In addition to the transmembrane protein, GP₁,₂, the Ebola virus glycoprotein gene encodes the soluble glycoproteins sGP and Δ-peptide. Two more soluble proteins, GP₁ and GP₁,₂ΔTM, are generated from ...GP₁,₂ as a result of disulfide-bond instability and proteolytic cleavage, respectively, and are shed from the surface of infected cells. The sGP glycoprotein is secreted as a disulfide-linked homodimer, but there have been conflicting reports on whether it is arranged in a parallel or antiparallel orientation. Off-line HPLC-MALDI-TOF MS (MS/MS) was used to identify the arrangement of all disulfide bonds and simultaneously determine site-specific information regarding N-glycosylation. Our data prove that sGP is a parallel homodimer that contains C53-C53' and C306-C306' disulfide bonds, and although there are six predicted N-linked carbohydrate sites, only five are consistently glycosylated. The disulfide bond arrangement was confirmed by using cysteine to glycine mutations at amino acid positions 53 and 306. The mutants had a reduced ability to rescue the barrier function of TNF-α-treated endothelial cells--a function previously reported for sGP. This indicates that these disulfide bonds are critical for the proposed anti-inflammatory function of sGP.
The identification of a novel β coronavirus, nCoV, as the causative agent of severe respiratory illness in humans originating in Saudi Arabia, Qatar and Jordan has raised concerns about the ...possibility of a coronavirus pandemic similar to that of SARS-CoV. As a definitive treatment regimen has never been thoroughly evaluated for coronavirus infections, there is an urgent need to rapidly identify potential therapeutics to address future cases of nCoV. To determine an intervention strategy, the effect of interferon-α2b and ribavirin on nCoV isolate hCoV-EMC/2012 replication in Vero and LLC-MK2 cells was evaluated. hCoV-EMC/2012 was sensitive to both interferon-α2b and ribavirin alone in Vero and LLC-MK2 cells, but only at relatively high concentrations; however, when combined, lower concentrations of interferon-α2b and ribavirin achieved comparable endpoints. Thus, a combination of interferon-α2b and ribavirin, which are already commonly used in the clinic, may be useful for patient management in the event of future nCoV infections.
•Camelids have heavy chain only antibodies. The VHH region of heavy chain only antibodies can be used as single domain antibody called a nanobody.•Dromedary camels use somatic hypermutation to ...increase T cell receptor diversity in ꝩδ T cells.•Alpacas have a subset of ꝩδ T cells, called Vꝩ9Vδ2 T cells, that recognize phophoantigens. Previously, these were only identified in primates.
Members of the Camelidae have unique adaptive immunological features that are not widely observed in other species. All camelids are known to have three distinct IgG isotypes – IgG1, IgG2 and IgG3. While IgG1 has a conventional antibody structure, both IgG2 and IgG3 are devoid of light chains and instead possess hypervariable regions in their heavy chain (VHH), while lacking the typical CH1 domain found in heavy chains. VHH domains are increasingly being utilized as “next generation” antibodies, as they have unique biochemical and structural properties including high pH stability as well as a lower molecular weight allowing for increased tissue penetration. These features of VHH domains offer a number of advantages for both biotechnology and clinical applications and are commonly termed “nanobodies”. A second unique aspect of the camelid adaptive response is involves T cell-mediated immunity. Characterization of gamma delta (ꝩδ) T cells in camelid species has found they use somatic hypermutation in their T cell receptor gamma (TRG) and delta (TRD) loci to increase the structural stability of their ꝩδ T receptor. The use of somatic hyper mutation to increase the diversity of their T cell repertoire, is a feature that has not been observed in other mammalian species. In addition, in alpacas there is a unique subset of ꝩδ T cells called Vꝩ9Vδ2 T cells. Activation of these cells is dependent upon phosphoantigen (PAg)–mediated interaction with B7-like butyrophilin molecules (BTN-3). This makes alpacas the first species outside of primates to be identified with this unique subset and activation mechanism. Here we review some fundamentals of camelid adaptive immunity that make them distinct from other vertebrate species and their potential applications to human therapies.
Animal models for COVID-19 Muñoz-Fontela, César; Dowling, William E; Funnell, Simon G P ...
Nature (London),
10/2020, Letnik:
586, Številka:
7830
Journal Article
Recenzirano
Odprti dostop
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel ...coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.
Most ebolaviruses can cause severe disease in humans and other primates, with high case fatality rates during human outbreaks. Although these viruses have been studied for almost 4 decades, little is ...know regarding the mechanisms by which they cause disease and what is important for protection or treatment after infection. Because of the sporadic nature of the outbreaks and difficulties accessing the populations affected by ebolaviruses, little is also known about what constitutes an appropriate immune response to infection in humans that survive infection. Such knowledge would allow a targeted approach to therapies. In contrast to humans, rodents are protected from disease on infection with ebolaviruses, although adapted versions of some of the viruses are lethal in mice, hamsters and guinea pigs. Using the recently described hamster model, along with T-cell depletion strategies, we show that CD4⁺ T cells are required for natural immunity to Ebola virus infection and that CD4-dependent antibody responses are required for immunity in this model.
An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane ...fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using nonspecific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high-throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.
The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) is of global concern: the virus has caused severe respiratory illness, with 111 confirmed cases and 52 deaths at the time of ...this article's publication. Therapeutic interventions have not been evaluated in vivo; thus, patient management relies exclusively on supportive care, which, given the high case-fatality rate, is not highly effective. The rhesus macaque is the only known model organism for MERS-CoV infection, developing an acute localized to widespread pneumonia with transient clinical disease that recapitulates mild to moderate human MERS-CoV cases. The combination of interferon-α2b and ribavirin was effective in reducing MERS-CoV replication in vitro; therefore, we initiated this treatment 8 h after inoculation of rhesus macaques. In contrast to untreated, infected macaques, treated animals did not develop breathing abnormalities and showed no or very mild radiographic evidence of pneumonia. Moreover, treated animals showed lower levels of systemic (serum) and local (lung) proinflammatory markers, in addition to fewer viral genome copies, distinct gene expression and less severe histopathological changes in the lungs. Taken together, these data suggest that treatment of MERS-CoV infected rhesus macaques with IFN-α2b and ribavirin reduces virus replication, moderates the host response and improves clinical outcome. As these two drugs are already used in combination in the clinic for other infections, IFN-α2b and ribavirin should be considered for the management of MERS-CoV cases.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve to give rise to variants of concern that can escape vaccine-induced immunity. As such, more effective ...vaccines are urgently needed. In this study, we evaluated virus-like particle (VLP) as a vaccine platform for SARS-CoV-2. The spike, envelope, and membrane proteins of the SARS-CoV-2 Wuhan strain were expressed by a single recombinant baculovirus BacMam and assembled into VLPs in cell culture. The morphology and size of the SARS-CoV-2 VLP as shown by transmission electron microscopy were similar to the authentic SARS-CoV-2 virus particle. In a mouse trial, two intramuscular immunizations of the VLP BacMam with no adjuvant elicited spike-specific binding antibodies in both sera and bronchoalveolar lavage fluids. Importantly, BacMam VLP-vaccinated mouse sera showed neutralization activity against SARS-CoV-2 spike pseudotyped lentivirus. Our results indicated that the SARS-CoV-2 VLP BacMam stimulated spike-specific immune responses with neutralization activity.IMPORTANCEAlthough existing vaccines have significantly mitigated the impact of the COVID-19 pandemic, none of the vaccines can induce sterilizing immunity. The spike protein is the main component of all approved vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due primarily to its ability to induce neutralizing antibodies. The conformation of the spike protein in the vaccine formulation should be critical for the efficacy of a vaccine. By way of closely resembling the authentic virions, virus-like particles (VLPs) should render the spike protein in its natural conformation. To this end, we utilized the baculovirus vector, BacMam, to express virus-like particles consisting of the spike, membrane, and envelope proteins of SARS-CoV-2. We demonstrated the immunogenicity of our VLP vaccine with neutralizing activity. Our data warrant further evaluation of the virus-like particles as a vaccine candidate in protecting against virus challenges.
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