Project description:Comparison of unmodified and modified mRNA platforms for Andes virus mRNA vaccine in rodent models Kuzmin IV, Soto-Acosta R, Pruitt L, Wasdin PT, Kedarinath K, Hernandez KR, Gonzales KA, Hill K, Weidner NG, Mire C, Engdahl TB, Moon WJ, Popov V, Crowe JE, Georgiev IS, Garcia-Blanco MA, Abbott RK, Bukreyev A. This SuperSeries is composed of the SubSeries listed below.
Project description:A total of 10 cDNA libraries were constructed, representing 3 and 2 independent biological replicates of S. aureus strain KES34 (unmodified ribosomes) and KES30 (m6A2058-modified ribosomes), respectively. Translational efficiency (RPF/mRNA ratios), total mRNA abundance and ribosome density of each ORFs were calculated.
Project description:Andes virus (ANDV) is a rodent-borne zoonotic orthohantavirus endemic in South America that causes hantavirus pulmonary syndrome in humans, with up to a 40% case fatality rate. We developed ANDV mRNA vaccines based on the M segment of the viral genome that codes for glycoproteins Gn and Gc in a single open reading frame of glycoprotein precursor (GPC). We generated RNAs either with regular uridine (U-mRNA) or N1-methylpseudouridine (m1Ψ-mRNA). Mice immunized by either ANDV U-mRNA or m1Ψ-mRNA developed similar germinal center responses in lymph nodes. Single cell RNA and BCR sequencing of germinal center B cells from vaccinated mice demonstrated similar levels of activation, except an additional cluster of cells exhibiting strong interferon response that was present in animals vaccinated with U-mRNA but not m1Ψ-mRNA. Furthermore, similar immunoglobulin class-switching and somatic hypermutations were observed for the two vaccines. Golden Syrian hamsters were immunized intramuscularly with 2 doses of the vaccines on days 0 and 21. The titers of Gn/Gc-binding antibodies were moderately greater for U-mRNA construct than for m1Ψ-mRNA construct, however, the titers of ANDV-neutralizing antibodies were equivalent. Vaccinated animals were challenged with a lethal dose of ANDV at 21 days after the boost, along with the naïve control group. All control animals succumbed to infection whereas all vaccinated animals survived without any detectable disease or viral load. The data demonstrate the development of effective vaccines against ANDV and the lack of a significant effect of m1Ψ mRNA modification on immunogenicity and protection in the hamster model.
Project description:Andes virus (ANDV) is a rodent-borne zoonotic orthohantavirus endemic in South America that causes hantavirus pulmonary syndrome in humans, with up to a 40% case fatality rate. We developed ANDV mRNA vaccines based on the M segment of the viral genome that codes for glycoproteins Gn and Gc in a single open reading frame of glycoprotein precursor (GPC). We generated RNAs either with regular uridine (U-mRNA) or N1-methylpseudouridine (m1Ψ-mRNA). Mice immunized by either ANDV U-mRNA or m1Ψ-mRNA developed similar germinal center responses in lymph nodes. Single cell RNA and BCR sequencing of germinal center B cells from vaccinated mice demonstrated similar levels of activation, except an additional cluster of cells exhibiting strong interferon response that was present in animals vaccinated with U-mRNA but not m1Ψ-mRNA. Furthermore, similar immunoglobulin class-switching and somatic hypermutations were observed for the two vaccines. Golden Syrian hamsters were immunized intramuscularly with 2 doses of the vaccines on days 0 and 21. The titers of Gn/Gc-binding antibodies were moderately greater for U-mRNA construct than for m1Ψ-mRNA construct, however, the titers of ANDV-neutralizing antibodies were equivalent. Vaccinated animals were challenged with a lethal dose of ANDV at 21 days after the boost, along with the naïve control group. All control animals succumbed to infection whereas all vaccinated animals survived without any detectable disease or viral load. The data demonstrate the development of effective vaccines against ANDV and the lack of a significant effect of m1Ψ mRNA modification on immunogenicity and protection in the hamster model.
Project description:The high-throughput sequencing has become a standard tool for analyzing gene expression. Usually a cDNA/DNA library is constructed with 5' and 3' linkers and then sequenced. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 3' methylation, affecting the linker addition during cloning processes. Small RNA is expressed at much lower levels than mRNA, making it more difficult to clone small RNA using a small amount of total RNA. Here we have developed a new strategy to clone modified/unmodified small RNA in an all-liquid-based reaction in a single PCR tube using as little as 20 ng total RNA. The 7-hour cloning process only needs ~1 hour labor time. Moreover, this method can be used to clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA. Since the linkers used are derived from the Illumina Truseq linkers for DNA cloning, the PCR primers based on the linker sequences can be used to obtain amplicons derived from small RNA/mRNA/DNA. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, accurate and versatile. Moreover, the all-liquid-based reaction can be performed in an automated manner.
Project description:We longitudinally profiled plasma proteomes in 54 adults vaccinated with the BNT162b2 (Pfizer-BioNTech) or ChAdOx1-S (Oxford-AstraZeneca) vaccines. Blood was collected pre-vaccination (V0) and 1-7 days after the 1st doses (BNT162b2 or mRNA-1273, V1) to assess innate and early adaptive responses. We identified key differences in the immune responses induced by the ChAdOx1-S and BNT162b2 vaccines that were correlated with subsequent antigen-specific antibody and T cell responses or vaccine reactogenicity. We observed that vaccination with ChAdOx1-S but not BNT162b2 induced a memory-like response after the first dose, which was correlated with the expression of several proteins involved in complement and coagulation. The COVID-19 Vaccine Immune Responses Study (COVIRS) thus represents a major resource to understand the immunogenicity and reactogenicity of these COVID-19 vaccines.
Project description:Objectives: To investigate the differences in efficacy and safety between the vector vaccine ChAdOx1 nCoV-19/AZD1222 (Oxford-AstraZeneca) and mRNA-based vaccine mRNA-1273 (Moderna), and evaluate the impact of anti-rheumatic medications on immunogenicity in patients with autoimmune rheumatic diseases (AIRD). Methods: From September 16 to November 15, 2021, we consecutively enrolled participants aged≥20 years with AIRD who received COVID-19 vaccination. The level of serum IgG antibodies to the SARS-Cov-2 receptor-binding domain on the spike protein S1 subunit was quantified by electrochemiluminescence immunoassay at 4-6 weeks after vaccination. The immunogenicity and adverse reactions between ChAdOx1 nCov-19/AZD1222 and mRNA-1273 were compared. Results: Of the 243 rheumatic disease patients who received COVID-19 vaccines, 113 and 130 were immunized with AZD1222 and mRNA-1273, respectively. The anti-SARS-CoV-2 IgG seropositivity rate was 78.8% (89/113) for AZD1222 and 83.1% (108/130) for mRNA-1273. The level of anti-SARS-Cov-2 IgG was higher in patients who received mRNA-1273 than in those who received AZD1222. Prednisolone-equivalent dose >5 mg/day, methotrexate (MTX), non-anti-tumor necrosis factor (TNF)-a biologics, and Janus kinase (JAK) inhibitor use were associated with inferior immunogenicity. All reported adverse reactions were minor. More localized pain at the injection site and less fever and chills were observed in patients receiving mRNA-1273 compared with those receiving AZD1222. Rheumatic disease activities after vaccination remained stable in most patients. Conclusions: mRNA-1273 and AZD1222 vaccines exhibited differential immunogenicity and adverse reaction profiles. Our study findings support temporary discontinuation of daily prednisolone dose >5 mg, MTX, non-anti-TNF-a biologics, or JAK inhibitors after COVID-19 vaccination.