Development of Human-Murine Chimeric Immunoglobulin G for Use in the Serological Detection of Human Flavivirus and Alphavirus Antibodies

Diagnosis of human arboviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the indirect IgG ELISA. Broad application of these assays is hindered by the lack of standardized positive human control sera that react with a wide variety of flaviviruses (e.g., dengue, West Nile, yellow fever, Japanese encephalitis, Saint Louis encephalitis, and Powassan viruses), or alphaviruses (e.g., Eastern equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis, and chikungunya viruses) that can cause human disease. We have created human-murine chimeric monoclonal antibodies (cMAbs) by combining the variable regions of flavivirus (6B6C-1) or alphavirus (1A4B-6) broadly cross-reactive murine MAbs (mMAbs) with the constant region of human IgG1. These cMAbs may be used as standardized reagents capable of replacing human infection-immune-positive control sera in indirect IgG ELISA for diagnosis of all human flaviviral or alphaviral infections. The IgG cMAbs secreted from plasmid-transformed Sp2/0-Ag14 cells had serological activity identical to that of the parent mMAbs, as measured by ELISA using multiple flaviviruses or alphaviruses.Arthropod-borne viruses (arboviruses) are responsible for a number of medically important human diseases. These viruses are maintained in nature through biological transmission between susceptible vertebrate hosts by blood-feeding arthropods, primarily mosquitoes and ticks. Although over 150 arboviruses are known to cause disease in humans, the majority of medically important arboviruses are found in three separate families, the Flaviviridae, the Togaviridae (genus Alphavirus), and the Bunyaviridae (24). Transmission of arboviruses can vary by season, a consequence of the feeding patterns of their respective arthropod vectors, as well as by specific geographic location, as is seen for dengue fever virus (DENV) and Japanese encephalitis virus (JEV) (20, 24). The primary clinical manifestation of arboviral disease in North America is encephalitis, although some arboviruses, such as yellow fever virus (YFV) are capable of causing severe hemorrhagic disease as well. Prior to the 1999 outbreak of West Nile virus (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 (26, 28, 33). Since 1999, the distribution of WNV has rapidly expanded from New York to the rest of the United States and into Canada, Central America, and South America. As of April 2009, a total of 29,598 human WNV cases in the United States had been reported to the Centers for Disease Control and Prevention, of which 1,159 resulted in death (http://www.cdc.gov/ncidod/dvbid/westnile/surv&control.htm).Given the globalization of commerce and travel, virus-infected people, animals, and arthropod vectors are able to move more easily between locations with great speed (16). 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. Therefore, a rapid and standardized approach to identification of arboviral infections is needed worldwide for the diagnosis and tracking of current and reemerging arboviral diseases.In the past, identification of antiviral antibody relied on four tests: the hemagglutination inhibition test, the complement fixation test, the plaque reduction neutralization test, and the indirect fluorescent antibody (IFA) test. Positive identification of a viral infection required a 4-fold increase in titer between acute- and convalescent-phase serum samples in these assays (20). Rapid serologic assays, such as the IgM capture enzyme-linked immunosorbent assay (MAC-ELISA) and IgG ELISA are now routinely used in diagnosis soon after infection. Early in infection, IgM antibody is more specific, while later in infection, IgG antibody is more cross-reactive. Inclusion of murine monoclonal antibodies (mMAbs) with defined virus specificities in these solid-phase assays has permitted a level of assay standardization that was not previously possible (30). In the diagnostic laboratory, the MAC-ELISA and the IgG ELISA are often used in tandem to identify positive specimens based on a 4-fold increase in titer between acute- and convalescent-phase serum samples and have replaced the more time-consuming and labor-intensive assays (11, 16, 21).Application of the ELISA in serodiagnosis of arboviral infection is most 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 by pooling small volumes of antibody-positive diagnostic serum specimens. The specimens are typically obtained for only the most prevalent arboviral agents (20, 21). Lot-to-lot variability of these serum pools can be high, and constant recollection and recalibration of antibody-positive and -negative control sera are necessary to ensure that test parameters remain valid (10, 21). Of even greater concern is the lack of antibody-positive control sera that can be used in diagnostic ELISAs to identify arboviruses that currently cause rare or infrequent human infections (20).The replacement of variably reactive human control sera with group-specific human IgG antibodies would be a tremendous asset in the serological diagnosis of arboviral infections. Although a number of mMAbs demonstrating flaviviral, alphaviral, or bunyaviral group reactivity exist, they are unsuitable for use as positive serum controls in ELISAs designed to detect the presence of human antibodies. Moreover, the capture or detector antibodies used in these assays are often designed to react with other murine components of the ELISA, leading to an overwhelming false-positive response if mMAbs are employed as positive controls.Fortunately, advances in the humanization of mMAbs have made it possible to overcome these limitations (31). One such method involves the incorporation of the heavy (H)- and light (L)-chain variable (V) regions of a given mMAb into an expression plasmid containing the constant (Cμ) region of human IgM (10). Upon transfection of cells, the resulting plasmid construct expresses a human-murine hybrid IgM chimeric MAb (cMAb) molecule that retains the specificity of the “parent” mMAb but reacts like human IgM in the MAC-ELISA (10, 12, 32).We have previously reported on the construction and evaluation of an IgM cMAb with the specificity of the broadly flavivirus cross-reactive mMAb 6B6C-1. The 6ME2 IgM cMAb reacted with each flaviviral suckling mouse brain (SMB) or virus-like particle (VLP) antigen tested in the MAC-ELISA and displayed a strong preference for the WNV VLP antigen. The use of cell culture viral seed in place of the SMB or VLP antigens in the MAC-ELISA format resulted in enhanced reactivity, as measured by the maximum dilution of cMAb yielding a positive P/N value (positive/negative ratio; see below for details) against WNV, SLEV, DENV serotype 2 (DENV-2), and YFV. In this report we describe the development and characterization of two new IgG cMAbs for use in the indirect IgG ELISA. These cMAbs were created by incorporating the V regions of 6B6C-1 or the alphavirus group-specific mMAb 1A4B-6 into a plasmid construct containing the human IgG γ1 chain. The alpha- or flaviviral group reactivity of each cMAb was confirmed and subsequently evaluated in the standard indirect IgG ELISA. The cMAb demonstrating alphaviral (1GD5) or flaviviral (6GF4) group reactivities were selected for further use and were satisfactory replacements for antibody-positive human control sera against all alphaviruses or flaviviruses tested.