Infected Cell Cultures Research Articles

Rapid Development of Modified Vaccinia Virus Ankara (MVA)-Based Vaccine Candidates Against Marburg Virus Suitable for Clinical Use in Humans

Background/Objectives: Marburg virus (MARV) is the etiological agent of Marburg Virus Disease (MVD), a rare but severe hemorrhagic fever disease with high case fatality rates in humans. Smaller outbreaks have frequently been reported in countries in Africa over the last few years, and confirmed human cases outside Africa are, so far, exclusively imported by returning travelers. Over the previous years, MARV has also spread to non-endemic African countries, demonstrating its potential to cause epidemics. Although MARV-specific vaccines are evaluated in preclinical and clinical research, none have been approved for human use. Modified Vaccinia virus Ankara (MVA), a well-established viral vector used to generate vaccines against emerging pathogens, can deliver multiple antigens and has a remarkable clinical safety and immunogenicity record, further supporting its evaluation as a vaccine against MARV. The rapid availability of safe and effective MVA-MARV vaccine candidates would expand the possibilities of multi-factored intervention strategies in endemic countries. Methods: We have used an optimized methodology to rapidly generate and characterize recombinant MVA candidate vaccines that meet the quality requirements to proceed to human clinical trials. As a proof-of-concept for the optimized methodology, we generated two recombinant MVAs that deliver either the MARV glycoprotein (MVA-MARV-GP) or the MARV nucleoprotein (MVA-MARV-NP). Results: Infections of human cell cultures with recombinant MVA-MARV-GP and MVA-MARV-NP confirmed the efficient synthesis of MARV-GP and MARV-NP proteins in mammalian cells, which are non-permissive for MVA replication. Prime-boost immunizations in C57BL/6J mice readily induced circulating serum antibodies binding to recombinant MARV-GP and MARV-NP proteins. Moreover, the MVA-MARV-candidate vaccines elicited MARV-specific T-cell responses in C57BL/6J mice. Conclusions: We confirmed the suitability of our two backbone viruses MVA-mCherry and MVA-GFP in a proof-of-concept study to rapidly generate candidate vaccines against MARV. However, further studies are warranted to characterize the protective efficacy of these recombinant MVA-MARV vaccines in other preclinical models and to evaluate them as vaccine candidates in humans.

Decomposition of L-glutamine and accumulation of ammonium in cell culture media inhibit infectivity of influenza viruses

Cationic lysosomotropic molecules such as ammonium salts and chloroquine inhibit influenza A virus (IAV) infection in cell culture by counteracting endosomal acidification and hampering viral-endosomal fusion. Here, we studied the effects of storage of L-glutamine-supplemented cell culture media on accumulation of ammonium and inhibition of IAV infection. The storage-related inhibitory effect was observed for DMEM and OptiMEM media but not for RPMI medium, and was more pronounced for IAV with pH-stable hemagglutinin. Our results highlight the importance to consider potential presence of virus-inhibiting lysosomotropic agents and/or their production in culture media with negative effect on influenza virus infection.

Neutralisation resistance of SARS-CoV-2 spike-variants is primarily mediated by synergistic receptor binding domain substitutions

ABSTRACT The evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested that changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence.

Infiltrating monocytes drive cardiac dysfunction in a cardiomyocyte-restricted mouse model of SARS-CoV-2 infection.

Cardiovascular manifestations of coronavirus disease 2019 (COVID-19) include myocardial injury, heart failure, and myocarditis and are associated with long-term disability and mortality. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antigens are found in the myocardium of COVID-19 patients, and human cardiomyocytes are susceptible to infection in cell or organoid cultures. While these observations raise the possibility that cardiomyocyte infection may contribute to the cardiac sequelae of COVID-19, a causal relationship between cardiomyocyte infection and myocardial dysfunction and pathology has not been established. Here, we generated a mouse model of cardiomyocyte-restricted infection by selectively expressing human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-2 receptor, in cardiomyocytes. Inoculation of Myh6-Cre Rosa26loxP-STOP-loxP-hACE2 mice with an ancestral, non-mouse-adapted strain of SARS-CoV-2 resulted in viral replication within the heart, accumulation of macrophages, and moderate left ventricular (LV) systolic dysfunction. Cardiac pathology in this model was transient and resolved with viral clearance. Blockade of monocyte trafficking reduced macrophage accumulation, suppressed the development of LV systolic dysfunction, and promoted viral clearance in the heart. These findings establish a mouse model of SARS-CoV-2 cardiomyocyte infection that recapitulates features of cardiac dysfunctions of COVID-19 and suggests that both viral replication and resultant innate immune responses contribute to cardiac pathology.IMPORTANCEHeart involvement after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection occurs in multiple ways and is associated with worse outcomes in coronavirus disease 2019 (COVID-19) patients. It remains unclear if cardiac disease is driven by primary infection of the heart or immune response to the virus. SARS-CoV-2 is capable of entering contractile cells of the heart in a culture dish. However, it remains unclear how such infection affects the function of the heart in the body. Here, we designed a mouse in which only heart muscle cells can be infected with a SARS-CoV-2 strain to study cardiac infection in isolation from other organ systems. In our model, infected mice show viral infection, worse function, and accumulation of immune cells in the heart. A subset of immune cells facilitates such worsening heart function. As this model shows features similar to those observed in patients, it may be useful for understanding the heart disease that occurs as a part of COVID-19.

Quasineutral multistability in an epidemiological-like model for defective-helper betacoronavirus infection in cell cultures

It is well known that, during replication, RNA viruses spontaneously generate defective viral genomes (DVGs). DVGs are unable to complete an infectious cycle autonomously and depend on coinfection with a wild-type helper virus (HV) for their replication and/or transmission. The study of the dynamics arising from a HV and its DVGs has been a longstanding question in virology. It has been shown that DVGs can modulate HV replication and, depending on the strength of interference, result in HV extinctions or self-sustained persistent fluctuations. Extensive experimental work has provided mechanistic explanations for DVG generation and compelling evidences of HV-DVGs virus coevolution. Some of these observations have been captured by mathematical models. Here, we develop and investigate an epidemiological-like mathematical model specifically designed to study the dynamics of betacoronavirus in cell culture experiments. The dynamics of the model is governed by several degenerate normally hyperbolic invariant manifolds given by quasineutral planes - i.e., filled by equilibrium points. Three different quasineutral planes have been identified depending on parameters and involving: (i) persistence of HV and DVGs; (ii) persistence of non-infected cells and DVG-infected cells; and (iii) persistence of DVG-infected cells and DVGs. Key parameters involved in these scenarios are the maximum burst size (B), the fraction of DVGs produced during HV replication (β), and the replication advantage of DVGs (δ). More precisely, in the case 0<B<1+β the system displays tristability, where all three scenarios are present. In the case 1+β<B<1+β+δ this tristability persists but attracting scenario (ii) is reduced to a well-defined half-plane. For B>1+β+δ, the scenario (i) becomes globally attractor. Scenarios (ii) and (iii) are compatible with the so-called self-curing since the HV is removed from the population. Sensitivity analyses indicate that model dynamics largely depend on DVGs production rate (β) and their replicative advantage (δ), and on both the infection rates and virus-induced cell deaths. Finally, the model has been fitted to single-passage experimental data using an artificial intelligence methodology based on genetic algorithms and key virological parameters have been estimated.

Inhibitors of the small membrane (M) protein viroporin prevent Zika virus infection.

Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small Membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics (MD) simulations. Models contained a predicted lumenal rimantadine binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted-therapies.

Polyallylamine antiviral activity against influenza and acute respiratory viral infection in various cell cultures

Acute respiratory viral infections, including influenza, comprise the most common group of seasonal viral infections. Influenza is the most common and dangerous viral infection. The only way to control it is influenza vaccination. Regarding infections caused by parainfluenza and respiratory syncytial virus, only symptomatic treatment is available. Influenza virus quickly bypasses post-vaccination immunity due to its ability to antigenic drift and genetic reassortment. Available antiviral drugs quickly lose effectiveness, especially in relation to highly contagious influenza virus strains. The aim of the study was to create chemotherapeutic agent with a multi-layered effect on all viral structures: surface proteins, lipid membrane and ribonucleoprotein. Such drugs include polyelectrolytes (PE), particularly, polyallylamine (PAA), which showed strong virus-inhibiting effect in combination with low cytotoxicity against several influenza strains in MDCK cell culture and as well as measles virus in Vero cell culture. Materials and methods. In this work, an extended study on PAA antiviral activity and cytotoxicity was carried out using three influenza virus strains in A549 cell line, parainfluenza virus type 3 (HPIV-3) and respiratory syncytial virus (RSV) in A549, HEp-2, Vero, L-41, MA-104 cell lines. Results. It was shown that influenza and RSV were the most sensitive to PAA,so that virus activity decreased by 3 orders of magnitude in human lung carcinoma cells A549. The lowest antiviral activity was registered in Vero cells, which may be because it lacks interferon production system. Based on the results of in vitro experiments, PAA can be considered as a broad-spectrum antiviral drug not only against influenza, but also other human respiratory viruses.

Comparative phylogenetic analysis of Siberian isolates of bovine pestiviruses

The results of comparative phylogenetic analysis of 52 isolates of bovine pestiviruses of three species circulating among highly productive dairy cattle in Siberia, as well as those present in the samples of commercial fetal serum, transfected cell culture lines and live vaccines on four genes: Npro, Erns, E1 and E2 are presented. The obtained data were compared with the results of the 5'UTR gene sequencing obtained earlier. The results confirmed the circulation in Siberia of eleven subtypes of BVDV-1 (a,b,c,d,f,g,i,j, k,p,r), three subtypes of BVDV-2 (a,b,c) and one BVDV -3(a). The genetic profiles of the isolates for the 5'UTR, Npro, Erns and E1 genes matched completely. The exceptions were three BVDV-1a isolates (R/FBS/96, N/MDBK/08 and SA/FBS/08) detected in infected cell cultures that clustered in a clade formed by BVDV-1j reference sequences. The remaining isolates previously shown to have 100% sequence identity of the 5'UTR had nucleotide sequence similarities of the Npro, Erns, E1, and E2 genes ranging from 96-99%, which also implies that they are closely related. The results of the BVDV-2 isolates sequencing showed a complete correspondence with the genome sequences identified in the previous studies. Comparative sequencing also confirmed the circulation of H Italian-Brazilian pestivirus isolates (BVBD-3a) in Siberia, but all the isolates were divided into two subclades. The first included the isolates isolated from fetal sera and cell cultures, while the second included those isolated from the vaccines and internal organs of diseased animals during outbreaks in farms. The genetic diversity of bovine pestiviruses, their origin and variability are discussed.

A Micronemal Protein, Scot1, Is Essential for Apicoplast Biogenesis and Liver Stage Development in Plasmodium berghei.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

The Topical Novel Formulations of Interferon α-2в Effectively Inhibit HSV-1 Keratitis in the Rabbit Eye Model and HSV-2 Genital Herpes in Mice.

Herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) are widespread human pathogens that establish chronic latent infections leading to recurrent episodes. Current treatments are limited, necessitating the development of novel antiviral strategies. This study aimed to assess the antiviral efficacy of novel topical formulations containing interferon alpha-2b (IFN α-2b) against HSV-1 and HSV-2. The formulations, Oftalmoferon® forte (eye drops) and Interferon Vaginal Tablets, demonstrated potent antiviral effects against HSV-1 and HSV-2 in Vero cells, respectively, with concentration-dependent inhibition of viral replication. Subsequently, their efficacy was tested in animal models: HSV-1 keratitis in the rabbit eye model and HSV-2 genital herpes in mice. Oftalmoferon® forte effectively treated HSV-1 keratitis, reducing clinical symptoms and ulcerations compared to virus control. Interferon Vaginal Tablets showed promising results in controlling HSV-2 genital herpes in mice, improving survival rates, reducing clinical signs, weight loss and viral replication. The novel IFN α-2b formulations exhibited significant antiviral activity against HSV infections in cell culture and animal models. These findings suggest the potential of these formulations as alternative treatments for HSV infections, particularly in cases resistant to current therapies. Further studies are warranted to optimize treatment regimens and assess clinical efficacy in humans.

Chlamydia trachomatis induces disassembly of the primary cilium to promote the intracellular infection.

Chlamydia trachomatis is a clinically important bacterium that infects epithelial cells of the genitourinary and respiratory tracts and the eye. These differentiated cells are in a quiescent growth state and have a surface organelle called a primary cilium, but the standard Chlamydia cell culture infection model uses cycling cells that lack primary cilia. To investigate if these differences are relevant, we performed infections with host cells that have a primary cilium. We found that C. trachomatis caused progressive loss of the primary cilium that was prevented by disrupting Aurora A (AurA), HDAC6 or calmodulin, which are components of the cellular cilia disassembly pathway. Stabilization of the primary cilium by targeting this pathway caused a large reduction in infectious progeny although there were no changes in chlamydial inclusion growth, chlamydial replication or the ultrastructural appearance of dividing and infectious forms (RBs and EBs, respectively). Thus, the presence of a primary cilium interfered with the production of infectious EBs at a late step in the developmental cycle. C. trachomatis infection also induced quiescent cells to re-enter the cell cycle, as detected by EdU incorporation in S-phase, and Chlamydia-induced cilia disassembly was necessary for cell cycle re-entry. This study therefore describes a novel host-pathogen interaction in which the primary cilium limits a productive Chlamydia infection, and the bacterium counteracts this host cell defense by activating the cellular cilia disassembly pathway.

A versatile automated pipeline for quantifying virus infectivity by label-free light microscopy and artificial intelligence

Virus infectivity is traditionally determined by endpoint titration in cell cultures, and requires complex processing steps and human annotation. Here we developed an artificial intelligence (AI)-powered automated framework for ready detection of virus-induced cytopathic effect (DVICE). DVICE uses the convolutional neural network EfficientNet-B0 and transmitted light microscopy images of infected cell cultures, including coronavirus, influenza virus, rhinovirus, herpes simplex virus, vaccinia virus, and adenovirus. DVICE robustly measures virus-induced cytopathic effects (CPE), as shown by class activation mapping. Leave-one-out cross-validation in different cell types demonstrates high accuracy for different viruses, including SARS-CoV-2 in human saliva. Strikingly, DVICE exhibits virus class specificity, as shown with adenovirus, herpesvirus, rhinovirus, vaccinia virus, and SARS-CoV-2. In sum, DVICE provides unbiased infectivity scores of infectious agents causing CPE, and can be adapted to laboratory diagnostics, drug screening, serum neutralization or clinical samples.

Insights of potential trypanocidal effect of the synthetic derivative (2E)-1-(4-aminophenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one: in vitro assay, MEV analysis, quantum study, molecular docking, molecular dynamics, MPO analysis, and predictive ADMET.

This study aims to evaluate the antitrypanosomiasis activity of a synthetic dichloro-substituted aminochalcone via in vitro assays against infected cell cultures, as well as a theoretical characterization of pharmacokinetics and pharmacodynamics against the protein targets of the evolutionary cycle of T. cruzi. The in vitro evaluation of parasite proliferation inhibition was performed via cytotoxicity analysis on mammalian host cells, effect on epimastigote and trypomastigote forms, and cell death analysis, while computer simulations characterized the electronic structure of (2E)-1-(4-aminophenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (DCl), the mechanism of action against the proteins of the evolutionary cycle of T. cruzi: Cruzain, Trypanothione reductase, TcGAPDH, and CYP51 by molecular docking and dynamics and predictive pharmacokinetics by MPO-based ADMET. The in vitro tests showed that the DCl LC50 in order of 178.9 ± 23.9 was similar to the BZN, evidencing the effectiveness of chalcone against Trypomastigotes. Molecular docking and dynamics simulations suggest that DCl acts on the active site of the CYP51 receptor, with hydrogen interactions that showed a high degree of occupation, establishing a stable complex with the target. MPO analysis and ADMET prediction tests suggest that the compound presents an alignment between permeability and hepatic clearance, although it presents low metabolic stability. Chalcone showed stable pharmacodynamics against the CYP51 target, but can form reactive metabolites from N-conjugation and C = C epoxidation, as an indication of controlled oral dose, although the estimated LD50 rate > 500mg/kg is a indicative of low incidence of lethality by ingestion, constituting a promising therapeutic strategy.

Characterization of SARS-CoV-2 replication in human H1299/ACE2 cells: A versatile and practical infection model for antiviral research and beyond

A range of cell culture infection models have been used to study SARS-CoV-2 and perform antiviral drug research. Commonly used African green monkey Vero, human lung-derived Calu-3 and ACE2+TMPRSS2-expressing A549 cells, each have their limitations. Here, we describe human ACE2-expressing H1299 lung cells as a more efficient and robust model for SARS-CoV-2 research. These cells are as easy to handle as Vero cells, support SARS-CoV-2 replication to high titers, display a functional innate immune response and are suitable for plaque assays, microscopy, the production of (genetically stable) virus stocks and antiviral assays. H1299/ACE2-based (CPE reduction) assays can be performed without adding a P-gP drug efflux pump inhibitor, which is often required in Vero-based assays. Moreover, H1299/ACE2 cells allowed us to perform CPE reduction assays with omicron variants that did not work in Vero-based assays. In summary, H1299/ACE2 cells are a versatile infection model to study SARS-CoV-2 replication in the context of antiviral drug development and virus-host interaction studies.

Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.

EcoHIV Infection of Primary Murine Brain Cell Cultures to Model HIV Replication and Neuropathogenesis.

EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.

JFH1-based Core-NS2 genotype variants of HCV with genetic stability in vivo and in vitro: Important tools in the evaluation of virus neutralization.

HCV infection continues to be a major global health burden despite effective antiviral treatments. The urgent need for a protective vaccine is hindered by the scarcity of suitable HCV-permissive animal models tractable in vaccination and challenge studies. Currently, only antibody neutralization studies in infectious cell culture systems or studies of protection by passive immunization of human liver chimeric mice offer the possibility to evaluate the effect of vaccine-induced antibodies. However, differences between culture-permissive and in vivo-permissive viruses make it a challenge to compare analyses between platforms. To address this problem, we aimed at developing genotype-specific virus variants with genetic stability both in vitro and in vivo. We demonstrated infection of human liver chimeric mice with cell culture-adapted HCV JFH1-based Core-NS2 recombinants of genotype 1-6, with a panel of 10 virus strains used extensively in neutralization and receptor studies. Clonal re-engineering of mouse-selected mutations resulted in virus variants with robust replication both in Huh7.5 cells and human liver chimeric mice, with genetic stability. Furthermore, we showed that, overall, these virus variants have similar in vitro neutralization profiles as their parent strains and demonstrated their use for in vivo neutralization studies. These mouse-selected HCV recombinants enable the triage of new vaccine-relevant antibodies in vitro and further allow characterization of protection from infection in vivo using identical viruses in human liver chimeric mice. As such, these viruses will serve as important resources in testing novel antibodies and can thus guide strategies to develop an efficient protective vaccine against HCV infection.

Immunogenicity and efficacy of VLA2001 vaccine against SARS-CoV-2 infection in male cynomolgus macaques

BackgroundThe fight against COVID-19 requires mass vaccination strategies, and vaccines inducing durable cross-protective responses are still needed. Inactivated vaccines have proven lasting efficacy against many pathogens and good safety records. They contain multiple protein antigens that may improve response breadth and can be easily adapted every year to maintain preparedness for future seasonally emerging variants.MethodsThe vaccine dose was determined using ELISA and pseudoviral particle-based neutralization assay in the mice. The immunogenicity was assessed in the non-human primates with multiplex ELISA, neutralization assays, ELISpot and intracellular staining. The efficacy was demonstrated by viral quantification in fluids using RT-qPCR and respiratory tissue lesions evaluation.ResultsHere we report the immunogenicity and efficacy of VLA2001 in animal models. VLA2001 formulated with alum and the TLR9 agonist CpG 1018™ adjuvant generate a Th1-biased immune response and serum neutralizing antibodies in female BALB/c mice. In male cynomolgus macaques, two injections of VLA2001 are sufficient to induce specific and polyfunctional CD4+ T cell responses, predominantly Th1-biased, and high levels of antibodies neutralizing SARS-CoV-2 infection in cell culture. These antibodies also inhibit the binding of the Spike protein to human ACE2 receptor of several variants of concern most resistant to neutralization. After exposure to a high dose of homologous SARS-CoV-2, vaccinated groups exhibit significant levels of protection from viral replication in the upper and lower respiratory tracts and from lung tissue inflammation.ConclusionsWe demonstrate that the VLA2001 adjuvanted vaccine is immunogenic both in mouse and NHP models and prevent cynomolgus macaques from the viruses responsible of COVID-19.

Crimean–Congo haemorrhagic fever virus uses LDLR to bind and enter host cells

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean–Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.