Peachman, Rao et al. 2006) and the possible paradox infection-enhancing effect of low levels of neutralizing antibodies on Fc gamma bearing cells (Pierson, Xu et al. 2007), the NThigh group was used as surrogate marker for the strength of protection. Most prominently, this paradoxical antibody-dependent enhancement might occur in the case of a dengue virus infection, another flavivirus infection, due to preformed antibodies generated against a different serotype of the dengue virus. In the case of a second infection with a different serotype, these antibodies promote a severe (haemorrhagic) reaction in cells bearing the Fc-γ receptor (Morens and Halstead 1990). Furthermore, as antibody production is the net result of complex interaction of a variety of cell types (antigen-presenting cells, T and B cells), it represents the distinct coordination of a variety of players in the immune system. However, as specific neutralizing antibodies were appearing rather late, it might not adequately represent the early phases of the immune response.
Thus, the second parameter that was used as a surrogate marker for the immunogenicity of the vaccine was the absence or presence of viral RNA in peripheral blood. Interestingly, viremia could only be detected in 8 of 18 vaccinees and only between day 3 and day 7, which is consistent with other findings where transient viremia could be observed from day 2 until day 6 after vaccination (Reinhardt, Jaspert et al. 1998). The absence before day 3 clearly indicates that the observed viremia is dependent on active viral replication, which can be assumed to be equal in all participants. Thus the presence of viral RNA in the blood of only some of the donors points to differences in the extent of early antiviral immunity, which is orchestrated mainly by the innate immune system.
6.1 Innate Immune Mechanisms Following Yellow Fever
pathogen entry and migrate to the secondary lymphoid tissues, thereby down-regulating their antigen sampling and transit to a rather immune-stimulatory state.
A clear increase in the frequency of mDC in the peripheral blood of vaccinees peaking at day 7 could be observed. Interestingly, these cells also showed signs of recent activation/ maturation, as they showed an increased expression of MHC class II on their surface. Following activation and consecutive maturation, these mDCs are sufficient to initiate an adoptive immune response. Also pDC numbers were increased in the peripheral blood at day 1 and day 7, however to a lesser extent and here the expression of MHC class II was not altered. Interestingly, the early rise in pDCs complements previous reports generated in rhesus monkeys, where after occulation of dengue virus, a yellow fever related flavivirus, an increased frequency of pDCs has been detected.
Furthermore, in humans a rise in pDC number could be observed in less severe dengue fever, whereas in haemorrhagic dengue fever, a life threatening form, this rise in pDC frequency was absent (Pichyangkul, Endy et al. 2003). In contrast to pDCs, mDC have been shown to be rather reduced during acute dengue infection, yet also for them a correlation between low cell numbers and worse clinical outcome with detected virus replication has been described in the case of an HIV infection (Donaghy, Pozniak et al.
2001). In contrast to the low cell numbers of mDCs described during dengue fever infection, the number of mDCs following YFV 17D vaccination rather increases. This might be explained by the fact that, although the YFV 17D successfully infects DCs, this infection does not support viral replication that would then lead to cell death and reduced cell numbers (Barba-Spaeth, Longman et al. 2005).
The general mobilisation of dendritic cell subsets might indicate the activation of DCs in the peripheral tissue by cytokines, which initially leads to their migration through lymphatic vessels towards the secondary lymph nodes or through the blood stream to the spleen and bone marrow. Interestingly, the final destination for pDCs might preferentially be the lymph nodes (Diacovo, Blasius et al. 2005), whereas activated peripheral blood mDCs have been shown to migrate preferentially into the spleen and bone marrow (Cavanagh, Bonasio et al. 2005). This mechanism could also explain why lower pDC and mDC cell numbers have been shown to be correlated with a worse clinical outcome, as insufficient DC mobilisation might result in an insufficient further activation of the immune system. The detected up-regulation of MHC class II in mDCs, but not in pDCs, following vaccination with YFV 17D most probably reflects their distinctive characteristic with different susceptibility of infection with the live-attenuated
virus. In monocyte derived DCs, YFV 17D could be monitored by intracellular FACS staining 24 h after infection (Barba-Spaeth, Longman et al. 2005) whereas, although not divided into distinct DC subsets, the work of Palmer et al. could not detect intracellular replicative RNA of the YFV vaccine after infection of DCs (Palmer, Fernandez et al.
2007). Nevertheless, after infection with YFV 17D activation and maturation only of mDCs, but not pDCs, was shown to lead to an up-regulation of MHC class II and CD86 (Querec, Bennouna et al. 2006). In contrast, pDCs were the main cytokine producers especially of IFNα (Querec, Bennouna et al. 2006). This distinct difference between m- and pDCs has also been shown following dengue virus infection. There, only in mDCs an infection detected by an anti-viral monoclonal antibody was observed. Furthermore, only mDCs, but not pDCs, obtained additional up-regulation of activation as well as maturation markers like e.g. CD40 and CD86. Nevertheless, pDCs were able to produce cytokines like e.g. IFNα with titers 10 times higher than that of mDC (Sun, Fernandez et al. 2009). Taken together, this might suggest a disparity between DC subsets, with mDCs functioning as antigen-presenting cells, while pDCs are the principal Type-I IFN producing cells.
The drop in monocytes at day 14 could indicate refreshment of the mDC/ pDC pool, as monocytes have been shown to have the ability to differentiate into macrophages or dendritic cells depending on the cytokine milieu (Randolph, Beaulieu et al. 1998;
Randolph, Inaba et al. 1999; Chomarat, Banchereau et al. 2000).
Other direct effectors of the innate immunity in an antiviral response are NK cells that can detect and lyse virus-infected cells. In humans, these can be subdivided into rather cytotoxic CD56dim CD16+ and CD56bright CD16- NK cellswith a rather high potential for the production of cytokines. Interestingly, CD56bright and cd56dim NK cells behaved quite differently during vaccination.
CD56bright NK cells showed an unchanged pattern of absolute cell counts in peripheral blood despite a short decrease at day 7 with normalization at later time-points. The significant drop of CD56bright NK cells observed at day 7 could be due to a possible homing of these cells into the lymph node, as it is known that CD56bright NK cells do have the capability to enter secondary lymphoid organs by the expression of CD62L, CXCR3 and CCR7 (Frey, Packianathan et al. 1998; Campbell, Qin et al. 2001), enabling them to get into close contact with DCs (Fehniger, Cooper et al. 2003). In an in vitro cross-talk between NK cells and DC subsets, it has been shown by several authors
that their interaction leads to an activation and cytokine production of both cell types.
Mature myeloid DCs are the major source of the secretion of IL-12, which is known to enhance NK cell activation and IFNγ secretion (Trinchieri 2003). In contrast, as it is known that CD56bright cells have the ability to respond to low doses of IL-2 by the expression of their high affinity interleukin-2 receptor (IL-2R) (Frey, Packianathan et al.
1998; Campbell, Qin et al. 2001), these IL-2 activated NK cells have the reciprocal ability to induce maturation of DCs and to activate IFNα secretion from pDCs (Gerosa, Gobbi et al. 2005). Plasmacytoid DCs, on the other hand are also known to be early producers of IL-2 (Granucci, Vizzardelli et al. 2001), which enables them to provide further activating and survival signals for NK cell populations. Furthermore, as CD56bright NK cells could be found in the T-cell-rich area of lymph nodes, they are also known to interact with T cells (Fehniger, Cooper et al. 2003). As CD56bright NK cells produce large amounts of IFNγ, these cells could promote a Th1 polarization of helper CD4+ T cells.
Nevertheless, CD56bright NK cells are also capable of producing Th2 polarizing cytokines like IL-10 (Cooper, Fehniger et al. 2001).
In contrast to the CD56bright NK cell subset, there was a constant drop of CD56dim NK cells detectable during the course of vaccination, with lowest levels at day 28, the latest time-point of the analysis. Though difficult to compare, these observations are also at least partially in line with previous reports about a drop of NK cell subsets at day 15 during vaccination with YFV 17D, however this analysis was solely based on frequencies in peripheral blood and NK cell subset definitions were different (Martins, Silva et al. 2008). Nevertheless, a decreased frequency of CD56dimCD16+ NK cells in some donors at time points similar to those used in the present study has been reported after influenza vaccination (Long, Michaelsson et al. 2008). As DCs are one of the first targets for an YFV 17D infection, their cytokine production might be a potent stimulus to recruit NK cells from blood to the site of infection. Recently, da Costa Neves et al. have confirmed the activation of NK cells, as measured by the increase of surface activation markers like CD69, CD25 and MHC class II, soon after YFV vaccination. This activation seemed to be dependent on viral recognition by NK cells at the site of infection provoking their engagement of TLR 3 and 9. Thereby also the cytotoxicity of NK cells, as measured by the up-regulation of the activation marker CD38 and the stimulatory high-affinity Fc receptor CD16, is increased (Neves, Matos et al. 2009).
This notwithstanding, not only the recruitment of NK cells to the site of infection but also the consumption of NK cells during the fight against the pathogen could be interpreted
as an explanation for the constant drop in the number of CD56dim NK cells. As the CD56dim subset appears to be nearly unsusceptible to IL-2-mediated proliferative signals (Baume, Robertson et al. 1992), the proliferative capacity of these cells is believed to be low. Therefore, these cells have to be recovered from other sources, but until now it is not clear what pre-cursor cells are used for the CD56dim and CD56bright subsets, to what extent this occurs and how time-consuming this is (Parrish-Novak, Dillon et al. 2000). Nevertheless, as the reduction of CD56dim NK cells is rather prolonged, this could point to a substantially longer persistence of the virus than measured by serum PCR. As previously assumed, also the subset of CD56dim cells could have preferentially migrated to tissues like for example the liver with further ongoing, although not detectable, YFV 17D replication (Monath 2001; Lefeuvre, Contamin et al. 2006).