 to receive HBsAg with AS01B, AS01E, AS03, AS04, or Alum/Al(OH)3 at days 0 and 30 (ClinicalTrials.gov: NCT00805389). Blood protein, cellular, and mRNA innate responses were assessed at early time-points and up to 7 days after vaccination, and used with reactogenicity symptoms in linear regression analyses evaluating their correlation with HBs-specific CD4+ T-cell and antibody responses at day 44. All AS induced transient innate responses, including interleukin (IL)-6 and C-reactive protein (CRP), mostly peaking at 24 h post-vaccination and subsiding to baseline within 1-3 days. After the second but not the first injection, median interferon (IFN)-γ levels were increased in the AS01B group, and IFN-γ-inducible protein-10 levels and IFN-inducible genes upregulated in the AS01 and AS03 groups. No distinct marker or signature was specific to one particular AS. Innate profiles were comparable between AS01B, AS01E, and AS03 groups, and between AS04 and Alum groups. AS group rankings within adaptive and innate response levels and reactogenicity prevalence were similar (AS01B ≥ AS01E > AS03 > AS04 > Alum), suggesting an association between magnitudes of inflammatory and vaccine responses. Modeling revealed associations between adaptive responses and specific traits of the innate response post-dose 2 (activation of the IFN-signaling pathway, CRP and IL-6 responses). In conclusion, the ability of AS01 and AS03 to enhance adaptive responses to co-administered HBsAg is likely linked to their capacity to activate innate immunity, particularly the IFN-signaling pathway.

Central Role of CD169+ Lymph Node Resident Macrophages in the Adjuvanticity of the QS-21 Component of AS01.

Saponins represent a promising class of vaccine adjuvant. Together with the TLR4-ligand MPL, QS-21 is part of the Adjuvant System AS01, a key component of the malaria and zoster candidate vaccines that display demonstrated clinical efficacy. However, the mechanism of action of QS-21 in this liposomal formulation is poorly understood. Upon intra-muscular immunisation, we observed that QS-21 rapidly accumulated in CD169+ resident macrophages of the draining lymph node where it elicited a local innate immune response. Depletion of these cells abrogated QS-21-mediated innate cell recruitment to the lymph node, dendritic cell (DC) phenotypic maturation as well as the adjuvant effect on T-cell and antibody responses to co-administered antigens. DCs rather than lymph node-resident macrophages were directly involved in T-cell priming by QS-21, as revealed by the decrease in antigen-specific T-cell response in Batf3-/- mice. Further analysis showed that the adjuvant effect of QS-21 depended on the integration of Caspase-1 and MyD88 pathways, at least in part through the local release of HMGB1. Taken together, this work unravels the key role of lymph node sentinel macrophage in controlling the adjuvant effect of a molecule proven to improve vaccine response in humans.

Adjuvant System AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity.

AS03 is an Adjuvant System (AS) containing α-tocopherol and squalene in an oil-in-water (o/w) emulsion. AS03 has been considered for the development of pandemic and seasonal influenza vaccines. Key features of AS03's mode of action were investigated in vivo in mice and ex vivo in human cells. AS03's adjuvant activity was superior to that of aluminium hydroxide and required the spatio-temporal co-localisation of AS03 with the antigen. This requirement coincided with AS03 triggering a transient production of cytokines at the injection site and in the draining lymph nodes (dLNs). The nature of the cytokines produced was consistent with the enhanced recruitment of granulocytes and of antigen-loaded monocytes in the dLNs. The presence of α-tocopherol in AS03 was required for AS03 to achieve the highest antibody response. The presence of α-tocopherol also modulated the expression of some cytokines, including CCL2, CCL3, IL-6, CSF3 and CXCL1; increased the antigen loading in monocytes; and increased the recruitment of granulocytes in the dLNs. Hence, AS03's promotion of monocytes as the principal antigen-presenting cells, and its effects on granulocytes and cytokines, may all contribute to enhancing the antigen-specific adaptive immune response.

Inhibition of mRNA export and dimerization of interferon regulatory factor 3 by Theiler's virus leader protein.

Theiler's murine encephalomyelitis virus (TMEV or Theiler's virus) is a neurotropic picornavirus that can persist lifelong in the central nervous system of infected mice, causing a chronic inflammatory demyelinating disease. The leader (L) protein of the virus is an important determinant of viral persistence and has been shown to inhibit transcription of type I interferon (IFN) genes and to cause nucleocytoplasmic redistribution of host proteins. In this study, it was shown that expression of the L protein shuts off synthesis of the reporter proteins green fluorescent protein and firefly luciferase, suggesting that it induces a global shut-off of host protein expression. The L protein did not inhibit transcription or translation of the reporter genes, but blocked cellular mRNA export from the nucleus. This activity correlated with the phosphorylation of nucleoporin 98 (Nup98), an essential component of the nuclear pore complex. In contrast, the data confirmed that the L protein inhibited IFN expression at the transcriptional level, and showed that transcription of other chemokine or cytokine genes was affected by the L protein. This transcriptional inhibition correlated with inhibition of interferon regulatory factor 3 (IRF-3) dimerization. Whether inhibition of IRF-3 dimerization and dysfunction of the nuclear pore complex are related phenomena remains an open question. In vivo, IFN antagonism appears to be an important role of the L protein early in infection, as a virus bearing a mutation in the zinc finger of the L protein replicated as efficiently as the wild-type virus in type I IFN receptor-deficient mice, but had impaired fitness in IFN-competent mice.

La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts.

La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the host's IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.

Neurons produce type I interferon during viral encephalitis.

Type I interferons, also referred to as IFN-alpha/beta, form the first line of defense against viral infections. Major IFN-alpha/beta producers in the periphery are the plasmacytoid dendritic cells (pDCs). Constitutive expression of the IFN regulatory factor (IRF)-7 enables pDCs to rapidly synthesize large amounts of IFN-alpha/beta after viral infection. In the central nervous system (CNS), pDCs are considered to be absent from the parenchyma, and little is known about the cells producing IFN-alpha/beta. The study presented here aimed to identify the cells producing IFN-alpha/beta in the CNS in vivo after infection by neurotropic viruses such as Theiler's virus and La Crosse virus. No cells with high constitutive expression of IRF-7 were detected in the CNS of uninfected mice, suggesting the absence of cells equivalent to pDCs. Upon viral infection, IFN-beta and some subtypes of IFN-alpha, but not IFN-epsilon or IFN-kappa, were transcriptionally up-regulated. IFN-alpha/beta was predominantly produced by scattered parenchymal cells and much less by cells of inflammatory foci. Interestingly, in addition to some macrophages and ependymal cells, neurons turned out to be important producers of both IFN-alpha and IFN-beta. However, only 3% of the infected neurons produced IFN-alpha/beta, suggesting that some restriction to IFN-alpha/beta production existed in these cells. All CNS cell types analyzed, including neurons, were able to respond to type I IFN by producing Mx or IRF-7. Our data show that, in vivo, neurons take an active part to the antiviral defense by being both IFN-alpha/beta producers and responders.

The leader protein of Theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins.

The leader protein of Theiler's virus was previously shown to block the production of alpha/beta interferon by infected cells. Here, we observed that expression of the leader protein in infected cells triggered subcellular redistribution of a nucleus-target green fluorescent protein. It enhanced redistribution of the nuclear polypyrimidine tract-binding protein but had no influence on the localization of the nuclear splicing factor SC-35. The leader protein also interfered with trafficking of the cytoplasmic interferon regulatory factor 3, a factor critical for transcriptional activation of alpha/beta interferon genes.