Following the anthrax attacks in the fall of 2001, discussions about the potential use of other virulent pathogens as biological weapons have increased. The threat of a smallpox outbreak elicits great concerns because of its acute and significant morbidity and mortality rates and the lack of significant immunity against poxviruses among civilians born after 1970, despite a large vaccination campaign effort among the military and medical first responders (17
). Discussions about the widespread use of the present FDA-approved vaccine Dryvax have resulted in much debate in the medical community. While the vaccine is efficacious, the complications associated with Dryvax make the development of a safer second-generation smallpox vaccine a medical priority.
For the present study, we evaluated the efficacy and immune response of a highly attenuated vaccinia virus, MVA, by use of a mouse model. Investigations of this vaccinia virus strain began in the late 1950s after attenuation of the parent strain CVA in chicken embryo fibroblasts. Since that time, MVA has been evaluated in several animal models as well as in humans (22
). During the 1970s, more than 120,000 people were immunized with MVA (37
). Reports reflect a good safety profile for MVA, including safe administration to immunocompromised animals (38
). However, the specific determinants of MVA-induced immunity compared with those induced by replication-competent vaccinia virus have only recently been studied (5
We demonstrated the ability of a single dose of MVA to protect against both intradermal and intranasal challenges with vaccinia virus. In particular, we demonstrated that MVA immunization can protect against a lethal respiratory challenge with a molecularly modified recombinant vaccinia virus expressing murine IL-4. While previous studies have shown a dose-related effect following single-dose escalation (4
), we showed that a multidose schedule of MVA may enhance the overall immune response and provide added protection against vaccinia virus replication. For both the intradermal and intranasal models, virologic protection, as judged by the presence or absence of pox lesions, and vaccinia virus titers in the lungs improved in mice that received two or three immunizations (Table and Fig. ). While clinical illness was reduced in all MVA-immunized mice after an intranasal challenge, the vaccinia virus titers in the lungs were higher for mice that received a single immunization. This enhanced protection correlated with the magnitude of both the humoral and cellular immune responses, as judged by the neutralization activity and intracellular IFN-γ production in vaccinia virus-specific T cells, respectively (Fig. ). In mice receiving multiple immunizations, the immune responses were enhanced, suggesting that strategies to optimize the protection of humans against poxviruses such as variola virus and monkeypox virus may necessitate a multidose immunization regimen. Although it was not evaluated in these studies, the route of immunization is also important. For example, MVA was administered intramuscularly in our studies, and the responses compared favorably to those induced by replication-competent vaccinia virus given by the intramuscular or intradermal route. However, when vaccinia virus is given by intraperitoneal injection, much higher frequencies of CD4+
- and CD8+
-T-cell responses can be achieved in splenocytes (20
). Therefore, additional exploration of the route of poxvirus immunization may be warranted.
With the threat of bioterrorism, there is concern not only about the potential release of virulent pathogens, but also about the purposeful manipulation of pathogens in an effort to augment their virulence. Importantly, we have demonstrated that MVA immunization can protect against a lethal pulmonary challenge with a molecularly modified recombinant vaccinia virus expressing murine IL-4. Prior studies have demonstrated the importance of cytokine balance in the pathogenesis of viral, bacterial, and parasitic diseases. The Th1 cytokine IFN-γ plays a key role in the control of vaccinia virus infection (24
). Disruption of the Th1-Th2 balance has been shown to adversely affect viral clearance and protection from dissemination. As previously described (24
), we demonstrated that the virulence of vaccinia virus is enhanced in the setting of excess IL-4 production, with a prolonged elevation of lung viral titers and death in mice infected with vSC8-mIL4 (Fig. ). Despite this enhanced virulence, MVA immunization protected mice from a lethal pulmonary challenge with vSC8-mIL4 and eliminated clinical illness and weight loss. The ability to protect against a lethal molecularly modified vaccinia virus lends further support to MVA as a candidate vaccine against smallpox.
While they are effective against vaccinia virus challenges, the determinants of immunity elicited by MVA and other vaccinia viruses have not been clearly defined. The immunization of human subjects with Dryvax has been shown to elicit both humoral and cellular immunity (15
). Historical reports suggest that both arms of the immune system are relevant to protection from smallpox. Investigations of villages during outbreaks of smallpox correlated the vaccine take and antibody response with immunity (28
). More recent reports point to the importance of the cellular immune response for containing disseminated vaccinia virus (7
). It becomes important in the evaluation of novel vaccines to dissect the role of each component of the immune response in order to demonstrate similar patterns of response to both replication-competent and attenuated vaccines.
Recent work suggested that selected human CD8+
CTL epitopes are conserved between various poxviruses, including MVA and variola virus (11
). The selective depletion of either CD4+
T cells alone prior to vSC8-mIL4 challenge resulted in minimal weight loss and viral replication, which suggests that neither cell type is required for the containment of vaccinia virus replication (Fig. ). The depletion of both CD4+
T cells resulted in weight loss, which corresponds to enhanced viral replication and delayed clearance, but the weight loss was minimal. While this demonstrates the importance of cellular immunity for vaccinia virus clearance, it suggests that T cells are not the sole factor in the defense against poxvirus infection and that the immune system has evolved redundant mechanisms to control this important class of pathogens.
Contributions from the humoral immune response following immunization are also likely to be important for protection against vaccinia virus infection. We demonstrated a dose-dependent increase in the antibody response to MVA immunization, which correlated with protection. However, in the absence of neutralizing antibodies there was minimal illness in this murine model from an intranasal vaccinia virus challenge (Fig. ). While MVA-immunized B-cell-deficient mice demonstrated significant viral replication in the lungs on day 4 after the intranasal challenge, they lacked signs of clinical illness and cleared the vaccinia virus by day 8. As for many other virus infections, we believe that the data support an important role for vaccine-induced antibodies in protection from infection, but effective T-cell responses also appear to be important for protection against severe poxvirus-induced disease. These data are consistent with recent studies that evaluated the immune determinants of protection against the WR strain of vaccinia virus (5
) and that indicated that vaccine-induced protection from poxviral challenge should not be restricted to a single arm of the immune response, but should include a combination of both cellular and humoral immune responses.
We demonstrated a robust immune response following MVA immunization in a murine model. MVA not only protected mice from a standard vaccinia virus challenge, but it was also able to prevent illness and limit viral replication after the administration of a lethal molecularly modified strain of vaccinia virus. These data support the further development of MVA as a stand-alone vaccine against smallpox and infections caused by other orthopoxviruses.