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.

No abstract

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.

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.

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.

A sub-population of memory T cells, tissue resident memory T cells (TRM), have in recent years gained attention for their indispensable role in heterologous protection against respiratory viruses. For this reason, they are of great interest for vaccine research. However, the number of lung TRM decline within a few months after infection and researchers have been unable to determine how the TRM population, and protection, can be made to last long-term in the lung. We have previously shown that an Adenovirus expressing nucleoprotein (AdNP) from influenza A virus induce CD8 T cell mediated heterosubtypic immunity lasting up to at least 8 months post vaccination when administered locally and systemically, but the underlying reason was not investigated. Here, we show that the number of CD8 T cell in the lung induced by AdNP is significantly higher compared to cells induced by an influenza infection. Using AdNP, we show that CD8 TRM in the lung can be maintained for at least one year post-vaccination. Our results revealed that lung TRM continued to proliferate in-situ 8 months after AdNP vaccination. Importantly, this required pulmonary vaccination and antigen persistence in the lung, as non-respiratory vaccination routes failed to support lung TRM maintenance. Additionally, parabiosis experiments show that in AdNP vaccinated mice the lung TRM pool is sustained by continual replenishment from circulating memory CD8 T cells that differentiate into lung TRM, a phenomenon not observed in influenza infected parabiont partners. Concluding, these results demonstrates requirements for long-lived cellular immunity to influenza virus, knowledge that could be utilized in future vaccine design.