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.
The pandemic potential of zoonotic pathogens lies in their ability to become efficiently transmissible amongst humans. Here, we focus on contact-transmitted pathogens and discuss the factors, at the pathogen, host and environmental levels that promote or hinder their human-to-human transmissibility via the following modes of contact transmission: skin contact, sexual contact, respiratory contact and multiple route contact. Factors common to several modes of transmission were immune evasion, high viral load, low infectious dose, crowding, promiscuity, and co-infections; other factors were specific for a pathogen or mode of contact transmission. The identification of such factors will lead to a better understanding of the requirements for human-to-human spread of pathogens, as well as improving risk assessment of newly emerging pathogens.
Rift Valley fever (RVF) is an emerging zoonosis of major public health concern in Africa and Arabia. Previous outbreaks attributed camelids a significant role in the epidemiology of Rift Valley fever virus (RVFV), making them an important target species for vaccination. Using three alpacas as model-organisms for dromedary camels, the safety, immunogenicity and pathogenicity of the MP-12 vaccine were evaluated inthis study. To compare both acute and subacute effects, animals were euthanized at 3 and 31days post infection (dpi). Clinical monitoring, analysis of liver enzymes and hematological parameters demonstrated the tolerability of the vaccine, as no significant adverse effects were observed. Comprehensive analysis of serological parameters illustrated the immunogenicity of the vaccine, eliciting high neutralizing antibody titers and antibodies targeting different viral antigens. RVFV was detected in serum and liver of the alpaca euthanized 3dpi, whereas no viruswas detectable at 31dpi. Viral replication was confirmed by detection of various RVFV-antigens in hepatocytes by immunohistochemistry and the presence of mild multifocal necrotizing hepatitis. In conclusion, results indicate that MP-12 is a promising vaccine candidate but still has a residual pathogenicity, which requires further investigation.
Antibodies specific for Rift Valley fever virus (RVFV) can be detected by diverse methods, including ezyme-linked immunosortbent assay (ELISA) and virus neutralization test (VNT). The VNT is superior in sensitivity and specificity and is therefore considered the gold standard serological assay. Classical VNTs make use of virulent RVFV and therefore have to be performed in biosafety level 3 laboratories. Here, we report the development of a novel VNT that is based on an avirulent RVFV expressing the enhanced green fluorescent protein (eGFP), which can be performed safely outside level 3 biocontainment facilities. Evaluation with a broad panel of experimental sera and field sera demonstrated that this novel VNT is faster and more sensitive than the classical VNT.
Orthobunyaviruses are enveloped viruses that can cause human and animal diseases. A novel and major member is the Schmallenberg virus(SBV), the etiological agent of an emerging disease of ruminants that has been spreading all over Europe since 2011. The glycoproteins Gn and Gc of orthobunyaviruses mediate the viral entry, and specifically Gc is a major target for the humoral immune response. For example, the N terminal subdomain of the SBV glycoprotein Gc is targeted by neutralizing monoclonal antibodies that recognize conformational epitopes. Here, we determined the structural features of the N terminus of Gc, and analysed its interaction with monoclonal antibodies. We were able to demonstrate that one of two N-glycosylation sites is essential for secretion and interaction with a subset of Gc-specific monoclonal antibodies. Furthermore, four disulfide bonds (S-S) were identified and the deletion of the third S-S blocked reactivity with another subset of mAbs with virus-neutralizing and non-neutralizing activity. The mutagenesis of the N-glycosylation sites and the disulfide bonds strongly indicated the independent folding of two subdomains within the SBV Gc N terminus. Further, the epitopes recognized by a panel of mAbs could be grouped into two clusters, as revealed by fine mapping using chimeric proteins. Combining the disulfide bonding and epitope mapping allowed us to generate a structural model of the SBV Gc N-terminus. This novel information about the role and structure of the amino terminal region of SBV Gc is of general relevance for the design of antivirals and vaccines against this virus.
Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.