We have used a highly lethal model of WEEV infection to evaluate the utility of CLDC as a prophylactic immunotherapy, capable of protecting mice from WEEV encephalitis. We found with this model that CLDC treatment may also have therapeutic potential for the post-exposure treatment of WEEV infection. These results might further extend to other encephalitic alphaviruses such as VEEV and EEEV.
It is likely that the acute virulence of the challenge virus used in our model affected our ability to observe protection using CLDC immunotherapy. Following s.c. infection, the incubation period is approximately 3-4 days, after which animals progress to severe clinical illness within 24-48 hours. As indicated, we saw no example in these studies of CLDC treatment facilitating the recovery from clinical illness. This contrasts with the human infection, which by the natural route may have an incubation time of 5-10 days. For those who progress to severe disease, it can still be up to a week from the time clinical symptoms appear until death. The longer disease course in humans may provide a larger therapeutic window, allowing for more successful post-exposure treatment, than we have with the current model. Using a wild-type virus strains such as Montana-64, with 70% mortality and MTD of 6.9 days (Logue et al. 2009
), two days longer than McMillan, may give a better representation of the human condition and improve the success of post-exposure treatment.
Despite the challenge presented by the virulence of this model, by changing the site of CLDC administration from the thigh to the neck, a significant treatment benefit was observed. It is not clear from the current data whether administering the CLDC at the back of the neck resulted in a more potent systemic immunostimulatory effect or whether the effect is graduated with immunostimulatory effect decreasing as distance from the site of administration increases. If the latter is true, then the most potent effect was localized more closely to the head and brain, which may have led to more efficient prevention of neuroinvasion. Further studies on the role of the site of treatment are warranted to fully evaluate this positional phenomenon.
In addition to the survival analyses, we also sought to gain an understanding of the immune response to WEEV infection in the presence and absence of CLDC treatment. IL-12 and IFN-α are produced in response to many viral infections, primarily by plasmacytoid DCs. Alphaviruses have been shown to block the induction of type I interferon, thus limiting upregulation of dependent antiviral gene products (Barry et al. 2009
; Burke et al. 2009
). Further, signaling through the IFNα/β receptor was shown to be necessary for the induction of the antiviral state and protection from VEEV challenge following treatment with viral replicon particles (VRP) (Konopka et al. 2009
). As with VRP, CLDC administration, and subsequent type-I IFN production, allows for an established antiviral state in advance of disseminating infection, and circumvents the requirement for the infected cell to produce IFN-α/β. Although alphaviruses can vary in their sensitivity to an established antiviral state generated through IFN-α/β priming (Simmons et al. 2009
), WEEV has been shown to be exquisitely sensitive to interferon in vivo (Julander et al. 2007
). However, virulence can vary greatly among isolates of WEEV (Logue et al. 2009
). It is possible that this virulence variation could involve viral suppression of both type I and type II interferon responses through host macromolecular shutoff and/or inhibition of Jak/STAT phosphorylation, as shown with the prototypic alphavirus Sindbis (Ryman et al. 2007
). Despite this, infection with acutely lethal WEEV McMillan strain was found to be controlled through the immunomodulatory effects of CLDC treatment.
The possibility for other TLR agonists to provide similar protection remains open. It is curious that administration of poly(I:C), a TLR3 agonist, was previously shown to elicit very low levels of IFN-γ and IL-12 when compared to that of CLDC (Dow et al. 1999
). However, in that study the poly(I:C) was not complexed to liposomes, thus limiting the presentation of the TLR agonist from the endosomal compartments where TLR3 is found. When poly(I:C) was complexed to liposomes it was equally efficient to CLDC in cross-priming CD8+ T cells (Zaks et al. 2006
). When poly(I:C) was complexed to the same liposome formulation as used for the CLDC in this study, up to 80% of animals were protected from WEEV-McMillan infection. As with CLDC, this protection was dependent on time and route of poly(I:C) administration (Phillips and Olson, unpublished data).
Among the cytokines induced by CLDC treatment, IFN-γ presents great potential for aiding in the control of WEEV infection. We observed two different IFN-γ responses in our study. CLDC-treated animals produced increased serum IFN-γ relative to untreated animals. This may play a role in the prevention of the neuroinvasion and acute encephalitis and/or the resolution of any neuroinvasion that does occur. Untreated animals showed increased IFN-γ in the CNS relative to CLDC-treated animals. That this peaked at the same time that virus titers in the brain peaked suggest that it is in response to the neuroinvasion and virus replication within the CNS. Several lines of evidence imply a role for IFN-γ in resolution of both peripheral and CNS WEEV infection. IFN-γ was shown to have direct antiviral effects that appear in advance of the adaptive immune response (Schroder et al. 2004
). These antiviral properties include reduced viral protein synthesis, inhibited viral transcription, and recovery of host protein synthesis (Burdeinick-Kerr and Griffin 2005
). IFN-γ was shown to aid in the noncytolytic clearance of alphavirus infection from neurons (Burdeinick-Kerr and Griffin 2005
), an observation critically relevant to WEEV, as neuronal death is a prominent feature in WEEV pathogenesis. Further, cellular protein synthesis in neurons can be restored by an IFN-γ-dependent mechanism (Burdeinick-Kerr et al. 2009
). Finally, the critical role of IFN-γ in the protective effects of CLDC to infection by other intracellular pathogens (Goodyear et al. 2009
) suggest that the mechanistic details of this cytokine’s actions may provide many avenues of study of WEEV pathogenesis and therapeutics.
Upregulated IFN-stimulated genes have previously been utilized as IFN response markers (Satoh et al. 2006
) and may provide insight into the effects of CLDC treatment and the host response to WEEV infection. MCP-1 was dramatically upregulated in virulent SINV infection of mouse brains (Johnston et al. 2001
), as well as the brain tissue of untreated infected animals of the current study. MCP-1 is highly induced in a variety of diseases that feature monocyte-rich cellular infiltrates and was shown to be critical in protection from intracellular pathogens (Goodyear et al. 2010
). Monocyte chemotactic proteins were shown to inhibit the production of IL-12 by macrophages but are ineffective for inhibition in DCs, thus suggesting that different G-protein-coupled receptors are involved in the regulation of IL-12 production by DCs and by phagocytes (Aliberti et al. 2000
; Trinchieri 2003
). IL-12 induces T cells and NK cells to produce cytokines such as GM-CSF and TNF-α and it remarkably efficient at inducing the production of IFN-γ (Murphy et al. 1994
). The particular immunomodulation and physiological effects of high MCP-1 expression require further investigation, and the contribution of MCP-1 expression to WEEV pathogenesis in the CNS, remains to be elucidated.
The contribution of IL-10 to the overall outcome of WEEV infection of the CNS requires further investigation. TH
1 responses generally suppress TH
2 responses through the production of cytokines such as IL-12 and IFN-γ, and that IL-10 is involved in limiting the effectiveness of the TH
1 response. IL-10 and IFN-γ are antagonistic; with IL-10 acting through suppression of IL-12 (Fiorentino et al. 1989
). Conversely, IFN-γ inhibits IL-10 production from monocytes which leads to upregulation of TNF-α (Chomarat et al. 1993
; Donnelly et al. 1995
). Therefore, IL-10 is a potent inhibitor of monocyte/macrophage function and anti-inflammatory with regard to its antagonism IL-12. Interestingly, IL-10 was shown to increase MCP-1 expression in blood mononuclear cells, however this effect on MCP-1 expression is activation state and cell type dependent (Seitz et al. 1995
). It is interesting to speculate whether the increase in IL-10 observed in the brains of untreated animals is being produced in a vain effort to dampen the effects of the inflammatory cytokines present.
In addition to furthering our understanding of WEEV, and potentially VEEV and EEEV pathogenesis, clarifying the immunostimulatory effects of CLDC and which of those effects are important in protection/recovery from WEEV infection is essential to the refinement of CLDC as a therapeutic. The ultimate feasibility of CLDC as a potential therapeutic for WEEV infection remains to be determined. In these studies, the therapeutic window was relatively short and CLDC failed to protect from aerosol challenge. However, that CLDC were able to offer protection in such a challenging model, suggests that additional study is warranted. It is possible that the antiviral activity of CLDC could be enhanced in a pathogen non-specific manner by coupling it with another immunomodulatory compound or in a more pathogen-specific fashion with a targeted anti-viral therapeutic.