Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG, IgM, and IgA

PLOS ONE, Dec 2019

Asian elephant (Elephas maximus) immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV.

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Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG, IgM, and IgA

February Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG, IgM, and IgA Alan F. Humphreys 0 1 2 Jie Tan 0 1 2 RongSheng Peng 0 1 2 Susan M. Benton 0 1 2 Xiang Qin 0 1 2 Kim C. Worley 0 1 2 Rose L. Mikulski 0 1 2 Dar-Chone Chow 0 1 2 Timothy G. Palzkill 0 1 2 Paul D. Ling 0 1 2 0 1 Center for Comparative Medicine, Baylor College of Medicine , Houston , Texas, United States of America, 2 Department of Molecular Virology and Microbiology, Baylor College of Medicine , Houston , Texas, United States of America, 3 Department of Molecular and Human Genetics, Baylor College of Medicine , Houston , Texas, United States of America, 4 Human Genome Sequencing Center, Baylor College of Medicine , Houston , Texas, United States of America, 5 Department of Pharmacology, Baylor College of Medicine , Houston, Texas , United States of America 1 Funding: Support for these services was provided by NIH grant P30 CA125123. Support for these studies was also provided by a grant from the Houston zoo to PDL and in part by the Institute of Museum and Library Services grant MG-30-13-0086-13 2 Academic Editor: Alfred L. Roca, University of Illinois at Urbana-Champaign, UNITED STATES Asian elephant (Elephas maximus) immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV. - Competing Interests: The authors have declared that no competing interests exist. Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease, particularly in juvenile Asian elephants in captivity [1,2,3,4,5,6] and in range countries [7,8,9]. However, herpesviruses do not typically cause lethal infections in their natural hosts and factors contributing to EEHV-associated disease susceptibility remain enigmatic. The anti-EEHV antibody status of young elephants and normal healthy adults might help to address this issue and the ability to assess elephant immune responses will be needed to evaluate future EEHV vaccines. To our knowledge, specific serology assays for the detection of anti-EEHV antibody titers in Asian elephants have not been reported. Previous studies in African savanna elephants and Asian elephants indicated that both species appear to make IgG [10,11] but these studies failed to confirm the presence of IgM or IgA. Readily available reagents for the detection of elephant IgG are also limited [10,12,13]. A more recent study characterizing the genomic organization of African savanna elephant immunoglobulin genes indicates that in addition to IgG, African savanna elephants encode for a constant region [14]. To our knowledge, there is no publicly available sequence information for either Asian or African savanna elephant constant regions. Very little specific information is known regarding immunoglobulin genes in other members of the Afrotheria superorder, although there has been specific antisera generated towards IgG in manatees (Sirenia) and identification of cross-reacting commercial antisera towards IgG in elephant shrews (Macroscelidea) and hyraxes (Hyracoidea) [15,16]. In order to address some gaps in our understanding of elephant immunoglobulin isotypes, we sought to determine whether Asian elephants produce IgM or IgA and to use this information to generate or identify specific antibodies that could detect these immunloglobulin isotypes. Mice were also immunized with Asian elephant (...truncated)


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Alan F. Humphreys, Jie Tan, RongSheng Peng, Susan M. Benton, Xiang Qin, Kim C. Worley, Rose L. Mikulski, Dar-Chone Chow, Timothy G. Palzkill, Paul D. Ling. Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG, IgM, and IgA, PLOS ONE, 2015, Volume 10, Issue 2, DOI: 10.1371/journal.pone.0116318