Middle East respiratory syndrome coronavirus (MERS-CoV) uses the S1B domain of its spike protein to bind to dipeptidyl peptidase 4 (DPP4), its functional receptor, and its S1A domain to bind to sialic acids. The tissue localization of DPP4 in humans, bats, camelids, pigs, and rabbits generally correlates with MERS-CoV tropism, highlighting the role of DPP4 in virus pathogenesis and transmission. However, MERS-CoV S1A does not indiscriminately bind to all α2,3-sialic acids, and the species-specific binding and tissue distribution of these sialic acids in different MERS-CoV-susceptible species have not been investigated. We established a novel method to detect these sialic acids on tissue sections of various organs of different susceptible species by using nanoparticles displaying multivalent MERS-CoV S1A. We found that the nanoparticles specifically bound to the nasal epithelial cells of dromedary camels, type II pneumocytes in human lungs, and the intestinal epithelial cells of common pipistrelle bats. Desialylation by neuraminidase abolished nanoparticle binding and significantly reduced MERS-CoV infection in primary susceptible cells. In contrast, S1A nanoparticles did not bind to the intestinal epithelium of serotine bats and frugivorous bat species, nor did they bind to the nasal epithelium of pigs and rabbits. Both pigs and rabbits have been shown to shed less infectious virus than dromedary camels and do not transmit the virus via either contact or airborne routes. Our results depict species-specific colocalization of MERS-CoV entry and attachment receptors, which may be relevant in the transmission and pathogenesis of MERS-CoV.
IMPORTANCE MERS-CoV uses the S1B domain of its spike protein to attach to its host receptor, dipeptidyl peptidase 4 (DPP4). The tissue localization of DPP4 has been mapped in different susceptible species. On the other hand, the S1A domain, the N-terminal domain of this spike protein, preferentially binds to several glycotopes of α2,3-sialic acids, the attachment factor of MERS-CoV. Here we show, using a novel method, that the S1A domain specifically binds to the nasal epithelium of dromedary camels, alveolar epithelium of humans, and intestinal epithelium of common pipistrelle bats. In contrast, it does not bind to the nasal epithelium of pigs or rabbits, nor does it bind to the intestinal epithelium of serotine bats and frugivorous bat species. This finding supports the importance of the S1A domain in MERS-CoV infection and tropism, suggests its role in transmission, and highlights its potential use as a component of novel vaccine candidates.
Copyright © 2019 American Society for Microbiology.
Antimicrobial resistance (AMR) is currently the most alarming issue for human health. AMR already causes 700,000 deaths/year. It is estimated that 10 million deaths due to AMR will occur every year after 2050. This equals the number of people dying of cancer every year in present times. International institutions such as G20, World Bank, World Health Organization (WHO), UN General Assembly, European Union, and the UK and USA governments are calling for new antibiotics. To underline this emergency, a list of antibiotic-resistant “priority pathogens” has been published by WHO. It contains 12 families of bacteria that represent the greatest danger for human health. Resistance to multiple antibiotics is particularly relevant for the Gram-negative bacteria present in the list. The ability of these bacteria to develop mechanisms to resist treatment could be transmitted with genetic material, allowing other bacteria to become drug resistant. Although the search for new antimicrobial drugs remains a top priority, the pipeline for new antibiotics is not promising, and alternative solutions are needed. A possible answer to AMR is vaccination. In fact, while antibiotic resistance emerges rapidly, vaccines can lead to a much longer lasting control of infections. New technologies, such as the high-throughput cloning of human B cells from convalescent or vaccinated people, allow for finding new protective antigens (Ags) that could not be identified with conventional technologies. Antibodies produced by convalescent B cell clones can be screened for their ability to bind, block, and kill bacteria, using novel high-throughput microscopy platforms that rapidly capture digital images, or by conventional technologies such as bactericidal, opsono-phagocytosis and FACS assays. Selected antibodies expressed by recombinant DNA techniques can be used for passive immunization in animal models and tested for protection. Antibodies providing the best protection can be employed to identify new Ags and then used for generating highly specific recombinant Fab fragments. Co-crystallization of Ags bound to Fab fragments will allow us to determine the structure and characteristics of new Ags. This structure-based Ag design will bring to a new generation of vaccines able to target previously elusive infections, thereby offering an effective solution to the problem of AMR.
Infectious disease outbreaks recapitulate biology: they emerge from the multi-level interaction of hosts, pathogens, and environment. Therefore, outbreak forecasting requires an integrative approach to modeling. While specific components of outbreaks are predictable, it remains unclear whether fundamental limits to outbreak prediction exist. Here, adopting permutation entropy as a model independent measure of predictability, we study the predictability of a diverse collection of outbreaks and identify a fundamental entropy barrier for disease time series forecasting. However, this barrier is often beyond the time scale of single outbreaks, implying prediction is likely to succeed. We show that forecast horizons vary by disease and that both shifting model structures and social network heterogeneity are likely mechanisms for differences in predictability. Our results highlight the importance of embracing dynamic modeling approaches, suggest challenges for performing model selection across long time series, and may relate more broadly to the predictability of complex adaptive systems.
The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.
Zoonoses are infectious diseases that can be transmitted to humans from animals (and vice versa). IMI's ZAPI project is working to create new platforms and technologies that will facilitate a fast, coordinated, and practical response to new infectious diseases as soon as they emerge. In the run-up to World Immunisation Week 2019, the IMI Programme Office caught up with ZAPI project coordinator Jean-Christophe Audonnet for an update on the project's progress so far.
Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV–exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries—apart from conventional antibodies—have relatively unique, heavy chain–only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV–infected dromedaries, we identified several MERS-CoV–specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs—composed of the camel VHH linked to a human Fc domain lacking the CH1 exon—had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.
The availability of vaccines in response to newly emerging infections is impeded by the length of time it takes to design, manufacture, and evaluate vaccines for clinical use. Historically, the process of vaccine development through to licensure requires decades; however, clinicians and public health officials are often faced with outbreaks of viral diseases, sometimes of a pandemic nature that would require vaccines for adequate control. New viral diseases emerge from zoonotic and vectorborne sources, such as Middle East Respiratory Syndrome coronavirus and Chikungunya, and while these diseases are often detected in resource-rich countries, they usually begin in low- and mid-income countries.1 Therefore, part of the timeline for a vaccine involves surveillance and detection of new pathogens in remote areas and transfer of specimens to laboratories capable of vaccine development.
Rift Valley fever virus (RVFV) is a member of the family Bunyaviridae and can lead to severe diseases in humans and livestock. Although most human infections proceed as mild flu-like illness, severe manifestations as retinitis, meningoencephalitis or even hemorrhagic fever syndromes due to fulminant hepatitis do occur in about 1-2% of the cases. Infections of adult ruminants and camels rarely lead to manifest and lethal hepatitis, but are rather observed as febrile diseases. However, so called ‘abortion storms’ are characteristic for RVFV infections of pregnant ruminants, leading to an up to 100% mortality rate in new borne animals. While human infections are mostly caused by contact to viremic animals, the transmission through RVFV-infected mosquitoes is of major importance for livestock and wildlife. To date RVFV was found in more than 30 mosquito species. Currently RVFV is widely endemic in Africa, recurrently causing substantial outbreaks. Significant losses in human and animal populations highlight the major impact of the pathogen for both healthcare and animal husbandry. For mitigation and monitoring of these impacts, knowledge of the specific infection ecology is of particular importance. To address these issues, a cross-regional serological and molecular screening of livestock sera in Cameroon and Mauritania was implemented. The findings in Cameroon demonstrated considerable inter-species differences, reflected by a significantly higher seroprevalence of cattle compared to small ruminants. Additionally, striking regional variabilities of seropositivity were observed, implicating a decline from north to south Cameroon. Apart from general seroconversion, acute infections were detected for the first time in three cattle and one small ruminant, harboring RVFV-specific IgM antibodies. Moreover, virus derived RNA was detected in one IgM positive cattle, indicating the existence of low-level circulation of RVFV. By providing first evidence of acute infections, both the existence of an ongoing enzootic cycle and the potential for severe outbreaks in future was demonstrated. Although recurrent RVFV outbreaks in Mauritania led to massive losses in the past, serological and molecular investigations during inter-epidemic periods are absent to date. Therefore, samples of small ruminants, cattle and camels that were collected during inter-epidemic periods from 2012-2013, were analyzed. Comparative analyses demonstrated a significant difference in small ruminants, showing a strong decline of seroprevalence during inter-epidemic periods. In contrast, the rate of seropositivity in camels and cattle was almost identical to those detected during epidemics. Obtained data do therefore clarify the significant role of small ruminants as important sentinels for RVFV, as a remarkable increase of seroconversion will indicate a possible introduction of RVFV into the herds. Furthermore the evidence of an IgM positive cattle harboring viral RNA illustrated the presence of an enzootic cycle. Camelids play a yet neglected but pivotal role in transmission and spread of RVFV and associations with human infections highlight the eminent need for effective vaccines for this species. For this purpose alpacas were chosen as model organisms for camelids and were immunized with the live attenuated MP-12 vaccine, evaluating its safety, immunogenicity and pathogenicity. The application of MP-12 proved to be safe as no shedding of vaccine virus was recorded and no persisting alterations in hematology and clinical chemistry were observed. Additionally the vaccine was highly immunogenic, as stable neutralizing antibody titers were generated by a single application. A detailed investigation of antigen-specific reactivity demonstrated a significant generation of antibodies directed against NSs, NP and Gn proteins. A minimal residual pathogenicity was demonstrated in alpacas 3 dpi as a replicative potential was verified in serum and liver. In addition pathological examinations revealed a mild, multifocal, acute necrotizing hepatitis with antigenic presence of NP, Gn, Gc and NSm. In contrast, hepatic lesions 31 dpi displayed a lymphohistiocytic character, indicating the efficient immunological clearance and absence of sequelae. Furthermore, next generation sequencing of recovered MP-12 confirmed the genetic stability of the vaccine. Therefore MP-12 is a safe and immunogenic vaccine for camelids, yet with considerable residual pathogenicity. In summary, the here presented results elucidate characteristics of the RVFV infection ecology in Cameroon, present comparative analyses during inter-epidemic periods in Mauritania and evaluate the suitability of the RVFV vaccine MP-12 for camelids. The obtained data can be used for awareness raising and risk assessment of Rift Valley fever as well as for the development of prevention strategies.
Reverse genetics is a critical tool to decrypt the biological properties of arboviruses. However, whilst reverse genetics methods have been usually applied to vertebrate cells, their use in insect cells remains uncommon due to the conjunction of laborious molecular biology techniques and of specific difficulties surrounding the transfection of such cells. To leverage reverse genetics studies in both vertebrate and mosquito cells, we designed an improved DNA transfection protocol for insect cells and then demonstrated that the simple and flexible ISA (Infectious Subgenomic Amplicons) reverse-genetics method can be efficiently applied to both mammalian and mosquito cells to generate in days recombinant infectious positive-stranded RNA viruses belonging to genera Flavivirus (Japanese encephalitis, Yellow fever, West Nile and Zika viruses) and Alphavirus (Chikungunya virus). This method represents an effective option to potentially overcome technological issues related to the study of arboviruses.
Middle East respiratory syndrome coronavirus (MERS-CoV) still causes outbreaks despite public awareness and implementation of health care measures, such as rapid viral diagnosis and patient quarantine. Here we describe the current epidemiological picture of MERS-CoV, focusing on humans and animals affected by this virus and propose specific intervention strategies that would be appropriate to control MERS-CoV. One-third of MERS-CoV patients develop severe lower respiratory tract infection and succumb to a fatal outcome; these patients would require effective therapeutic antiviral therapy. Because of the lack of such intervention strategies, supportive care is the best that can be offered at the moment. Limiting viral spread from symptomatic human cases to health care workers and family members, on the other hand, could be achieved through prophylactic administration of MERS-CoV neutralizing antibodies and vaccines. To ultimately prevent spread of the virus into the human population, however, vaccination of dromedary camels – currently the only confirmed animal host for MERS-CoV – may be the best option to achieve a sustained drop in human MERS cases in time. In the end, a One Health approach combining all these different efforts is needed to tackle this zoonotic outbreak.