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SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis in 2019. Currently, the main method for identification of SARS-CoV-2 is a reverse transcription (RT)-PCR assay designed to detect viral RNA in oropharyngeal (OP) or nasopharyngeal (NP) samples. While the PCR assay is considered highly specific and sensitive, this method cannot determine the infectivity of the sample, which may assist in evaluation of virus transmissibility from patients and breaking transmission chains. Thus, cell-culture-based approaches such as cytopathic effect (CPE) assays are routinely employed for the identification of infectious viruses in NP/OP samples. Despite their high sensitivity, CPE assays take several days and require additional diagnostic tests in order to verify the identity of the pathogen. We have therefore developed a rapid immunofluorescence assay (IFA) for the specific detection of SARS-CoV-2 in NP/OP samples following cell culture infection. Initially, IFA was carried out on Vero E6 cultures infected with SARS-CoV-2 at defined concentrations, and infection was monitored at different time points. This test was able to yield positive signals in cultures infected with 10 pfu/ml at 12 hours postinfection (PI). Increasing the incubation time to 24 hours reduced the detectable infective dose to 1 pfu/ml. These IFA signals occur before the development of CPE. When compared to the CPE test, IFA has the advantages of specificity, rapid detection, and sensitivity, as demonstrated in this work.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Imunofluorescência , Humanos , Nasofaringe , Pandemias , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
The COVID-19 pandemic led to development of mRNA vaccines, which became a leading anti-SARS-CoV-2 immunization platform. Preclinical studies are limited to infection-prone animals such as hamsters and monkeys in which protective efficacy of vaccines cannot be fully appreciated. We recently reported a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). BALB/c mice demonstrated specific immunologic responses following RBD-hFc mRNA vaccination. Now, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model. Administration of an RBD-hFc mRNA vaccine to K18-hACE2 mice resulted in robust humoral responses comprising binding and neutralizing antibodies. In correlation with this response, 70% of vaccinated mice withstood a lethal SARS-CoV-2 dose, while all control animals succumbed to infection. To the best of our knowledge, this is the first nonreplicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.
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COVID-19 , Nanopartículas , Vacinas , Animais , Humanos , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pandemias , RNA Mensageiro/genética , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
This study presents the development of a new correlative workflow to bridge the gap between electron microscopy imaging and genetic analysis of viruses. The workflow enables the assignment of genetic information to a specific biological entity by harnessing the nanodissection capability of focused ion beam (FIB). This correlative workflow is based on scanning transmission electron microscopy (STEM) and FIB followed by a polymerase chain reaction (PCR). For this purpose, we studied the tomato brown rugose fruit virus (ToBRFV) and the adenovirus that have significant impacts on plant integrity and human health, respectively. STEM imaging was used for the identification and localization of virus particles on a transmission electron microscopy (TEM) grid followed by FIB milling of the desired region of interest. The final-milled product was subjected to genetic analysis by the PCR. The results prove that the FIB-milling process maintains the integrity of the genetic material as confirmed by the PCR. We demonstrate the identification of RNA and DNA viruses extracted from a few micrometers of an FIB-milled TEM grid. This workflow enables the genetic analysis of specifically imaged viral particles directly from heterogeneous clinical samples. In addition to viral diagnostics, the ability to isolate and to genetically identify specific submicrometer structures may prove valuable in additional fields, including subcellular organelle and granule research.
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Vírion , Humanos , Microscopia Eletrônica de Transmissão e Varredura , Vírion/genéticaRESUMO
Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many "antigen" detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false positive results. In this work, we developed a novel alternative spike-based antigen assay, comprising four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results. Schematic representation of sample collection and analysis. The figure was created using BioRender.com.
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Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/análise , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/análise , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Fosfoproteínas/análise , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
L chain allelic inclusion has been proposed as a B cell tolerance mechanism in addition to clonal deletion, clonal anergy, and receptor editing. It is said to rescue autoreactive B cells from elimination by diluting out the self-reactive BCR through the expression of a second innocuous L chain. In autoimmune animals, such as lupus-prone mice, allelically included B cells could be activated and produce pathogenic autoantibodies. We have previously shown that anti-DNA hybridomas from diseased New Zealand Black/New Zealand White F1 mice exhibit nearly perfect allelic exclusion. In the current study, we have analyzed single B cells from these and from nonautoimmune mice. In addition, we have cloned and expressed the Ig variable regions of several L chain-included B cells in cell culture. We find that although the number of L chain-included B cells increases as a result of receptor editing, the majority of such cells do not retain an autoreactive HxL chain combination and, therefore, allelic inclusion in itself does not serve as a B cell tolerance mechanism in these autoimmune mice.
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Alelos , Autoanticorpos/biossíntese , Predisposição Genética para Doença/genética , Cadeias Leves de Imunoglobulina/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Tolerância a Antígenos Próprios/genéticaRESUMO
The eradication of smallpox was officially declared by the WHO in 1980, leading to discontinuation of the vaccination campaign against the virus. Consequently, immunity against smallpox and related orthopoxviruses like Monkeypox virus gradually declines, highlighting the need for efficient countermeasures not only for the prevention, but also for the treatment of already exposed individuals. We have recently developed human-like monoclonal antibodies (mAbs) from vaccinia virus-immunized non-human primates. Two mAbs, MV33 and EV42, targeting the two infectious forms of the virus, were selected for in vivo evaluation, based on their in vitro neutralization potency. A single dose of either MV33 or EV42 administered three days post-infection (dpi) to BALB/c female mice provides full protection against lethal ectromelia virus challenge. Importantly, a combination of both mAbs confers full protection even when provided five dpi. Whole-body bioimaging and viral load analysis reveal that combination of the two mAbs allows for faster and more efficient clearance of the virus from target organs compared to either MV33 or EV42 separately. The combined mAbs treatment further confers post-exposure protection against the currently circulating Monkeypox virus in Cast/EiJ female mice, highlighting their therapeutic potential against other orthopoxviruses.
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Orthopoxvirus , Infecções por Poxviridae , Varíola , Vacínia , Humanos , Feminino , Animais , Camundongos , Anticorpos Monoclonais , Infecções por Poxviridae/prevenção & controle , Vaccinia virus , Anticorpos AntiviraisRESUMO
Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.
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Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3-4 h post infection (PI), and accumulates as the infection proceeds. By 10-24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle.
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COVID-19 , Vírus da Estomatite Vesicular Indiana , Humanos , Vírus da Estomatite Vesicular Indiana/genética , SARS-CoV-2 , Proteínas Virais/metabolismoRESUMO
The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.
Assuntos
Francisella tularensis , Tularemia , Humanos , Francisella tularensis/metabolismo , Hemocultura , Tularemia/diagnóstico , Tularemia/metabolismo , Tularemia/microbiologia , Ferro/metabolismo , Antibacterianos/farmacologiaRESUMO
SARS-CoV-2 surface spike protein mediates the viral entry into the host cell and represents the primary immunological target of COVID-19 vaccines as well as post-exposure immunotherapy. Establishment of the highly immunogenic B-cell epitope profile of SARS-CoV-2 proteins in general, and that of the spike protein in particular, may contribute to the development of sensitive diagnostic tools and identification of vaccine` candidate targets. In the current study, the anti-viral antibody response in transgenic K18-hACE-2 mice was examined by implementing an immunodominant epitope mapping approach of the SARS-CoV-2 spike. Serum samples for probing an epitope array covering the entire spike protein were collected from mice following infection with the original SARS-CoV-2 strain as well as the B.1.1.7 Alpha and B.1.351 Beta genetic variants of concern. The analysis resulted in distinction of six linear epitopes common to the humoral response against all virus variants inspected at a frequency of more than 20% of the serum samples. Finally, the universality of the response was probed by cross-protective in vitro experiments using plaque-reducing neutralization tests. The data presented here has important implications for prediction of the efficacy of immune countermeasures against emerging SARS-CoV-2 variants.
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Plague, caused by the human pathogen Yersinia pestis, is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca+2)- and iron (Fe+2)-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca+2 and Fe+2. The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an increased sensitivity and without any detectable compromise of the accuracy of the antibiotic susceptibility testing (E-test), critical for subsequent successful therapeutic interventions.
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Hyper-immune antisera from large mammals, in particular horses, are routinely used for life-saving anti-intoxication intervention. While highly efficient, the use of these immunotherapeutics is complicated by possible recipient reactogenicity and limited availability. Accordingly, there is an urgent need for alternative improved next-generation immunotherapies to respond to this issue of high public health priority. Here, we document the development of previously unavailable tools for equine antibody engineering. A novel primer set, EquPD v2020, based on equine V-gene data, was designed for efficient and accurate amplification of rearranged horse antibody V-segments. The primer set served for generation of immune phage display libraries, representing highly diverse V-gene repertoires of horses immunized against botulinum A or B neurotoxins. Highly specific scFv clones were selected and expressed as full-length antibodies, carrying equine V-genes and human Gamma1/Lambda constant genes, to be referred as "Centaur antibodies". Preliminary assessment in a murine model of botulism established their therapeutic potential. The experimental approach detailed in the current report, represents a valuable tool for isolation and engineering of therapeutic equine antibodies.
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Anticorpos , Região Variável de Imunoglobulina , Animais , Anticorpos/genética , Técnicas de Visualização da Superfície Celular , Cavalos , Humanos , Região Variável de Imunoglobulina/genética , Mamíferos , Camundongos , Neurotoxinas , Proteínas Recombinantes/genéticaRESUMO
Longevity of the immune response following viral exposure is an essential aspect of SARS-CoV-2 infection. Mild SARS-CoV-2 infection of K18-hACE2 mice was implemented for evaluating the mounting and longevity of a specific memory immune response. We show that the infection of K18-hACE2 mice induced robust humoral and cellular immunity (systemic and local), which persisted for at least six months. Virus-specific T cells and neutralizing antibody titers decreased over time, yet their levels were sufficient to provide sterile immunity against lethal rechallenge six months post-primary infection. The study substantiates the role of naturally induced immunity against SARS-CoV-2 infection for preventing recurring morbidity.
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Clonal anergy has been well recognized as an important mechanism of B cell immunologic tolerance. However, the properties of anergic B cells and especially their role in the development of autoimmune disease in susceptible animals have been controversial. Here we show that low-affinity anti-DNA anergic B cells populate the mature B-cell compartment in the mouse spleen in excessive numbers and display paradoxical behavior in response to a combined B-cell receptor/TLR9 activation. Surprisingly, B-cell anergy was maintained in aged NZB/NZW F1 mice that develop a systemic lupus erythematosus (SLE)-like autoimmune disease. In several parameters of anergy, such as calcium mobilization and antibody secretion, the lupus-prone mice appeared more anergic than their non-autoimmune counterparts. We conclude that low-affinity anergic B cells are unlikely to serve as precursors for the high-affinity autoreactive B cells that give rise to pathogenic anti-DNA auto-antibodies in SLE.
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Anticorpos Antinucleares/sangue , Autoimunidade , Linfócitos B/imunologia , Anergia Clonal , Lúpus Eritematoso Sistêmico/imunologia , Baço/imunologia , Animais , Autoimunidade/genética , Linfócitos B/metabolismo , Cálcio/metabolismo , Células Cultivadas , Anergia Clonal/genética , Modelos Animais de Doenças , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NZB , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/imunologia , Baço/metabolismo , Receptor Toll-Like 9/imunologiaRESUMO
Recent work on B cell tolerance and autoimmunity has suggested the L chain allelic inclusion is a property of autoreactive B cells and is closely linked to receptor editing. Allelic inclusion could rescue autoreactive B cells from clonal deletion by reducing their effective BCR surface density. We have investigated this phenomenon in anti-DNA producing hybridomas, derived from different strains of Ig gene-targeted, lupus-prone NZB/NZW mice. Our results indicate that isotype and allelic exclusion was strictly maintained in most high- and low-affinity, edited and nonedited, anti-DNA transgenic B cells. However, a substantial fraction of the anti-DNA hybridomas expressed a very restricted set of nonproductively rearranged L chain mRNA, in addition to the productive anti-DNA L chain. The aberrant L chains could have a role in the selection and survival of autoreactive B cells in these autoimmune mice.
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Anticorpos Antinucleares/genética , Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Antígenos de Linfócitos B/genética , Sequência de Aminoácidos , Animais , Anticorpos Antinucleares/imunologia , Autoimunidade , Sequência de Bases , Deleção Clonal , Citometria de Fluxo , Técnicas de Introdução de Genes , Rearranjo Gênico do Linfócito B/imunologia , Hibridomas , Cadeias Pesadas de Imunoglobulinas/imunologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos B/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This protocol describes the use of a biolayer interferometry platform for assessing antibody-antigen interactions. The protocol focuses on affinity determination and epitope binning, although the system can be utilized for measuring any protein-protein interaction. Readings are collected in real time, allowing the use of unlabeled molecules, and data can thus be obtained in a fast and easy manner. Experiments should be carefully designed, taking into consideration the tested interaction, available sensors, and suitable controls. For complete details on the use and execution of this protocol, please refer to Noy-Porat et al. (2021).
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Antígenos , Interferometria , Epitopos , Interferometria/métodosRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple, and rapid platform for immunization, and therefore have been employed in recent studies toward the development of a SARS-CoV-2 vaccine. Herein, we present the design of an mRNA vaccine, based on lipid nanoparticles (LNPs)-encapsulated SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc). Several ionizable lipids have been evaluated in vivo in a luciferase (luc) mRNA reporter assay, and two leading LNPs formulations have been chosen for the subsequent RBD-hFc mRNA vaccine strategy. Intramuscular administration of LNP RBD-hFc mRNA elicited robust humoral response, a high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. The data in the current study demonstrate the potential of these lipids as promising candidates for LNP-based mRNA vaccines in general and for a COVID19 vaccine in particular.
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COVID-19 , Nanopartículas , Vacinas , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , Humanos , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
HLA transgenic mice are instrumental for evaluation of human-specific immune responses to viral infection. Mice do not develop COVID-19 upon infection with SARS-CoV-2 due to the strict tropism of the virus to the human ACE2 receptor. The aim of the current study was the implementation of an adenovirus-mediated infection protocol for human ACE2 expression in HLA transgenic mice. Transient pulmonary expression of the human ACE2 receptor in these mice results in their sensitisation to SARS-CoV-2 infection, consequently providing a valuable animal model for COVID-19. Infection results in a transient loss in body weight starting 3 days post-infection, reaching 20-30% loss of weight at day 7 and full recovery at days 11-13 post-infection. The evolution of the disease revealed high reproducibility and very low variability among individual mice. The method was implemented in two different strains of HLA immunized mice. Infected animals developed strong protective humoral and cellular immune responses specific to the viral spike-protein, strictly depending on the adenovirus-mediated human ACE2 expression. Convalescent animals were protected against a subsequent re-infection with SARS-CoV-2, demonstrating that the model may be applied for assessment of efficacy of anti-viral immune responses.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The continued spread of SARS-CoV-2 increases the probability of influenza/SARS-CoV-2 coinfection, which may result in severe disease. In this study, we examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV-infected mice to developing severe disease upon coinfection with SARS-CoV-2 two days later. In contrast to nonfatal influenza and lower mortality rates due to SARS-CoV-2 alone, this coinfection results in severe morbidity and nearly complete mortality. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic.
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COVID-19/imunologia , COVID-19/virologia , Coinfecção/imunologia , Coinfecção/virologia , Imunidade , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Antivirais/imunologia , COVID-19/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/genética , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regulação para Cima/genética , Carga Viral/imunologiaRESUMO
The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.