ABSTRACT The Middle East respiratory syndrome coronavirus (MERS-CoV) recently spread from an animal reservoir to infect humans, causing sporadic severe and frequently fatal respiratory disease. Appropriate public health and control measures will require discovery of the zoonotic MERS coronavirus reservoirs. The relevant animal hosts are liable to be those that offer optimal MERS virus cell entry. Cell entry begins with virus spike (S) protein binding to DPP4 receptors. We constructed chimeric DPP4 receptors that have the virus-binding domains of indigenous Middle Eastern animals and assessed the activities of these receptors in supporting S protein binding and virus entry. Human, camel, and horse receptors were potent and nearly equally effective MERS virus receptors, while goat and bat receptors were considerably less effective. These patterns reflected S protein affinities for the receptors. However, even the low-affinity receptors could hypersensitize cells to infection when an S-cleaving protease(s) was present, indicating that affinity thresholds for virus entry must be considered in the context of host-cell proteolytic environments. These findings suggest that virus receptors and S protein-cleaving proteases combine in a variety of animals to offer efficient virus entry and that several Middle Eastern animals are potential reservoirs for transmitting MERS-CoV to humans. IMPORTANCE MERS is a frequently fatal disease that is caused by a zoonotic CoV. The animals transmitting MERS-CoV to humans are not yet known. Infection by MERS-CoV requires receptors and proteases on host cells. We compared the receptors of humans and Middle Eastern animals and found that human, camel, and horse receptors sensitized cells to MERS-CoV infection more robustly than goat and bat receptors. Infection susceptibility correlated with affinities of the receptors for viral spike proteins. We also found that the presence of a cell surface lung protease greatly increases susceptibility to MERS-CoV, particularly in conjunction with low-affinity receptors. This cataloguing of human and animal host cell factors allows one to make inferences on the distribution of MERS-CoV in nature.
ABSTRACT Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.
Complex interactions between effector T cells and Foxp3+ regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4+Foxp3− T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.
The highly pathogenic coronaviruses SARS-CoV-2 and SARS-CoV have led to the COVID-19 pandemic and SARS outbreak, respectively. The receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2, particularly the Omicron variant, has frequent mutations, resulting in the reduced efficiency of current COVID-19 vaccines against new variants. Here, we designed two lipid nanoparticle-encapsulated mRNA vaccines by deleting the mutant RBD of the SARS-CoV-2 Omicron variant (SARS2-S (RBD-del)) or by replacing this mutant RBD with the conserved and potent RBD of SARS-CoV (SARS2-S (SARS-RBD)). Both mRNA vaccines were stable at various temperatures for different time periods. Unlike SARS2-S (RBD-del) mRNA, SARS2-S (SARS-RBD) mRNA elicited effective T-cell responses and potent antibodies specific to both SARS-CoV-2 S and SARS-CoV RBD proteins. It induced strong neutralizing antibodies against pseudotyped SARS-CoV-2 and SARS-CoV infections and protected immunized mice from the challenge of the SARS-CoV-2 Omicron variant and SARS-CoV by significantly reducing the viral titers in the lungs after Omicron challenge and by completely preventing SARS-CoV-induced weight loss and death. SARS2-S (SARS-RBD)-immunized serum antibodies protected naïve mice from the SARS-CoV challenge, with its protective efficacy positively correlating with the neutralizing antibody titers. These findings indicate that this mRNA vaccine has the potential for development as an effective vaccine against current and future SARS-CoV-2 variants and SARS-CoV.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.
Three novel zoonotic coronaviruses that have jumped the species barrier and caused lethal disease in humans have focused global public health attention in the past two decades: severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012,1Memish ZA Perlman S Van Kerkhove MD Zumla A Middle East respiratory syndrome.Lancet. 2020; 395: 1063-1077Summary Full Text Full Text PDF PubMed Scopus (301) Google Scholar and SARS-CoV-2 in late December 20192—the cause of the ongoing COVID-19 pandemic.2Perlman S Another decade, another coronavirus.N Engl J Med. 2020; 382: 760-762Crossref PubMed Scopus (600) Google Scholar All these coronaviruses are featured in the WHO Blueprint list of priority pathogens for research and development, because they threaten global health security, they have epidemic potential, and there are no effective treatments or vaccines.3WHOPrioritizing diseases for research and development in emergency contexts. World Health Organization, 2020https://www.who.int/activities/prioritizing-diseases-for-research-and-development-in-emergency-contextsDate accessed: June 27, 2020Google Scholar In Africa, only SARS-CoV-2 has caused significant outbreaks and disruption to health services. While African governments implement national mitigation and containment strategies4Nachega JB Grimwood A Mahomed H et al.From easing lockdowns to scaling-up community-based COVID-19 screening, testing, and contact tracing in Africa—shared approaches, innovations, and challenges to minimize morbidity and mortality.Clin Infect Dis. 2020; (published online May 31.)https://doi.org/10.1093/cid/ciaa695Crossref Scopus (48) Google Scholar to control the spread of COVID-19, MERS-CoV continues to circulate in the Middle East causing intermittent outbreaks with up to 34% mortality.1Memish ZA Perlman S Van Kerkhove MD Zumla A Middle East respiratory syndrome.Lancet. 2020; 395: 1063-1077Summary Full Text Full Text PDF PubMed Scopus (301) Google Scholar MERS-CoV is highly prevalent in dromedary camels on the Arabian peninsula and, they are the main sources of primary human MERS-CoV infections.5El-Kafrawy SA Corman VM Tolah AM et al.Enzootic patterns of Middle East respiratory syndrome coronavirus in imported African and local Arabian dromedary camels: a prospective genomic study.Lancet Planet Health. 2019; 3: e521-e528Summary Full Text Full Text PDF PubMed Scopus (45) Google Scholar However, there have not been any reported clinical cases from Africa of confirmed human MERS, despite 70% of the world's dromedaries residing in Africa (with high MERS-CoV infection rates) and frequent occupational and domestic contact between dromedaries and humans.6Farag E Sikkema RS Mohamedani AA et al.MERS-CoV in camels but not camel handlers, Sudan, 2015 and 2017.Emerg Infect Dis. 2019; 25: 2333-2335Crossref PubMed Scopus (21) Google Scholar Little serological evidence of human infection exists.7Ka Pun Mok C Zhu A Zhao J et al.T-cell responses to MERS coronavirus infection in people with occupational exposure to dromedary camels in Nigeria: an observational cohort study.Lancet Infect Dis. 2020; (published online Oct 6.)https://doi.org/10.1016/S1473-3099(20)30599-5Google Scholar, 8Zhao J Alshukairi AN Baharoon SA et al.Recovery from the Middle East respiratory syndrome is associated with antibody and T-cell responses.Sci Immunol. 2017; 2aan5393Crossref Scopus (211) Google Scholar This anomalous disparity between human MERS-CoV infections in the Arabian peninsula and Africa has not been easily explained and there has been a longstanding need for further studies of MERS-CoV at the animal–human interface. In The Lancet Infectious Diseases, Chris Ka Pub Mok and colleagues7Ka Pun Mok C Zhu A Zhao J et al.T-cell responses to MERS coronavirus infection in people with occupational exposure to dromedary camels in Nigeria: an observational cohort study.Lancet Infect Dis. 2020; (published online Oct 6.)https://doi.org/10.1016/S1473-3099(20)30599-5Google Scholar present data that indicate that human MERS-CoV infection is an occupational hazard in dromedary abattoir workers in Nigeria. They focus our attention on the potential for MERS-CoV transmission and human infection and consequent threat to people living in Africa and to public health services. Their observational cohort study of people working at a slaughterhouse in Kano, Nigeria, identified MERS-CoV-specific CD4+ or CD8+ T cells in PBMCs obtained from 61 workers with close contact to dromedaries, but not in 20 other workers from the same slaughterhouse with no dromedary contact. These results agree with others showing that MERS-CoV virus-specific T-cell responses are more sensitive than serological tests for detecting past infection.8Zhao J Alshukairi AN Baharoon SA et al.Recovery from the Middle East respiratory syndrome is associated with antibody and T-cell responses.Sci Immunol. 2017; 2aan5393Crossref Scopus (211) Google Scholar Thus, zoonotic MERS-CoV infections of dromedary-exposed individuals are probably taking place in Nigeria, and, by extrapolation, the incidence of human MERS infections in all regions of Africa with dromedaries has probably been underestimated. Further studies are required to determine the extent of human MERS-CoV infections across Africa. Genetic and phenotypic differences in west African viruses might also be relevant to zoonotic outbreak potential. The great diversity of MERS-CoV lineages, and the large number of MERS-CoV infected dromedaries in Africa,5El-Kafrawy SA Corman VM Tolah AM et al.Enzootic patterns of Middle East respiratory syndrome coronavirus in imported African and local Arabian dromedary camels: a prospective genomic study.Lancet Planet Health. 2019; 3: e521-e528Summary Full Text Full Text PDF PubMed Scopus (45) Google Scholar might, with time, lead to MERS-CoV adapting and transmitting more efficiently, with epidemic potential. The findings of Ka Pub Mok and colleagues7Ka Pun Mok C Zhu A Zhao J et al.T-cell responses to MERS coronavirus infection in people with occupational exposure to dromedary camels in Nigeria: an observational cohort study.Lancet Infect Dis. 2020; (published online Oct 6.)https://doi.org/10.1016/S1473-3099(20)30599-5Google Scholar also have implications for ongoing efforts on COVID-19 diagnostics development, testing, and tracing in Africa and for vaccine development in general.4Nachega JB Grimwood A Mahomed H et al.From easing lockdowns to scaling-up community-based COVID-19 screening, testing, and contact tracing in Africa—shared approaches, innovations, and challenges to minimize morbidity and mortality.Clin Infect Dis. 2020; (published online May 31.)https://doi.org/10.1093/cid/ciaa695Crossref Scopus (48) Google Scholar Mild cases of MERS and COVID-19 induce antibody responses that are transient or wane rapidly.8Zhao J Alshukairi AN Baharoon SA et al.Recovery from the Middle East respiratory syndrome is associated with antibody and T-cell responses.Sci Immunol. 2017; 2aan5393Crossref Scopus (211) Google Scholar Specific diagnostic tests for MERS-CoV and SARS-CoV-2 infected individuals based on relevant antigen-specific CD4+ or CD8+ T-cell responses could allow for more sensitive detection of past or present infection than serological tests and identify additional human MERS and COVID-19 cases. T-cell responses appear to be more stable and useful for detection of previous infection or vaccine response in settings where virus-specific antibody titres are absent. It would also be interesting to know if dromedary-exposed workers with MERS-CoV-specific T-cell responses are protected from developing severe MERS on rechallenge, and by extrapolation, whether T-cell responses are protective against severe COVID-19 disease, even if virus-specific antibody is not detectable. To confirm these results, these studies should be repeated at other sites in Africa and with larger numbers of samples. Furthermore, no MERS-specific T-cell responses were detected in abattoir workers with no dromedary exposure. By contrast, low numbers of SARS-CoV-2-specific T cells have been detected in uninfected individuals.9Grifoni A Weiskopf D Ramirez SI et al.Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals.Cell. 2020; 181: 1489-1501Summary Full Text Full Text PDF PubMed Scopus (2526) Google Scholar These apparent differences in pre-existing immunity to the two coronaviruses are unexpected and need to be reconciled. The results also suggest that COVID-19 and MERS vaccines should be formulated to induce T-cell responses to maximise the likelihood of long-term protection. COVID-19 and MERS provide unique opportunities for African and Middle Eastern countries with relevant stakeholders to align and synergise ongoing COVID-19 and MERS surveillance, diagnostics, vaccine development, and evaluation, with basic science and translational research activities.1Memish ZA Perlman S Van Kerkhove MD Zumla A Middle East respiratory syndrome.Lancet. 2020; 395: 1063-1077Summary Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 10Perlman S Azhar EI Memish ZA Hui DS Zumla A Confronting the persisting threat of the Middle East respiratory syndrome to global health security.Lancet Infect Dis. 2020; 20: 158-160Summary Full Text Full Text PDF PubMed Scopus (6) Google Scholar African and Middle Eastern countries must invest more in surveillance and urgent priority research to fill major knowledge gaps in the epidemiology, transmission, pathogenesis, and evolution of MERS-CoV and SARS-CoV-2, especially because they are co-circulating. The need for an effective One Human-Environmental-Animal Health multidisciplinary consortium across Africa, Middle East, and Asia to tackle the ever-present threat of lethal coronaviruses and other emerging infections remains a global priority.10Perlman S Azhar EI Memish ZA Hui DS Zumla A Confronting the persisting threat of the Middle East respiratory syndrome to global health security.Lancet Infect Dis. 2020; 20: 158-160Summary Full Text Full Text PDF PubMed Scopus (6) Google Scholar We have a special interest in coronaviruses and infectious diseases with epidemic potential. We declare no competing interests. T-cell responses to MERS coronavirus infection in people with occupational exposure to dromedary camels in Nigeria: an observational cohort studyZoonotic infection of dromedary-exposed individuals is taking place in Nigeria and suggests that the extent of MERS-CoV infections in Africa is underestimated. MERS-CoV could therefore adapt to human transmission in Africa rather than the Arabian Peninsula, where attention is currently focused. Full-Text PDF
Trabajo presentado en la Conference on the Cooperation and Collaboration on Prevention and Control of Animal Diseases, celebrada en Hangzhou (China), del 21 al 23 de mayo de 2019
Abstract Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag ( Nanosota-1C-Fc ) bound to SARS-CoV-2 RBD with a K d of 15.7picomolar (∼3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND 50 of 0.16microgram/milliliter (∼6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-F c documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic. Impact statement Potent and low-cost Nanosota-1 drugs block SARS-CoV-2 infections both in vitro and in vivo and act both preventively and therapeutically.
Background: Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 RNA can be detected by real-time reverse-transcription polymerase chain reaction (RT-PCR) for several weeks after infection. Discerning persistent RT-PCR positivity versus reinfection is challenging and the frequency of COVID-19 reinfections is unknown. We aimed to determine the frequency of clinically suspected reinfection in our center and confirm reinfection using viral whole-genome sequencing (WGS). Methods: The University of Iowa Hospitals and Clinics (UIHC) is an 811-bed academic medical center. Patients with respiratory complaints undergo COVID-19 RT-PCR using nasopharyngeal swabs. The RT-PCR (TaqPath COVID-19 Combo kit) uses 3 targets (ORF1ab, S gene, and N gene). We identified patients with previous laboratory-confirmed COVID-19 who sought care for new respiratory complaints and underwent a repeated SARS-CoV-2 test at least 45 days from their first positive test. We then identified patients with median RT-PCR cycle threshold (Ct) values. Results: During the study period, 13,603 patients had a SARS-CoV-2– positive RT-PCR. Of these, 296 (2.2%) had a clinical visit for new onset of symptoms and a repeated RT-PCR assay >45 days from the first test. Moreover, 29 patients (9.8%) had a positive RT-PCR assay in the repeated testing. Ct values were available for samples from 25 patients; 7 (28%) had Ct values. Conclusions: In patients with a recent history of COVID-19 infection, repeated testing for respiratory symptoms was infrequent. Some had a SARS-CoV-2–positive RT-PCR assay on repeated testing, but only 1 in 4 had Ct values suggestive of a reinfection. We confirmed 1 case of reinfection using WGS. Funding: No Disclosures: None
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