MGF100 but not MGF300 family is a potential multigene-deleted target for ASFV attenuation and live attenuated vaccine development

Genotype II Live‐Attenuated ASFV Vaccine Bearing 24 Genes Deletion in 3 Independent Regions Is Able to Provide Complete Protection Against Homologous Lethal Challenge

African swine fever (ASF) is an acute, febrile, and highly contagious infectious disease of swine with the etiological agent of African swine fever virus (ASFV). The mortality rate of virulent strains is as high as 100%. Strengthening biosafety is so far the most effective way to prevent and control ASF. Therefore, it is urgent to develop a safe and effective vaccine. In this study, a Genotype II live‐attenuated ASF vaccine bearing 24 genes deletion in 3 independent regions was constructed based on the highly virulent Eurasian strain ASFV CN/GS 2018 backbone. The resulting mutant ASFV‐Δ24 is characterized by complete deletion of 24 genes distributed in 3 genomic positions of 852 to 11 468, 19 732 to 22 929, and 179 519 to 180 617, among which MGF100 and whole MGF300 families are pioneeringly removed. The ASFV‐Δ24 displayed a delayed and reduced replication kinetics as well as aberrant icosahedral empty particles devoid of a nucleoid when compared to the parental virus. Animal experiments showed that ASFV‐Δ24 was completely attenuated in animals as evidenced by stable body temperature and no ASF‐compatible clinical signs in vaccinated pigs. The ASFV‐Δ24 could provide complete homologous protection against lethal challenge, as vaccinated pigs demonstrated boosted antibody response, transient but low levels of viremia in blood and virus titers in organs as well as almost undetectable viral shedding. Gene deletions in multiple regions are helpful for prevention of virulence reversion. These results indicate that ASFV‐Δ24 can be used as an effective and promising candidate vaccine to control the spread of ASFV.

Integrated multi-omics strategy for screening novel protective antigens against Brucella melitensis and analysis of their immunogenicity

Through multi-omics integration analysis, this study successfully identified a novel candidate antigen, BMEI1943, with strong Th1-type immunogenicity, providing an experimental basis for the development of safe and efficient subunit vaccines against brucellosis.

A Live Attenuated AP2X-1-Deficient Toxoplasma Strain Confers Protective Immunity Against Acute and Chronic Toxoplasmosis in a Murine Model

Simple Summary Toxoplasmosis, caused by the zoonotic parasite Toxoplasma gondii, is a major concern for immunocompromised individuals and the livestock industry. Research has shown that AP2X-1 is important for growth, conversion between tachyzoites and bradyzoites, and sexual stage development of T. gondii. In the present study, we found that all mice infected with the PruΔap2X-1 knockout strain survived even at the highest dose (5 × 106 tachyzoites), and no brain cysts were detected. Vaccinated mice were fully protected against both acute disease caused by the type II Pru and ToxoDB#9 PYS strains, and chronic infection induced by tissue cysts, whereas vaccinated mice had modestly extended survival time without conferring complete protection against challenge with the highly virulent RH strain. Moreover, vaccination induced a strong Th1-biased cellular and humoral immune response. Thus, PruΔap2X-1 represents a potential live-attenuated vaccine candidate against toxoplasmosis, with a favorable safety profile observed under the experimental conditions.

The Q653R substitution in the spike protein is associated with attenuation of a GVI-1 infectious bronchitis virus strain.

Live-attenuated Toxoplasma gondii RHΔddx6 mutant establishes comprehensive immunity against acute, chronic, and congenital toxoplasmosis

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite causing severe disease in immunocompromised individuals and congenitally infected infants. Despite decades of research, no licensed human vaccine exists. This study evaluates a novel live-attenuated vaccine candidate based on depletion of DDX6, a conserved DEAD-box RNA helicase involved in post-transcriptional gene regulation. A Δddx6 strain was generated in the type I virulent RH strain using CRISPR/Cas9, and its phenotype was comprehensively assessed. Although the mutant displayed only subtle defects in certain in vitro assays, it exhibited almost complete avirulence in mice even at high inoculums (106 tachyzoites), representing a dramatic attenuation phenotype not fully predicted by in vitro analyses. A single immunization with 1 × 106 Δddx6 tachyzoites induced robust Th1-biased immunity characterized by high T. gondii-specific IgG titers, CD4+/CD8+ T-cell activation, and IFN-γ production. Vaccinated mice achieved 100% survival against lethal RH challenge and showed a 99.5% reduction in brain cyst burden following PRU strain infection. Furthermore, the vaccine completely prevented vertical transmission in a pregnancy model. Transcriptomic analysis revealed widespread dysregulation in the Δddx6 strain, particularly downregulation of key virulence factors (ROPs, SAGs) and genes involved in intracellular transport. These findings demonstrate that targeting DDX6 generates a highly attenuated yet strongly immunogenic strain with exceptional cross-stage protective efficacy against acute, chronic, and congenital toxoplasmosis, representing a promising vaccine candidate worthy of further development.

A nanoluciferase-expressing attenuated CHIKV vaccine strain enables rapid and visual drug screening in vitro and in vivo

ABSTRACT Climate change has expanded the range of vectors for the chikungunya virus (CHIKV), thereby increasing its global threat. This situation underscores the urgent need for antiviral therapies. However, the development of drugs is limited by the need for Biosafety Level 3 (BSL-3) containment and the lack of tools for real-time infection monitoring. To address these challenges, we developed a replication-competent reporter virus called CHIKV-ΔnsP3-Nluc. We inserted the Nano-luciferase (Nluc) gene into the attenuation site of the FDA-approved live-attenuated vaccine strain VLA1553 (IXCHIQ). This modified virus retains an attenuated phenotype, replicating more slowly than wild-type CHIKV while maintaining high genetic stability. Nluc activity correlates well with viral titer (R2 = 0.9689), enabling precise quantification of viral replication. In interferon-deficient A6 mice, CHIKV-ΔnsP3-Nluc caused a lethal yet attenuated infection. Real-time bioluminescence imaging revealed that the spleen and kidneys were the primary replication sites, with luminescence intensity closely matching viral titers (R2 = 0.8388). We confirmed the platform’s potential for rapid antiviral screening using the nucleoside analog 4′-fluorouridine (4′-FlU). This compound inhibited viral replication in vitro (EC50= 0.063 µM) and significantly reduced viremia and mortality in vivo at a dose of 10 mg/kg, resulting in 100% survival. CHIKV-ΔnsP3-Nluc is a BSL-2-compliant tool that combines the safety of a vaccine candidate with a sensitive reporter function. It has the potential to accelerate studies of CHIKV pathogenesis and assist in developing new antiviral therapies. IMPORTANCE The expanding range of CHIKV outbreaks poses a threat to global public health, and there is currently a lack of suitable tools for the development of antiviral drugs. In this study, we developed a Nano-luciferase reporter virus based on the attenuated vaccine strain VLA1553 to track viral infection. This viral assay can effectively evaluate the inhibitory and protective effects of candidate compounds both in vitro and in vivo, and its luciferase signal reliably reflects the level of viral replication. Our research provides a safe and stable tool that serves as a suitable platform for the development of antiviral drugs targeting CHIKV. The expanding range of CHIKV outbreaks poses a threat to global public health, and there is currently a lack of suitable tools for the development of antiviral drugs. In this study, we developed a Nano-luciferase reporter virus based on the attenuated vaccine strain VLA1553 to track viral infection. This viral assay can effectively evaluate the inhibitory and protective effects of candidate compounds both in vitro and in vivo, and its luciferase signal reliably reflects the level of viral replication. Our research provides a safe and stable tool that serves as a suitable platform for the development of antiviral drugs targeting CHIKV.