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Molecular Therapy : the Journal of the... Feb 2024Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated...
Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8 T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.
Topics: Mice; Animals; Lymphocytic choriomeningitis virus; CD8-Positive T-Lymphocytes; Neoplasms; Antigens, Neoplasm; Cancer Vaccines; Autoantigens; Tumor Microenvironment
PubMed: 38058126
DOI: 10.1016/j.ymthe.2023.11.026 -
Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses.Scientific Reports Nov 2023Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on...
Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean-Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.
Topics: Cricetinae; Female; Male; Guinea Pigs; Animals; Mice; Hemorrhagic Fever Virus, Crimean-Congo; Dengue Virus; Models, Animal; Arenaviruses, New World; Cyclophilin A; RNA
PubMed: 37938597
DOI: 10.1038/s41598-023-45740-w -
Journal of Infection and Public Health Dec 2023Multiplex real-time PCR is a quick and cost effective method for detection of various gene simultaneously. HFSV (Hemorrhagic Fever Syndrome Virus) is a newly emerging...
BACKGROUND
Multiplex real-time PCR is a quick and cost effective method for detection of various gene simultaneously. HFSV (Hemorrhagic Fever Syndrome Virus) is a newly emerging infectious disease because of globalization and climate change. We tried to develop a molecular diagnostic technique for various causative viruses and evaluate its usefulness for improving public health.
METHODS
Molecular diagnostic test method that qualitatively detects viruses causing viral hemorrhagic fevers hired Taq-Man Real-time RT-PCR technique. The Ct value was experimentally observed three or more times at the RNA concentration before and after the detection limit. After designing a multiplex real-time RT-PCR test for target gene of selected 17 viruses, the detection limit for each target and the presence or absence of cross-reaction and interference reaction were evaluated to determine its availability.
RESULTS
Six kinds of viruses, including Crimean-Congo hemorrhagic fever virus, Omsk hemorrhagic fever virus, Sabia virus, Chapare virus, Yellow fever virus, and Variola virus (A4L gene, B12R gene), were able to confirm the detection limit of 0.5 copies/μl, and other Ebola virus, Marburg virus, Rift Valley fever virus, Kyasanur Forest disease virus, Junin virus, Guanarito virus, Machupo virus, Chikungunya virus, Hantavirus, Dengue virus types 1-4, and Lassa virus (L gene, GPC gene), and 11 kinds of viruses, the detection limit was confirmed at 5 copies/μl. No cross-reaction or interference between detected genes was observed.
CONCLUSION
The virus test method developed through this study using multiplex is expected to be used for public health and quarantine as a test method that can be used when a hemorrhagic fever virus of unknown cause is introduced.
Topics: Animals; Humans; Hemorrhagic Fever Virus, Crimean-Congo; Dengue Virus; Viruses; Arenaviruses, New World; Orthohantavirus; Polymerase Chain Reaction
PubMed: 37866271
DOI: 10.1016/j.jiph.2023.10.012 -
Nature Microbiology Mar 2024Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many...
Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.
Topics: Humans; Lassa Fever; Genome-Wide Association Study; Seroepidemiologic Studies; Lassa virus; Fever; Human Genetics
PubMed: 38326571
DOI: 10.1038/s41564-023-01589-3 -
Health Science Reports Oct 2023The Lassa virus is an RNA virus belonging to the family. It is responsible for Lassa fever, an acute viral zoonosis of the severe hemorrhagic fever type with... (Review)
Review
The Lassa virus is an RNA virus belonging to the family. It is responsible for Lassa fever, an acute viral zoonosis of the severe hemorrhagic fever type with manifestations of fever, muscle pain, sore throat, nausea, vomiting, and chest and abdominal pain. Lassa fever is endemic in West Africa, where the first case was reported in 1969 in Lassa, a town in Nigeria, more than 50 years ago, and it is estimated that nearly 5000 deaths occur in West Africa each year. Nigeria is one of the endemic hotspots and has experienced numerous recurrent outbreaks of Lassa fever due to the increased multiplication of the host reservoir, . For the Lassa epidemics in 2022 and January 2023 alone, Nigeria accounts for a quarter of the annual deaths from this disease. Poor lifestyle and hygiene, difficulty in diagnosis due to nonspecific symptomatology, lack of effective treatment based on clinical evidence, an ineffective human immunization program combined with a health system that is not adapted or equipped to control and prevent recurrent deadly epidemics, and an outdated regional disease surveillance system in West Africa are some of the challenges that must be overcome to rapidly and effectively eradicate this disease, whose area of spread is constantly expanding as a result of the movement of populations in the context of economic and socio-cultural activities.
PubMed: 37885466
DOI: 10.1002/hsr2.1628 -
Emerging Infectious Diseases Nov 2023Lassa fever, caused by Lassa virus (LASV), is endemic to West Africa, where ≈300,000 illnesses and ≈5,000 deaths occur annually. LASV is primarily spread by infected...
Lassa fever, caused by Lassa virus (LASV), is endemic to West Africa, where ≈300,000 illnesses and ≈5,000 deaths occur annually. LASV is primarily spread by infected multimammate rats via urine and fomites, highlighting the need to understand the environmental fate of LASV. We evaluated persistence of LASV Josiah and Sauerwald strains on surfaces, in aqueous solutions, and with sodium hypochlorite disinfection. Tested strains were more stable in deionized water (first-order rate constant [k] for Josiah, 0.23 days; for Sauerwald, k = 0.34 days) than primary influent wastewater (Josiah, k = 1.3 days; Sauerwald, k = 1.9 days). Both strains had similar decay rates on high-density polyethylene (Josiah, k = 4.3 days; Sauerwald, k = 2.3 days) and stainless steel (Josiah, k = 5.3 days; Sauerwald, k = 2.7 days). Sodium hypochlorite was highly effective at inactivating both strains. Our findings can inform future risk assessment and management efforts for Lassa fever.
Topics: Animals; Rats; Lassa virus; Lassa Fever; Disinfection; Sodium Hypochlorite; Africa, Western
PubMed: 37877545
DOI: 10.3201/eid2911.230678 -
Nature Communications Nov 2023During infection, virus-specific CD8 T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral...
During infection, virus-specific CD8 T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8 T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8 T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8 T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8 T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.
Topics: CD8-Positive T-Lymphocytes; Cell Cycle; Cullin Proteins; Lymphocytic choriomeningitis virus; Ubiquitin-Protein Ligases; Proto-Oncogene Proteins c-myc
PubMed: 37925424
DOI: 10.1038/s41467-023-42765-7 -
Blood Dec 2023Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the...
Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies it is found that arenaviruses, such as Lassa, Machupo, and Guanarito viruses cause HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito, and Lassa viruses is associated with cutaneomucosal, cerebral, digestive, and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation, and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic arenaviruses.
Topics: Animals; Arenaviridae; Arenavirus; Hemorrhagic Fevers, Viral; Hemorrhage; Hemostasis; Hemostatics; Macaca
PubMed: 37699247
DOI: 10.1182/blood.2023020351 -
Nature Communications Jan 2024Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover,...
Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.
Topics: Animals; Guinea Pigs; Lassa virus; Single-Domain Antibodies; Lassa Fever; Antibodies, Viral; Antibodies, Neutralizing
PubMed: 38177144
DOI: 10.1038/s41467-023-44534-y -
Cell Death & Disease Dec 2023Acute infection and chronic infection are the two most common fates of pathogenic virus infections. While several factors that contribute to these fates are described,...
Acute infection and chronic infection are the two most common fates of pathogenic virus infections. While several factors that contribute to these fates are described, the critical control points and the mechanisms that underlie infection fate regulation are incompletely understood. Using the acute and chronic lymphocytic choriomeningitis virus (LCMV) infection model of mice, we find that the early dynamic pattern of the IFN-I response is a differentiating trait between both infection fates. Acute-infected mice generate a 2-wave IFN-I response while chronic-infected mice generate only a 1-wave response. The underlying cause is a temporal difference in CD8 T cell-mediated killing of splenic marginal zone CD169+ macrophages. It occurs later in acute infection and thus enables CD169+ marginal zone macrophages to produce the 2nd IFN-I wave. This is required for subsequent immune events including induction of inflammatory macrophages, generation of effector CD8+ T cells and virus clearance. Importantly, these benefits come at a cost for the host in the form of spleen fibrosis. Due to an earlier marginal zone destruction, these ordered immune events are deregulated in chronic infection. Our findings demonstrate the critical importance of kinetically well-coordinated sequential immune events for acute infection control and highlights that it may come at a cost for the host organism.
Topics: Mice; Animals; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Persistent Infection; Mice, Inbred C57BL; CD8-Positive T-Lymphocytes; Macrophages
PubMed: 38110339
DOI: 10.1038/s41419-023-06374-y