Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4⁺ lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.The World Health Organization (WHO) announced the worldwide eradication of smallpox at the end of the 1970s. Nevertheless, the threat of an outbreak of smallpox or a smallpox-like disease, either by natural means or via bioterrorism, exists to this day. This danger was illustrated in 2003 by the mini-epidemic of monkeypox in the U.S. Midwest (33). Thus, a vaccine against smallpox is, even today, essential. Although vaccination against smallpox using vaccinia virus (VACV) was quite successful, the incidence of severe side effects prompted the WHO to discontinue the use of the vaccine. Therefore, there is currently still a need for an effective and safe vaccine against smallpox.Among the orthopoxviruses, VACV is frequently used to study poxvirus infection, since it displays many properties of variola virus, the etiologic agent of smallpox, including the capability of modulating and suppressing the immune system by means of expressing several immunoregulatory proteins (44). The assumed natural primary infection site of variola virus is considered to be the respiratory tract (5). Here, the virus encounters lung epithelial cells, conventional dendritic cells, and macrophages. More than three decades ago, viral antigen was detected in alveolar macrophages after a sublethal infection of rabbits with inhaled VACV by using immunofluorescence assays (3). Additionally, under the electron microscope, the cells obtained from repeated washing of rabbit lungs demonstrated that VACV infects and replicates exclusively in macrophages (18). Macrophages, the most abundant hematopoietic cell type in the lung, play a key role in antiviral immune defense through the phagocytosis of infectious particles and the production of reactive oxygen species as well as leukotrienes and inflammatory cytokines (14).The coordinated migration, differentiation, and activation of dendritic cells as well as lymphocytes are required for the efficient elimination of microbes, including viruses, from the lung (15). Activated alveolar macrophages, in particular, enhance the cellular immune response by triggering the immigration of several leukocyte types into the lung owing to the production of chemokines (35). The importance of alveolar macrophages in limiting the replication of a recombinant VACV strain Western Reserve (WR) was recently demonstrated with mice (38). It is likely that the stimulation of cytokine production contributed to the elimination of the virus. In summary, the infection of resident alveolar macrophages with orthopoxviruses and the subsequent upregulation of chemokine expression attract several different leukocyte types, representing a critical event in antiviral defense.A highly attenuated orthopoxvirus strain, modified VACV Ankara (MVA), is being considered as a candidate for the production of a vaccine against smallpox and as a viral vector in gene therapeutic protocols (9). MVA, administered intranasally (i.n.) or intramuscularly as a short-term immunization, has been proven to be protective against a lethal challenge with the virulent VACV strain WR in a mouse model. In contrast, the VACV strain Elstree failed to protect mice when administered intramuscularly 48 h before challenge but was effective when the lethal challenge occurred 14 days postimmunization. Interestingly, an elevated concentration of various types of leukocytes, including monocytes, granulocytes, and T lymphocytes, was present in the lung 48 h after i.n. immunization with MVA (46). This respiratory immigration of leukocytes was most likely triggered by a chemoattractant factor produced by resident lung cells due to infection with MVA. In order to elucidate the mechanism underlying the migration of leukocytes, pilot experiments were performed to test whether MVA triggers the expression of a soluble factor capable of attracting leukocytes, especially monocytes.