The genus Orthopoxvirus (OPV) comprises a large number of morphologically identical viruses, including the agent of human smallpox variola virus (VARV), the agent of mousepox ectromelia virus (ECTV), the vaccine species vaccinia virus (VACV), and monkeypox virus, a zoonotic virus with its natural reservoir in African rodents.
Smallpox, a grave systemic disease characterized by high mortality and responsible for the death of billions over at least three millennia (1
), was eradicated worldwide in the late 1970s through vaccination with VACV. However, there is fear that VARV could be used as a weapon. This would be devastating, because massive vaccination against smallpox was discontinued in 1978 and most of the human population is presently not immune. In addition, the reduced level of herd immunity against OPV increases the possibility of infection with zoonotic monkeypox virus, as exemplified by its current endemic status in central and west Africa, as well as a recent outbreak in the US Midwest (2
). Moreover, because there are many other OPVs in nature, there is a threat of emerging OPV zoonoses.
The current smallpox vaccine uses live VACV, which is normally poorly or nonpathogenic to humans but induces cross-protective immunity against other OPVs. However, because of cases of adverse effects, including generalized infection and death, the live VACV vaccine is not safe by current standards (1
). On the other hand, killed VACV induces antibodies but is not effective at preventing disease, perhaps because it does not induce a subset of antibodies important for protection (7
). Therefore, there is a need to replace the current VACV-based vaccine (Dryvax) with a safer vaccine that, ideally, should be noninfectious, such as subunit vaccines based on recombinant (Rec) viral proteins or DNA. However, it will be difficult to evaluate the efficacy of new vaccines in the absence of human smallpox or information regarding the correlates of immunity. Thus, the design and testing of new types of smallpox vaccines will be facilitated by advances in our understanding of the viral pathogenesis and immunology to lethal poxvirus infections.
OPVs and other poxviruses such as the Leporipoxvirus
myxoma virus (the agent of myxomatosis in the European rabbit) encode nonstructural proteins that play important roles in host specificity and virulence (13
). Some of these proteins are secreted immune response modifiers (IRMs) that may or may not bind to the surface of cells. These secreted IRMs can either mimic (virokines) or compete (viroreceptors) with the function of chemokines, cytokines, and growth factors (22
). Although we still lack a complete understanding of the role of IRMs in poxvirus pathogenesis, it is clear that some play an important role in the virulence of myxoma virus in rabbits and of VACV when inoculated intranasally to mice (25
), Because secreted IRMs are exposed to the extracellular milieu, it is very possible that they are targets of the host's antibody response. However, this has not been formally tested. Furthermore, antibodies to secreted IRMs important for virulence could be significant for protective immunity and be used as targets of novel vaccine strategies.
The OPV ECTV has host specificity for the mouse and, in susceptible strains such as BALB/c mice, produces systemic lethal mousepox when inoculated with as little as ~1 PFU through the footpad and other routes (24
). Moreover, mousepox is remarkably similar to human smallpox and can also be prevented by VACV inoculation (27
). Of interest, most of the IRMs in VARV are also present in ECTV. Consequently, it is very likely that they play similar roles in pathogenesis in their respective natural hosts. Thus, ECTV is an excellent model to understand the role of IRMs in pathogenesis and as possible candidates for new anti-OPV vaccines.
IFNs are proinflammatory cytokines produced in high quantities and during the early stages of viral infections. By inducing an antiviral state in cells (29
) and modulating the immune response (30
), IFNs are major mediators of the antiviral defense (31
). Type I IFN-α and -β use a common heterodimeric receptor (IFN-α/βR), composed of IFNAR1 and IFNAR2, that is ubiquitously expressed (32
). Type I IFNs can be produced by almost any infected cell, but in some viral infections are mostly produced by plasmacytoid dendritic cells (33
). The type II IFN-γ is mainly the product of NK cells and CD8+
T cells, and binds to IFN-γR at the surface of cells.
IFNs play an important role in resistance to OPV infections. Mice deficient in type I and II IFNR or IFN-β are highly susceptible to VACV infection (36
). Similarly, type I and II IFNs are essential for natural resistance to mousepox in 129 and C57BL/6 (B6) mice, as demonstrated in experiments of antibody depletion, or with mice deficient in IFN-γ, IFN α/βR, or IFN-γR (40
OPVs encode IRMs that are type I and II IFN binding proteins (bp's) that compete with type I or II IFNR for their ligands, respectively (13
). In ECTV, the type I IFN bp is encoded by ECTV Moscow open reading frame no. 166 (EVM166) and is 88% identical with both the VACV and the VARV orthologues (13
). However, whether the ECTV protein binds mouse IFN with similar or different affinity than the VACV protein is not known.
The role of IFN bp in OPV virulence is still not completely clear. For example, VACV deficient in the IFN-γ bp was not attenuated in mice (48
). However, this is not significant because VACV IFN-γ bp does not inhibit the biological activity of mouse IFN-γ (49
). On the other hand, deletion of the IFN-γ bp results in moderate attenuation of ECTV (50
). In the case of the type I IFN bp, its deletion in VACV (B19R, formerly B18R) results in ~100-fold attenuation of VACV in mice challenged through the intranasal route. However, whether the type I IFN bp plays any role in ECTV virulence has not been determined. A comparative analysis is of particular interest given the fact that ECTV is much more pathogenic for the mouse than VACV.
Vaccines provide immunity by mimicking some aspects of the natural infection. Immunity to OPV in humans and mice is characterized by the presence of circulating antiviral antibodies and relatively high frequencies of memory T and B lymphocytes specific for the virus (51
). Experiments of passive immunization showed that either preexisting antibody or memory CD8+
T cells can protect immunocompetent mice from mousepox (28
) However, neither antibody nor memory CD8+
T cells transfer reduced virus replication at the site of entry or completely abrogated systemic virus spread (56
). These findings are not opposed to the concept of protective immunity. Indeed, it is well established that VACV immunization does not completely prevent ECTV or VACV replication or spread in mice or in humans, respectively (28
). Therefore, although vaccines should ideally prevent infection, the fact that immunization with VACV does not prevent virus replication but was very effective at eradicating smallpox indicates that protective humoral immunity may use antibodies that do not neutralize the viral particles but that target secreted virulence factors. In this study, we demonstrate that the type I IFN bp is essential for ECTV virulence, a natural target of the antibody response, and constitutes an effective target for protective immunization.