The association between recent Zika virus (ZIKV) infection and neurological complications, microcephaly in the fetus, and Guillain–Barré syndrome in adults underscores the necessity for a protective vaccine. Rational vaccine development requires an in-depth understanding of the mechanisms which could protect against infection with this virus. However, so far, such an analysis has been hampered by the absence of a suitable small animal model. Unlike the situation in humans, ZIKV only replicates effectively in the peripheral organs of mice, if type I IFN signaling is interrupted. As type I IFN also impacts the adaptive immune response, mice with such a defect are not optimal for a comprehensive immunological analysis. In this report, we show that even in wild-type (WT) mice i.c. infection with low doses of virus causes marked local virus replication and lethal encephalitis in naïve mice. Furthermore, peripheral infection of WT mice with low doses of virus induces a significant immune response, which provides long-lasting protection of WT mice from a fatal outcome of subsequent i.c. challenge. Therefore, combining peripheral priming with later i.c. challenge represents a new approach for studying the adaptive immune response to ZIKV in mice with an intact type I IFN response. In this study, we focused on the mechanisms underlying resistance to reinfection. Using a combination of adoptive transfer, antibody-based cell depletion, and gene targeting, we show that the key protective factor in type I IFN replete mice is humoral immunity. CD8 T cells are not essential in mice with preformed specific antibodies, but under conditions where initial antibody levels are low, effector CD8 T cells may play a role as a back-up system. These results have important implications for our understanding of natural immunity to ZIKV infection and for Zika vaccine design.
While the brain is considered an immune-privileged site, the CNS may nevertheless be the focus of immune mediated inflammation in the case of infection and certain autoimmune diseases, e.g., multiple sclerosis. As in other tissues, it has been found that acute T-cell infiltration may be followed by establishment of persistent local T-cell memory. To improve our understanding regarding the regulation of putative tissue resident memory T (Trm) cells in CNS, we devised a new model system for studying the generation of Trm cells in this site. To this purpose, we exploited the fact that the CNS may be a sanctuary for adenoviral infection, and to minimize virus-induced disease, we chose replication-deficient adenoviruses for infection of the CNS. Non-replicating adenoviruses are known to be highly immunogenic, and our studies demonstrate that intracerebral inoculation causes marked local T-cell recruitment, which is followed by persistent infiltration of the CNS parenchyma by antigen specific CD8 + T cells. Phenotypical analysis of CNS infiltrating antigen specific CD8 + T cells was consistent with these cells being Trms. Regarding the long-term stability of the infiltrate, resident CD8 + T cells expressed high levels of the anti-apoptotic molecule Bcl-2 as well as the proliferation marker Ki-67 suggesting that the population is maintained through steady homeostatic proliferation. Functionally, memory CD8 + T cells from CNS matched peripheral memory cells with regard to capacity for ex vivo cytotoxicity and cytokine production. Most importantly, our experiments revealed a key role for local antigen encounter in the establishment of sustained CD8 + T-cell memory in the brain. Inflammation in the absence of cognate antigen only led to limited and transient infiltration by antigen specific CD8 + T cells. Together these results indicate that memory CD8 + T cells residing in the CNS predominantly mirror previous local infections and immune responses to local autoantigens.
The worldwide attention that the Zika virus (ZIKV) attracted, following its declaration as a Public Health Emergency of International concern by WHO in 2016, has led to a large collective effort by the international scientific community to understand its biology. Despite the mild symptoms caused by ZIKV in most infected people, the virus displays a number of worrying features, such as its ability to cause transplacental infection, fetal abnormalities and vector independent transmission through body fluids. In addition, the virus has been associated with the induction of Guillain-Barre syndrome in a number of infected individuals. With travelling, the virus has spread outside the original ZIKV endemic areas making it imperative to find ways to control it. Thus far, the large number of animal models developed to study ZIKV pathogenesis have proven to be valuable tools in understanding how the virus replicates and manifests itself in the host, its tissue tropism and the type of immune responses it induces. Still, vital questions, such as the molecular mechanisms of ZIKV persistence and the long-term consequences of ZIKV infection in the developing brain, remain unanswered. Here, we reviewed and discussed the major and most recent findings coming from animal studies and their implications for a ZIKV vaccine design.
Recently, we showed that combined intranasal and subcutaneous immunization with a non-replicating adenoviral vector expressing NP of influenza A, strain PR8, induced long-standing protection against a range of influenza A viruses. However, H-2b mice challenged with an influenza A strain mutated in the dominant NP366 epitope were not efficiently protected. To address this problem, we envision the use of a cocktail of adenovectors targeting different internal proteins of influenza A virus. Consequently, we investigated the possibility of using PB1 as a target for an adenovector-based vaccine against influenza A. Our results showed that PB1 is not as immunogenic as the NP protein. However, by tethering PB1 to the murine invariant chain we were able to circumvent this problem and raise quite high numbers of PB1-specific CD8+ T cells in the circulation. Nevertheless, mice immunized against PB1 were not as efficiently protected against influenza A challenge as similarly NP-vaccinated animals. The reason for this is not a difference in the quality of the primed cells, nor in functional avidity. However, under similar conditions of immunization fewer PB1-specific cells were recruited to the airways, and surface expression of the dominant PB1 peptide, PB1703, was less stable than in the case of NP366.
Zika virus (ZIKV), a mosquito-borne flavivirus, came into the spotlight in 2016 when it was found to be associated with an increased rate of microcephalic newborns in Brazil. The virus has further been recognized to cause neurologic complications in children and adults in the form of myelitis, encephalitis, acute disseminated encephalomyelitis (ADEM) and Guillain Barre Syndrome in a fraction of infected individuals. With the ultimate goal of identifying correlates of protection to guide the design of an effective vaccine, the study of the immune response to ZIKV infection has become the focus of research worldwide. Both innate and adaptive immune responses seem to be essential for controlling the infection. Induction of sufficient levels of neutralizing antibodies has been strongly correlated with protection against reinfection in various models, while the role of CD8 T cells as antiviral effectors in the CNS has been controversial. In an attempt to improve our understanding regarding the role of ZIKV-induced CD8 T cells in protective immunity inside the CNS, we have expanded on previous studies in intracranially infected mice. In a recent study, we have demonstrated that, peripheral ZIKV infection in adult C57BL/6 mice induces a robust CD8 T cell response that peaks within a week. In the present study, we used B cell deficient as well as wild-type mice to show that there is a race between CXCR3-dependent recruitment of the effector CD8 T cells and local ZIKV replication, and that CD8 T cells are capable of local viral control if they arrive in the brain early after viral invasion, in appropriate numbers and differentiation state. Our data highlight the benefits of considering this subset when designing vaccines against Zika virus.
Intracellular pathogens represent a serious threat during early life. Importantly, even though the immune system of newborns may be characterized as developmentally immature, with a propensity to develop Th2 immunity, significant CD8+ T-cell responses may still be elicited in the context of optimal priming. Replication deficient adenoviral vectors have been demonstrated to induce potent CD8+ T-cell response in mice, primates and humans. The aim of the present study was therefore to assess whether replication-deficient adenovectors could overcome the risk of overwhelming antigen stimulation during the first period of life and provide a pertinent alternative in infant vaccinology. To address this, infant mice were vaccinated with three different adenoviral vectors and the CD8+ T-cell response after early life vaccination was explored. We assessed the frequency, polyfunctionality and in vivo cytotoxicity of the elicited memory CD8+ T cells, as well as the potential of these cells to respond to secondary infections and confer protection. We further tested the impact of maternal immunity against our replication-deficient adenoviral vector during early life vaccination. Overall, our results indicate that memory CD8+ T cells induced by adenoviral vectors in infant mice are of good quality and match those elicited in the adult host.
Expression of programmed cell death-1 receptor (PD-1) has traditionally been linked to T-cell exhaustion, as signaling via PD-1 dampens the functionality of T-cells upon repetitive antigen exposures during chronic infections. However, resent findings pointing to the involvement of PD-1 both in T-cell survival and in restraining immunopathology, challenge the concept of PD-1 solely as marker for T-cell exhaustion. Tissue resident memory T cells (Trms) hold unique effector qualities, but within a delicate organ like the CNS, these protective abilities could potentially be harmful. In contrast to their counterparts in many other tissues, brain derived CD8+ Trms have been found to uniformly and chronically express PD-1. In this study we utilized a recently established model system for generating CNS Trms in order to improve our understanding regarding the role of PD-1 expression by Trms inside the CNS. By intracerebral (i.c.) inoculation with a non-replicating adeno-viral vector, we induced a PD-1hi CD8+ T cell memory population within the CNS. We found that PD-1 expression lowered the severity of clinical disease associated with the i.c. inoculation. Furthermore, high levels of PD-L1 expression were found on the infiltrating monocytes and macrophages as well as on the resident microglia, oligodendrocytes and astrocytes during the acute phase of the response. Additionally, we showed that the intensity of PD-1 expression correlates with local antigen encounter and found that PD-1 expression was associated with decreased CD8+ T cell memory formation in the CNS despite an increased number of infiltrating CD8+ T cells. Most importantly, our experiments revealed that despite expression of PD-1 and several additional markers linked to T-cell exhaustion, Tim-3, Lag-3 and CD39, the cells did not show signs of limited effector capacity. Collectively, these results endorse the increasing amount of evidence pointing to an immune-modifying role for PD-1 expression within the CNS, a mechanism we found to correlate with local antigen exposure.
Current influenza vaccines are inducing antibody-mediated immunity toward the surface proteins hemagglutinin and neuraminidase. High variability of these proteins results in costly annual evaluation and production of new vaccines, not providing protection against heterosubtypic or emerging pandemic strains. Consequently, there is a need for a new universal influenza vaccine strategy. Unlike the surface proteins, the internal proteins of influenza are highly conserved. By vaccinating systemically and locally with nucleoprotein encoded in an adenovirus (AdNP) we could induce a protective CD8 T-cell response, that lasted up to at least 8 month post vaccination. To increase the breadth of our vaccine by combining construct, we studied the vaccine potential of another influenza protein. We showed that an adenovirus encoding PB1 elicits high numbers of antigen-specific CD8 T cells in mice. However, in vivo killing of peptide loaded cells was lower in PB1-vaccinated mice than in NP-vaccinated mice. This correlated with a poorer protection against challenge. To explain this, T cells from vaccinated mice were co-incubated with naïve splenocytes loaded with decreasing concentrations of peptide. If T cells were immediately added to the co-cultures, similar dose/response curves regarding IFNγ production was observed. However, if the T cells were added 6 hours after loading, cells from PB1-vaccinated mice required a much higher initial concentration of peptide to be activated compared with NP specific cells, indicating a lower stability of the MHC-PB1peptide complex. These results underscore that not only are T-cell quality and quantity crucial to consider in future vaccine design, but also MHC-peptide interactions.
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