Development, characterization and use of monoclonal VP40-antibodies for the detection of Ebola virus

Lucht, A; Grunow, Roland; Möller, Peggy; Feldmann, Heinz; Becker, Stephan

doi:10.1016/s0166-0934(03)00131-9

Cited by 31 publications

(22 citation statements)

References 23 publications

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“…The NP and GP antigens were most useful for distinguishing sera from ZEBOV in comparison to Marburg virus infection, while results from the Marburg virus study sera indicated that Marburg VP40 induced a cross-reactive VP40 antibody response against all Ebola viruses. We also observed a general antibody cross-reactivity among Ebola virus NP and VP40 proteins, in a manner similar to results from previously reported ELISA studies (43)(44)(45), while GP exhibited the highest level of antibody specificity. Supporting the value of the GP mucin domain as a serological marker of infection, E. coli-expressed GP mucins for Zaire and Marburg filoviruses displayed species-specific antibody recognition similar to that of the multidomain GPs (⌬TM) that were produced from eukaryotic cells, based on assay results from the ZEBOV and MARV studies.…”

Section: Discussionsupporting

confidence: 90%

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Kamata¹,

Natarajan²,

Warfield³

et al. 2014

Clin. Vaccine Immunol.

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bInfectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses.

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Section: Discussionsupporting

confidence: 90%

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Kamata¹,

Natarajan²,

Warfield³

et al. 2014

Clin. Vaccine Immunol.

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“…For detection of NP, a specific chicken anti-NP antibody (kindly provided by R. Schade, Institut für Pharmakologie, Charité-Universitätsmedizin Berlin [37]) was used. For detection of VP40, a specific mouse monoclonal anti-VP40 antibody was used (38). For detection of GP, a specific chicken anti-GP antibody was used (38).…”

Section: Methodsmentioning

confidence: 99%

“…For detection of VP40, a specific mouse monoclonal anti-VP40 antibody was used (38). For detection of GP, a specific chicken anti-GP antibody was used (38). Anti-chicken and anti-mouse IRDye 680-or 800-conjugated antibodies from goat were used as secondary antibodies.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak

Dietzel

Schudt

Krähling

et al. 2017

J Virol

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The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. IMPORTANCEThe dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity.KEYWORDS West Africa, adaptive mutations, Ebola virus, glycoprotein, zoonotic infections E bola virus (EBOV) belongs to the family Filoviridae in the order Mononegavirales. The Ebola virus disease is characterized by severe fever accompanied by systemic inflammation and damage to the endothelial cell barrier leading to shock and multiorgan failure with high case/fatality rates (1). EBOV particles are filamentous (1 m in length, 80 nm in diameter) and composed of seven viral proteins. The nucleocapsid complex contains the viral RNA encapsidated by nucleoprotein NP and four additional viral proteins. The polymerase complex of VP35 and L is associated with NP via

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“…An ELISA method was standardized to screen the hybridoma supernatants. Briefly, the purified virus was solubilized with 1% SDS and diluted in carbonate-buffer (pH 9.6) (Lucht et al 2003). Then, the virus antigen was coated to the solid phase of 96-well high binding EIA/RIA plates (Costa) overnight at 4°C.…”

mentioning

confidence: 99%

Monoclonal antibodies developed for detection of an epizootic virus associated with mass mortalities of cultured scallop Chlamys farreri

Song

Li³

2005

Dis. Aquat. Org.

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Recently, an enveloped, spherical RNA virus was identified as the causative agent of mass mortalities among adult scallop Chlamys farreri, which is cultured on the northern coast of China. Hybridomas were prepared from mice immunized with highly purified virions. Four stable hybridomas secreting monoclonal antibodies (MAbs) of IgG isotype were obtained after screening by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). The specificity of the MAbs to this virus was confirmed by immunogold electron microscopy (IEM). All the selected MAbs recognized epitopes on the envelope spikes of the virions. Subsequently, the MAbs were used for in situ immunofluorescent detection of the virus in Davidson's fixed tissue sections. The results showed that the fluorescent cells were mostly observed in epithelia of different organs, but not in the epithelium of the digestive diverticulae. Cytopathological changes and focal lesions corresponding to virus-positive cells were clearly recognized in the affected epithelia, revealing a potential role of this virus in pathogenesis. KEY WORDS: Scallop · Chlamys farreri · Acute virus necrobiotic disease · Monoclonal antibodies Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [17][18][19][20][21][22] 2005 mass mortalities in mid-July 2001 and 2002 from several private farms in Qingdao and Rizhao (Shandong Province of China). The gills and mantles were removed from the scallops and stored at -85°C before use. Virus purification by centrifugation was performed according to C. M. . Tissues were homogenized in 8 volumes of TEN buffer (50 mM Tris-HCl; 10 mM EDTA; 360 mM NaCl, pH 7.8) and clarified by 2 low speed centrifugations (3500 × g for 15 min followed by 7500 × g for 15 min). All centrifugations were carried out at 4°C. The supernatants were then concentrated by sedimentation through a 6 ml cushion of 35% sucrose (w/w) at 113 000 × g for 90 min and the resulting pellets were resuspended in TEN buffer. After centrifugation at 8000 × g for 20 min, supernatants were layered onto a 30 to 60% (w/w) continuous sucrose density gradient. After centrifugation at 113 000 × g for 2.5 h, the opalescent virus containing band was collected. The virus was washed with TN buffer (50 mM Tris-HCl; 360 mM NaCl, pH 7.8) and resuspended in a small amount of TN buffer. The purity was checked by a transmission electron microscope (TEM, JEOL JEM-1200) using 2% phosphotungustic acid (PTA, pH 7.0) as the negative stain. The concentration of viral proteins was determined using the procedure described by Bradford (1976).Hybridoma production. Eight week old BALB/c mice were immunized intraperitoneally (i.p.) with purified virus (100 µg mouse -1 ) mixed with an equal volume of Freund's complete adjuvant (Gibco). Subsequent boosters with antigen in incomplete adjuvant were given on Days 14, 28, and 42. Three days before cell fusion, mice received the last immunization via intrasplenic injection (20 µg mouse -1 ) with the puri...

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Development, characterization and use of monoclonal VP40-antibodies for the detection of Ebola virus

Cited by 31 publications

References 23 publications

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak

Monoclonal antibodies developed for detection of an epizootic virus associated with mass mortalities of cultured scallop Chlamys farreri

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