Eri Nakayama

Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever.

Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.

Animal models for Ebola and Marburg virus infections

Ebola and Marburg hemorrhagic fevers (EHF and MHF) are caused by the Filoviridae family, Ebolavirus and Marburgvirus (ebolavirus and marburgvirus), respectively. These severe diseases have high mortality rates in humans. Although EHF and MHF are endemic to sub-Saharan Africa. A novel filovirus, Lloviu virus, which is genetically distinct from ebolavirus and marburgvirus, was recently discovered in Spain where filoviral hemorrhagic fever had never been reported. The virulence of this virus has not been determined. Ebolavirus and marburgvirus are classified as biosafety level-4 (BSL-4) pathogens and Category A agents, for which the US government requires preparedness in case of bioterrorism. Therefore, preventive measures against these viral hemorrhagic fevers should be prepared, not only in disease-endemic regions, but also in disease-free countries. Diagnostics, vaccines, and therapeutics need to be developed, and therefore the establishment of animal models for EHF and MHF is invaluable. Several animal models have been developed for EHF and MHF using non-human primates (NHPs) and rodents, which are crucial to understand pathophysiology and to develop diagnostics, vaccines, and therapeutics. Rhesus and cynomolgus macaques are representative models of filovirus infection as they exhibit remarkably similar symptoms to those observed in humans. However, the NHP models have practical and ethical problems that limit their experimental use. Furthermore, there are no inbred and genetically manipulated strains of NHP. Rodent models such as mouse, guinea pig, and hamster, have also been developed. However, these rodent models require adaptation of the virus to produce lethal disease and do not mirror all symptoms of human filovirus infection. This review article provides an outline of the clinical features of EHF and MHF in animals, including humans, and discusses how the animal models have been developed to study pathophysiology, vaccines, and therapeutics.

Characterization of H5N1 highly pathogenic avian influenza virus strains isolated from migratory waterfowl in Mongolia on the way back from the southern Asia to their northern territory

H5N1 highly pathogenic avian influenza (HPAI) viruses were isolated from dead wild waterfowl at Khunt, Erkhel, Doityn Tsagaan, Doroo, and Ganga Lakes in Mongolia in July 2005, May 2006, May 2009, July 2009, and May 2010, respectively. The isolates in 2005 and 2006 were classified into genetic clade 2.2, and those in 2009 and 2010 into clade 2.3.2. A/whooper swan/Mongolia/6/2009 (H5N1) experimentally infected ducks and replicated systemically with higher mortality than that of the isolates in 2005 and 2006. Intensive surveillance of avian influenza in migratory waterfowl flying from their nesting lakes in Siberia to Mongolia in every autumn indicate that HPAI viruses have not perpetuated at their nesting lakes until 2009. The present results demonstrate that wild waterfowl were sporadically infected with H5N1 HPAI viruses prevailing in domestic poultry in the southern Asia and died in Mongolia on the way back to their northern territory in spring.

Enzyme-Linked Immunosorbent Assay for Detection of Filovirus Species-Specific Antibodies

ABSTRACT Several enzyme-linked immunosorbent assays (ELISAs) for the detection of filovirus-specific antibodies have been developed. However, diagnostic methods to distinguish antibodies specific to the respective species of filoviruses, which provide the basis for serological classification, are not readily available. We established an ELISA using His-tagged secreted forms of the transmembrane glycoproteins (GPs) of five different Ebola virus (EBOV) species and one Marburg virus (MARV) strain as antigens for the detection of filovirus species-specific antibodies. The GP-based ELISA was evaluated by testing antisera collected from mice immunized with virus-like particles as well as from humans and nonhuman primates infected with EBOV or MARV. In our ELISA, little cross-reactivity of IgG antibodies was observed in most of the mouse antisera. Although sera and plasma from some patients and monkeys showed notable cross-reactivity with the GPs from multiple filovirus species, the highest reactions of IgG were uniformly detected against the GP antigen homologous to the virus species that infected individuals. We further confirmed that MARV-specific IgM antibodies were specifically detected in specimens collected from patients during the acute phase of infection. These results demonstrate the usefulness of our ELISA for diagnostics as well as ecological and serosurvey studies.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

Antibody-Dependent Enhancement of Marburg Virus Infection

BACKGROUND Marburg virus (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in primates. Earlier studies demonstrated that antibodies to particular epitopes on the glycoprotein (GP) of EBOV enhanced virus infectivity in vitro. METHODS To investigate this antibody-dependent enhancement (ADE) in MARV infection, we produced mouse antisera and monoclonal antibodies (mAbs) to the GPs of MARV strains Angola and Musoke. RESULTS The infectivity of vesicular stomatitis virus pseudotyped with Angola GP in K562 cells was significantly enhanced in the presence of Angola GP antisera, whereas only minimal ADE activity was seen with Musoke GP antisera. This difference correlated with the percentage of hybridoma clones producing infectivity-enhancing mAbs. Using mAbs to MARV GP, we identified 3 distinct ADE epitopes in the mucinlike region on Angola GP. Interestingly, some of these antibodies bound to both Angola and Musoke GPs but showed significantly higher ADE activity for strain Angola. ADE activity depended on epitopes in the mucinlike region and glycine at amino acid position 547, present in the Angola but absent in the Musoke GP. CONCLUSIONS These results suggest a possible link between ADE and MARV pathogenicity and provide new insights into the mechanisms underlying ADE entry of filoviruses.

Different Potential of C-Type Lectin-Mediated Entry between Marburg Virus Strains

ABSTRACT The glycoproteins (GPs) of filoviruses are responsible for virus entry into cells. It is known that GP interacts with cellular C-type lectins for virus attachment to cells. Since primary target cells of filoviruses express C-type lectins, C-type lectin-mediated entry is thought to be a possible determinant of virus tropism and pathogenesis. We compared the efficiency of C-type lectin-mediated entry between Marburg virus strains Angola and Musoke by using a vesicular stomatitis virus (VSV) pseudotype system. VSV pseudotyped with Angola GP (VSV-Angola) infected K562 cells expressing the C-type lectin, human macrophage galactose-type C-type lectin (hMGL), or dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) more efficiently than VSV pseudotyped with Musoke GP (VSV-Musoke). Unexpectedly, the binding affinity of the C-type lectins to the carbohydrates on GPs did not correlate with the different efficiency of C-type lectin-mediated entry. Site-directed mutagenesis identified the amino acid at position 547, which switched the efficiency of C-type lectin-mediated entry. In a three-dimensional model of GP, this amino acid was in close proximity to the putative site of cathepsin processing. Interestingly, the cathepsin inhibitors reduced the infectivity of VSV-Angola less efficiently than that of VSV-Musoke in C-type lectin-expressing K562 cells, whereas only a limited difference was found in control cells. The amino acid at position 547 was critical for the different effects of the inhibitors on the virus infectivities. These results suggest that the efficiency of C-type lectin-mediated entry of filoviruses is controlled not only by binding affinity between C-type lectins and GP but also by mechanisms underlying endosomal entry, such as proteolytic processing by the cathepsins.

Inhibition of Marburg Virus Budding by Nonneutralizing Antibodies to the Envelope Glycoprotein

ABSTRACT The envelope glycoprotein (GP) of Marburg virus (MARV) and Ebola virus (EBOV) is responsible for virus entry into host cells and is known as the only target of neutralizing antibodies. While knowledge about EBOV-neutralizing antibodies and the mechanism for the neutralization of infectivity is being accumulated gradually, little is known about antibodies that can efficiently regulate MARV infectivity. Here we show that MARV GP-specific monoclonal antibodies AGP127-8 (IgG1) and MGP72-17 (IgM), which do not inhibit the GP-mediated entry of MARV into host cells, drastically reduced the budding and release of progeny viruses from infected cells. These antibodies similarly inhibited the formation of virus-like particles (VLPs) consisting of GP, the viral matrix protein, and nucleoprotein, whereas the Fab fragment of AGP127-8 showed no inhibitory effect. Morphological analyses revealed that filamentous VLPs were bunched on the surface of VLP-producing cells cultured in the presence of the antibodies. These results demonstrate a novel mechanism of the antibody-mediated inhibition of MARV budding, in which antibodies arrest unformed virus particles on the cell surface. Our data lead to the idea that such antibodies, like classical neutralizing antibodies, contribute to protective immunity against MARV and that the “classical” neutralizing activity is not the only indicator of a protective antibody that may be available for prophylactic and therapeutic use.

C-type lectins do not act as functional receptors for filovirus entry into cells

Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

Serological Evidence of Ebola Virus Infection in Indonesian Orangutans

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae and cause severe hemorrhagic fever in humans and nonhuman primates. Despite the discovery of EBOV (Reston virus) in nonhuman primates and domestic pigs in the Philippines and the serological evidence for its infection of humans and fruit bats, information on the reservoirs and potential amplifying hosts for filoviruses in Asia is lacking. In this study, serum samples collected from 353 healthy Bornean orangutans (Pongo pygmaeus) in Kalimantan Island, Indonesia, during the period from December 2005 to December 2006 were screened for filovirus-specific IgG antibodies using a highly sensitive enzyme-linked immunosorbent assay (ELISA) with recombinant viral surface glycoprotein (GP) antigens derived from multiple species of filoviruses (5 EBOV and 1 MARV species). Here we show that 18.4% (65/353) and 1.7% (6/353) of the samples were seropositive for EBOV and MARV, respectively, with little cross-reactivity among EBOV and MARV antigens. In these positive samples, IgG antibodies to viral internal proteins were also detected by immunoblotting. Interestingly, while the specificity for Reston virus, which has been recognized as an Asian filovirus, was the highest in only 1.4% (5/353) of the serum samples, the majority of EBOV-positive sera showed specificity to Zaire, Sudan, Cote d’Ivoire, or Bundibugyo viruses, all of which have been found so far only in Africa. These results suggest the existence of multiple species of filoviruses or unknown filovirus-related viruses in Indonesia, some of which are serologically similar to African EBOVs, and transmission of the viruses from yet unidentified reservoir hosts into the orangutan populations. Our findings point to the need for risk assessment and continued surveillance of filovirus infection of human and nonhuman primates, as well as wild and domestic animals, in Asia.