Development of a Human-Murine Chimeric Immunoglobulin M Antibody for Use in the Serological Detection of Human Flavivirus Antibodies

Current diagnosis of human flaviviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Broad application of this assay is hindered by a lack of standardized human positive-control sera that react with the wide variety of flaviviruses that can cause human disease, e.g., dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and St. Louis encephalitis virus (SLEV). We have created a human-murine chimeric antibody combining the variable regions of the broadly flavivirus cross-reactive murine monoclonal antibody (MAb) 6B6C-1 and the constant region of human IgM to produce a standardized reagent capable of replacing human positive-control sera in a MAC-ELISA for the diagnosis of all human flaviviral infections. The human-murine chimeric IgM antibody secreted from plasmid-transformed Sp2/0-Ag14 cells had a level of serological activity identical to that of 6B6C-1 as measured by ELISA, immunoblotting, and MAC-ELISA for multiple members of the flavivirus genus, including WNV, SLEV, YFV, DENV, and JEV.Flaviviruses are positive-stranded RNA viruses, members of the family Flaviviridae, and are responsible for a number of medically important human diseases. Flaviviruses are arthropod-borne viruses (arboviruses) that are most commonly transmitted seasonally and in specific geographic locations. In the United States, flaviviruses are transmitted mainly by mosquitoes (17, 21). More than 70 different flaviviruses are known to exist; however, the majority are not currently associated with human disease (21). The tenuous nature of the status quo was proven by the emergence of West Nile virus (WNV) in the western hemisphere. Prior to the 1999 outbreak of WNV encephalitis in New York City, St. Louis encephalitis virus (SLEV) was the most important agent of epidemic viral encephalitis in North America, last causing a major epidemic in the mid-1970s (22, 24, 28). Since 1999, the distribution of WNV has rapidly expanded from New York to the rest of the United States and into Canada and Central and South America. As of August 2008, 27,841 human WNV cases in the United States have been reported to the Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount08_detailed.htm). Given the globalization of commerce and travel, virus-infected people, animals, and arthropod vectors are able to move easily between distant locations with great speed (13). Thus, it is likely that other arboviruses will follow the example of WNV, resulting in new or novel disease outbreaks in regions of the world outside their normal geographic ranges. Because of this, a rapid and standardized approach to the identification of arboviral infections is needed, worldwide, for the diagnosis and tracking of current and reemerging arboviral diseases.The most commonly employed serological technique for the diagnosis of human flaviviral infections is the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), which detects virus-reactive IgM, an effective marker of acute viral infection (17, 18, 21), in serum or cerebrospinal fluid from a person with a clinically compatible illness. In the diagnostic setting, the MAC-ELISA and a similar ELISA measuring virus-reactive human IgG are often used in tandem to provide clear diagnostic profiles (9, 13, 18).Application of the MAC-ELISA in the serodiagnosis of flaviviral infection is hampered by the limited availability of human infection-immune sera for use as virus-reactive, antibody-positive control specimens. For the most part, antibody-positive control sera are derived from small volumes of diagnostic serum specimens. The specimens are typically collected only from the most prevalent flaviviral agents (17, 18). The lot-to-lot variability of these specimens can be high, and constant recalibration of antibody-positive and negative-control sera is necessary to ensure that test parameters remain valid (8, 18). Of even greater concern is the lack of broadly cross-reactive antibody-positive control sera that can be used in the MAC-ELISA for the identification of atypical flaviviral infections (17).The availability of a flavivirus group-reactive human IgM antibody would be a tremendous asset in the serological diagnosis of flaviviral infections. Although a number of murine monoclonal antibodies (MAbs) demonstrating flavivirus group reactivity exist, they are unsuitable for use in the human MAC-ELISA. Fortunately, advances in the humanization of murine MAbs have made it possible to overcome these limitations (25). One such method, described by Hackett et al., involves the incorporation of the heavy (H)- and light (L)-chain variable (V) regions of a given murine MAb into an expression plasmid (pJH2-24-95B1; referred to below as pJH2) that contains the constant (Cμ) region of human IgM (8). Upon transfection of cells, the resulting plasmid construct expresses a human-murine hybrid (chimeric) IgM molecule that retains the specificity of the “parent” murine MAb but reacts like human IgM in the MAC-ELISA (8, 10).In this report we describe the development and characterization of such a human-murine chimeric IgM antibody prepared by using the IgM expression plasmid described by Hackett et al. (8). This chimeric IgM was created by incorporating the V regions of the broadly flavivirus cross-reactive murine MAb 6B6C-1 into a plasmid construct containing the human IgM μ chain. The murine MAb 6B6C-1 was originally raised against SLEV and is specific for the flaviviral envelope (E) protein (19, 23). The flavivirus group reactivity of chimeric 6B6C-1 IgM was confirmed, and the chimeric 6B6C-1 IgM was evaluated in the standard MAC-ELISA and shown to be a satisfactory replacement for antibody-positive human control sera against all flaviviruses tested.