We constructed a series of ECTV recombinants expressing murine IL-4 or non-functional IL-4D116/D119 mutant under the control of different promoters with varying strengths and temporal regulation. The expression of the murine IL-4 gene was consistent with the reported strengths of the tested promoters. Accordingly, the ECTV-7.5E-IL-4 and ECTV-11KL-IL-4 recombinants were observed to produce in vitro and in vivo the lowest and the highest levels of IL-4, respectively. There was no obvious difference in the pathogenesis of disease that could be attributed to promoter strength, unless virus replication was reduced by prophylaxis. In ECTV-7.5E-IL-4 infections, protection from death in the inherently susceptible A/NCR and Swiss strains could be reduced by Dryvax™ immunization or CDV treatment. Based on this observation, we selected the ECTV-11KL-IL-4 recombinant, which produced the highest level of IL-4, as the challenge virus for evaluation of the robustness of the various treatments.
The IL-4 levels generated in vitro
by the Jackson et al.
ECTV-IL-4 recombinant were not reported, but would likely be comparable to our ECTV-7.5E/L-IL-4 that utilized an identical promoter, but lacked a fully functional tk
gene and contained an insertion in the orthologue of the VACV F7L ORF. The IL-4 levels induced by all ECTV recombinants far exceeded that achieved by other vectors. For example, adenovirus and P. aeruginosa
vectors achieved maximal systemic levels of 150 pg/ml and ~100ng/ml of IL-4, respectively, following intravenous infection of mice (Giampietri, et al., 2000
;Kim, et al., 2000
). For the following reasons we conclude the lethality of the ECTV-IL-4 recombinants results from virus-expressed IL-4 signaling through the IL-4 and/or IL-13 receptor and not due to IL-4 toxicity or the contribution of host-expressed IL-4: 1. immunized BALB/c-Il4ratm1Sz
mice lacking a functional IL-4 receptor were resistant to lethal infection; 2. administration of 6.3 to 100 µg of exogenous IL-4 was lethal for ECTV-WT-infected, but not mock-infected, C57BL/6 mice; 3. the non-signaling ECTV-IL-4D116/D119
recombinants behaved as ECTV-WT; and 4. C57BL/6-Il4tm1Nnt
mice lacking the ability to make IL-4 were equally susceptible to lethal infection.
ECTV expressed IL-4 could exert its effect on cells in two distinct ways. The large amounts of biologically active IL-4 secreted from ECTV-IL-4 infected cells could induce physiological changes through the IL-4 receptor ubiquitously found on a large number of cell types. In addition, virus expressed IL-4 could be non-specifically trapped within the virion during assembly and released within the cell’s cytoplasm in the subsequent replication cycle as previously shown for chloramphenicol acetyl transferase (Franke & Hruby, 1987
). Indeed western blot analysis of sucrose gradient, band-purified virions from ECTV-IL-4 infected cells detected ~ 7 × 105
molecules of IL-4/virion (Figure S2
); however, for the virion associated IL-4 to have physiological effects, it would need to have access to recently synthesized IL-4 receptor or receptor on the cell surface as no IL-4 receptor independent pathways for IL-4 function has yet to be described.
The Jackson et al.
study reported ECTV-IL-4 recombinant infections associated with increased viral titers in spleens, and diminished splenic NK and T cell lytic and IFN-γ responses. In our studies we also observed increased viral titers in assayed tissues. Histopathologic examination of sacrificed mice revealed large areas of the liver showing confluent necrosis without associated inflammatory cells. Another major difference from ECTV-WT infections was the presence in the hepatic vessels of large numbers of macrophages and a progressive replacement of normal hematopoietic cells with macrophages in the bone marrow. This is consistent with a recent study that has shown that exogenously provided IL-4 can also drive expansion of macrophage populations in uninfected mice (Milner, et al., 2010
). In ECTV-7.5E/L-IL-4 infections, histiocytosis and hemorrhage in the red pulp of the spleen were more prominent and in general lymphopoiesis was reduced in examined lymphoid tissue. In summary, ECTV-IL-4 recombinants replicated to high titers in lymphoid organs and the liver correlating with extensive tissue necrosis, and the lack of a detectable protective inflammatory infiltrate. We speculate that a major factor contributing to the lethality of ECTV-IL-4 recombinants is its lymphoid tissue tropism, resulting in focal production of extremely high levels of IL-4 at the sites of primary and memory adaptive immune responses. This hypothesis may explain the failure of other virus-IL-4 recombinants to cause similar disease.
In spite of replicating to higher titers in examined tissues, surprisingly ECTV-11KL-IL-4 infected mice were less efficient in transmission to contacts suggesting an effect of IL-4 on virus replication in the skin, the primary source of transmissible virus. Based on severe immunosuppression induced in the ECTV-IL-4 recombinant infections and reduced transmissibility, we speculate that a VARV expressing human IL-4 may cause a disease similar to, or more severe than hemorrhagic smallpox. Hemorrhagic smallpox is thought to occur in subjects with varying degrees of immunodeficiency. As compared to ordinary smallpox, it presents with a shorter time to death and more severe disease manifestations that may result in earlier hospitalization and/or isolation, which could result in less efficient person-to-person transmission of VARV.
Treatments using the VACV-based Dryvax™ smallpox vaccine (prime or prime/boost formats) and VIG were thoroughly evaluated and found to be ineffective against ECTV-IL-4 recombinants producing the greatest amounts of IL-4. Prime/boost immunizations failed to protect even when challenges were initiated during a period when high levels of virus specific T cells and antibodies were present in blood. It was of interest to note that prior infection with ECTV-WT completely protected mice from lethal ECTV-IL-4 recombinant infections. This protective effect was also observed by Jackson et al.
where an attenuated, recombinant ECTV (ECTV-602) was able to protect 40% of immunized C57BL/6 or BALB/c mice from challenge with an ECTV-IL-4 recombinant employing the 7.5E/L promoter. This differential effect might reflect the differences in antigenicity of VACV as compared to ECTV (even though the genomes of the viruses share ~95% nucleotide identity) and as a consequence of the significantly greater systemic replication competence of ECTV as compared to VACV in mice (Chen, et al., 2003
;Bray, et al., 2000
;Neyts, et al., 2010
;Smee, et al., 2004
Although it can be problematic to extrapolate conclusions from one system to another, the biology of IL-4 appears to be quite similar in mouse and human species (Okada, et al., 2003
). Therefore, it is possible that a VARV-IL-4 recombinant would also break through the immunity generated by a vaccine based on VACV (e.g
. Dryvax™, ACAM2000, MVA or Lister). It is possible that a vaccine composed of key protective antigens from VARV could provide improved efficacy against such a VARV-IL-4 recombinant.
We also evaluated small molecule inhibitors of specific steps in the virus replication cycle for their efficacy against ECTV recombinants expressing high levels of IL-4. We confirmed the finding of Robbins et al
that the DNA polymerase inhibitor, CDV, was ineffective at protecting inherently susceptible mice from lethal ECTV-IL-4 infections (Robbins, et al., 2005
). Similarly, an oral lipid conjugate of CDV, CMX001®
, or the virus transport and release inhibitor, ST-246®
, failed to protect against lethal ECTV-11KL-IL-4 infections at doses and regimens used in this study; however, the combined administration of CDV or CMX001®
significantly protected against lethal IN or FP challenges with ECTV-11KL-IL-4. Although this combination of antivirals worked well, the added presence of memory immunity provided by the licensed smallpox vaccine might provide an additional margin of protection. Also useful would be a third antiviral that specifically targets transcription, which would prevent expression of all transgenes regardless of activity.
The failure of antiviral monotherapy to protect mice from ECTV-IL-4 recombinants is consistent with experimental and clinical data that suggests a functioning immune system is necessary for successful antiviral treatment of robust poxvirus infection. ST-246 protected against lethal challenges in all tested immunocompetent animal models, and provided full protection in some, but not all immunodeficient hosts infected with VACV strain WR (Grosenbach, et al., 2010
). Similarly, CDV treatment of vaccinia or cowpox virus infections of athymic nude, SCID, or cyclophosphamide treated mice failed to clear infections (Bray, et al., 2000
;Neyts, et al., 2010
;Smee, et al., 2004
). In 2007, a child with eczema vaccinatum, a life-threatening complication of VACV infection, was treated with VIG alone and then in combination with CDV and finally with a second antiviral, ST-246, prior to clinical progress and recovery was observed (Vora s et al 2008
). Similarly, in 2008, a case of progressive vaccinia, a rare and often fatal adverse event to vaccination with smallpox vaccine, was treated with VIG and then in combination with ST-246, Imiquimod, and CMX001 (CDC, 2009
). In both cases a monotherapy evolved into a combination therapy due to the lack of clinical response. These two clinical cases, which involved patients with varying degrees of immunodeficiency, the available antiviral studies using immunodeficient animal hosts, and our work here with a virus that induces a profound immunodeficient state suggest that more research is needed to evaluate combination antiviral therapies against poxviruses. This is particularly relevant as a portion of the population is immunosuppressed or immunocompromised (Handley, et al., 2009
). Combination antiviral therapy with compounds that target different pathways in virus replication have the added benefit of mitigating against the evolution of resistance, which is the inevitable consequence of monotherapy.
ECTV-IL-4 recombinants could be useful tools for evaluating the potency of candidate antivirals, since they induce a profound immunosuppression in all tested mouse strains whether genetically resistant or susceptible to severe mousepox. Furthermore, our studies indicate it should be possible to develop effective countermeasures against poxviruses expressing IL-4 or other molecules. And finally, the reduced transmissibility of ECTV-11KL-IL-4 for mice suggests that poxviruses expressing IL-4, while lethal for those infected, may or may not initiate an epidemic.