Ali Mirazimi

Crimean–Congo haemorrhagic fever virus (CCHFV) is an emerging tick-borne virus transmitted primarily by Hyalomma ticks, whose geographic distribution is expanding. CCHFV infection is associated with high case-fatality rates, and there are currently no widely approved vaccines available globally. We were the first to demonstrate that a genetic DNA vaccine (CCHF-N-pVAX1) targeting the nucleoprotein (N) of CCHFV can confer robust protection against lethal disease in mice and attenuate clinical symptoms in non-human primates (NHPs), providing proof-of-concept for a previously underexplored vaccine strategy through activation of NP-specific T cells and antibodies. The CCHF-N-pVAX1 vaccine was tested in a phase I clinical trial in 15 healthy volunteers (HVs) with three dose groups (0.45 mg, 0.90 mg, and 1.80 mg). Five HVs per group received vaccinations at day 0, 28, and 84 given intramuscularly using in vivo electroporation (EP; IGEA Medical), followed by 3 months of follow-up. The protocol-defined completed study reports the primary safety evaluation and secondary immunogenicity assessments through day 169. No power calculations were performed. All patients experienced temporary pain in close proximity to the vaccination/EP procedure. The vaccine was safe, and no serious adverse event (SAE) occurred. 139 other Adverse Events (AEs) occurred; 69 reactogenicity and 70 other. The AEs were mostly of mild grade (n = 123); 10 AEs were of moderate intensity, and 6 AEs were of severe intensity (diarrhea (n = 1), headache (n = 1), and fatigue (n = 4)). Most AEs (114/139) were likely or possibly related to the vaccine/EP. CCHFV N-specific antibodies and T cells appeared after the 2nd dose, and mean levels peaked after the 3rd dose at 1207 ± 2747 (endpoint titres) and 621 ± 561 (IFN-g spotforming cells), respectively. The T cell responses remained high during the 3-month follow-up. The data was consistent with our previous studies in NHPs. In conclusion, we show for the first time that a genetic nucleoprotein-based vaccine against CCHF is safe and immunogenic in humans, inducing strong T- and B-cell responses in most vaccine recipients, and suggesting a potential to confer protection against severe disease in humans. EU clinical trial number 2023-508556-18-00/IN, ClinicalTrials.gov identifier NCT06684431.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO-priority pathogen with high mortality and wide global distribution. A major challenge in CCHFV vaccine development is identifying antigens that induce functional cellular and humoral immunity. We previously showed that the CCHF nucleoprotein (NP) elicits protective responses in mice and non-human primates but does not induce neutralizing antibodies. We therefore systematically screened six chimeric DNA vaccines expressing different CCHF glycoproteins combined with NP. A construct comprising the ectodomain of the glycoprotein Gc and NP elicited robust antibody and T-cell responses to both antigens. The Gc-NP gene was subsequently developed as a lipid nanoparticle-formulated mRNA vaccine using a scalable manufacturing pipeline. Gc-NP mRNA-LNP induced robust antigen-specific humoral and cellular responses comparable to those elicited by the corresponding DNA vaccine, despite the substantially lower dose. Both vaccine approaches generated Gc-directed neutralizing antibody responses against CCHFV pseudovirus and protected mice against homologous Hoti and heterologous IbAr10200 strain challenge. These findings demonstrate that systematic antigen screening combined with genetic vaccine platforms can identify a potent multi-antigen CCHF vaccine candidate. HIGHLIGHTS: A systematic screening of DNA-based genes identified a highly immunogenic combination of the Gc protein ectodomain and NP. This novel vaccine candidate induced Gc-specific neutralizing antibodies and T-cell responses against both NP and Gc. An mRNA-LNP vaccine expressing the Gc-NP gene further improved immunogenicity and conferred protection against lethal challenge with both CCHFV Hoti and IbAr10200 strains in mice. GRAPHICAL

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus widely distributed across Asia, Africa, the Middle East, and Southern Europe, causing severe illness and hemorrhagic fever in humans. While sheep usually show no clinical symptoms, they develop viremia and can play a key role in transmitting the virus to ticks and humans. Despite their importance in transmission, our understanding of infection dynamics in these animals remains limited. To bridge this knowledge gap, we conducted a controlled study in which six Pomeranian Coarsewool sheep were experimentally infected with CCHFV, strain Kosovo Hoti, and monitored over 21 days. All sheep developed transient fever and viremia. Necropsies at 5–7 days post infection (dpi) revealed highest viral loads in liver, spleen, testicles, and lymphoid tissues. Seroconversion occurred by 7 dpi and virus-neutralizing antibodies were detectable by day 8. These findings enhance our understanding of CCHFV infection dynamics in sheep and will aid future research, including vaccine development.

The virus responsible for COVID-19, SARS-CoV-2, continues to spread through the world. The ongoing emergence of new variants with increased viral transmission and immune evasion continue to pose a challenge. Although large-scale genetic screens have identified numerous host factors required for SARS-CoV-2 infection, however the function of these hits remain incompletely understood. In this study, we performed a haploid forward genetic screen in chemically mutagenized mouse embryonic stem cells overexpressing human ACE2 and identified MED13 and DDX60 as important host factors involved in the modulation of SARS-CoV-2 infection. In this study, we have identified and characterized the function of these key element factors for SARS-CoV2 infection. Knockdown of CKM subunits—with the exception of CDK8—or the helicase DDX60 was sufficient to reduce SARS-CoV-2 infection across Vero E6, A549, and Calu-3 cells. During SARS-CoV-2 infection, MED13 was found to function downstream of the JAK/STAT interferon pathway, but also showed another function independently of the interferon response pathway. Surprisingly, while DDX60 is traditionally involved in the interferon response pathway, its knockdown reduces SARS-CoV-2 infection, suggesting DDX60 can promote SARS-CoV-2 infection Interestingly, while inactivation of MED13 or DDX60 markedly reduced SARS-CoV-2 and SARS-CoV infection, it did not affect MERS-CoV. Collectively, these results identify MED13 and DDX60 as critical host determinants for SARS-CoV-2 related-coronaviruses with pandemic potential. Impact statement: Using an unbiased haploid genetic screening approach, this study identifies for the first time MED13 and DDX60 as host factors that influence coronavirus replication through transcriptional and interferon-associated pathways. While DDX60 has previously been linked to antiviral signaling, our findings suggest it can promote coronavirus infection. Most interestingly, the effects of these factors extend beyond SARS-CoV-2 to other coronaviruses. These findings broaden the current understanding of coronavirus-host interaction by highlighting transcriptional control and interferon-associated pathways as modulators of infection rather than focusing solely on viral entry. The work provides mechanistic insight into how host regulatory networks influence coronavirus replication and suggests potential targets for host-directed therapies with activity against multiple present and future coronavirus threats. Data summary: The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Repositories: The data generated in this study are provided in the Supplementary Information/Source Data file. Sequencing data are available on NCBI Sequence Read Archive under the accession number BioProject PRJNA1271794. The next generation sequencing data generated in this study has been deposited in the NCBI Sequence Read Archive (SRA) under accession number SRX29485992, SRX29485993, SRX29485994, SRX29485995, SRX29485996, SRX29485997, SRX29485998, SRX29485999, SRX29486000, SRX29486001, SRX29486002, SRX29486003, SRX29486004, SRX29486005, SRX29486006, SRX29486007, SRX29486010, SRX29486011, SRX29486012, SRX29486013, SRX29486014, SRX29486015, SRX29486016, SRX29486017, SRX29486018, SRX29486019, SRX29486021, SRX29486022, SRX29486023, SRX29486024

In a time of climate change, population growth, and globalization, the risk of viral spread has significantly increased. The 21st century has already witnessed outbreaks of Severe Acute Respiratory Syndrome virus (SARS-CoV), Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2), Ebola virus and Influenza virus, among others. Viruses rapidly adapt and evade human immune systems, complicating the development of effective antiviral countermeasures. Consequently, the need for novel antivirals resilient to viral mutations is urgent. In this study, we developed a CRISPR-Cas13b system to target SARS-CoV-2. Interestingly, this system was also efficient against SARS-CoV, demonstrating broad-spectrum potential. Our findings highlight CRISPR-Cas13b as a promising tool for antiviral therapeutics, underscoring its potential in RNA-virus-associated pandemic responses.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches. IMPORTANCE: Ebola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

Nairoviridae is a family for negative-sense RNA viruses with genomes of about 17.2–21.1 kb. These viruses are maintained in and/or transmitted by arthropods among birds, reptiles and mammals. Norwaviruses and orthonairoviruses can cause febrile illness in humans. Several orthonairoviruses can infect mammals, causing mild, severe and sometimes, fatal diseases. Nairovirids produce enveloped virions containing two or three single-stranded RNA segments with open reading frames that encode a nucleoprotein (N), sometimes a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Nairoviridae, which is available at www.ictv.global/report/nairoviridae.

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.

While having already killed more than 7 million of people worldwide in 4 years, SARS-CoV-2, the etiological agent of COVID-19, is still circulating and evolving. Understanding the pathogenesis of the virus is of capital importance. It was shown that in vitro and in vivo infection with SARS-CoV-2 can lead to cell cycle arrest but the effect of the cell cycle arrest on the virus infection and the associated mechanisms are still unclear. By stopping cells in the G1 phase as well as targeting several pathways involved using inhibitors and small interfering RNAs, we were able to determine that the cell cycle arrest in the late G1 is beneficial for SARS-CoV-2 replication. This cell cycle arrest is independent of p53 but is dependent on the CDC25A-CDK2/cyclin E pathway. These data give a new understanding in SARS-CoV-2 pathogenesis and highlight some possible targets for the development of novel therapeutic approaches.