Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: In vitro expression analysis of hepatitis B surface antigen mRNA vaccines. LNP-encapsulated L mRNA, M mRNA, and S mRNA were transfected into 293T cells in different combinations, and the dosage of each single mRNA was 5 μg for each treatment. ( a ) The proportion of cells expressing pre-S2 antigen or S antigen on the surface was detected by FCM at 48 h and 96 h after transfection, respectively. The results of FCM were showed using density maps. ( b ) WB analysis of the expression or secretion of the L protein (42 kDa), M protein (31 kDa), and S protein (27 kDa) in the cell membrane or supernatants at 48 h after transfection.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: In Vitro, Expressing, Vaccines, Transfection, Membrane

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Humoral immune response analysis of HBV Tg mice immunized with the hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for the immunization of BALB/c HBV Tg mice with different combinations of mRNAs encoding hepatitis B surface antigens or with control vaccines. ( b ) CLIA detection of serum HBsAb levels in HBV Tg mice at weeks 0–32 of the immunization experiment. ( c ) Analysis of changes in the serum HBsAb concentration in mice whose HBsAb concentration was consistently less than 2000 IU/L and greater than 0 IU/L. ( d ) Correlation analysis between the serum levels of HBsAb and reduced HBsAg levels. ( e ) Indirect ELISA analysis of the binding ability of induced serum IgG to recombinant HBsAg, the pre-S peptide, or the LMS VLP after immunization with different vaccines. ( f ) Variable slope (four parameters) analysis via nonlinear regression (curve fit) was performed for the pre-S antigen-specific indirect ELISA. Representative results are presented as the means ± standard deviations (SDs).

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines, Control, Concentration Assay, Indirect ELISA, Binding Assay, Recombinant

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Serological and virological response analysis of pAAV HBV1.2 mice immunized with hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for immunization of pAAV HBV1.2 mice with LMS mRNA vaccines, S mRNA, or GFP mRNA. Changes in serum ( b ) HBsAg, ( c ) HBsAb, and ( d ) HBeAg from 0 to 5 weeks after immunization of pAAV HBV-1.2 mice with different vaccines. ( e ) Q–PCR detection of serum HBV DNA levels in immunized mice at week 0 and week 5. Representative results are presented as the means ± SDs.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Serological and virological response analysis of rAAV8 HBV1.3 mice immunized with hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for immunization of rAAV8 HBV1.3 (1 × 10 10 vg/mouse) mice with hepatitis B surface antigen mRNA vaccines in different combinations, S mRNA, or GFP mRNA. Changes in serum ( b ) HBsAg, ( c ) HBsAb, and ( d ) HBeAg from 0 to 9 weeks of immunization with different vaccines. ( e ) Q–PCR detection of serum HBV DNA levels in immunized mice at week 0 and week 9. Representative results are presented as the means ± SDs.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: The analysis of antigen-dependent T-cell responses activated by Hepatitis B surface antigen mRNA vaccines. ( a ) BALB/3T3 cells were transiently transfected with pD2531.L, pD2531.M, and pD2531.S plasmids (1:1:1 mass ratio) via Lipofectamine 3000, and FCM was performed to detect pre-S2 antigen and S antigen expression on the cell surface. ( b ) After immunization of BALB/c mice with different hepatitis B surface antigen mRNA vaccines or PBS, splenic CD4 + T cells were isolated and stimulated with BALB/3T3 cells transiently expressing the LMS antigen. Meanwhile, antigen-incubated T cells from PBS-immunized mice were used as negative control and CD3/CD28 bead-stimulated T cells from PBS-immunized mice were used as positive control. FCM was used to measure the level of IFN-γ in CD4 + T cells after the addition of brefeldin A.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines, Transfection, Expressing, Isolation, Incubation, Negative Control, Positive Control

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Schematic diagram of the mechanism by which the LMS mRNA therapeutic vaccine disrupts HBsAg-mediated immune tolerance and reactivates immune responses against HBV. The numbers 1–6 marked on the membrane structure of L protein represent intracellular structure of S antigen, T cell recognition epitopes of pre-S1 antigen, hepatocyte surface receptor NTCP binding site for pre-S1 antigen, S antigen (highly variable region) with a-antigen determinant, lipid membrane, hepatocyte binding region for pre-S2 antigen, membrane attachment region of HBV-infected hepatocytes, respectively.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Membrane, Binding Assay, Infection

Journal: Virology Journal

Article Title: Prevalence of a virus similar to human hepatitis B virus in swine

doi: 10.1186/1743-422x-7-60

Figure Lengend Snippet: Figure 3 Immunohistochemical analysis of HBsAg and HBcAg in liver tissues.(A) Strong HBsAg immune positivity was shown in hepatocytes (arrow). (B) Immunopositivity for HBsAg was mainly distributed in cytoplasm of hepatocytes. (C, D) HBcAg was distributed mainly distributed nucleus of hepatocytes. Spotty parenchymal cell degeneration, with necrosis and karyopyknosis (arrow) of hepatocytes were observed. Original magnification×400. (A, C: liver sample from CP74; B, D: liver sample from DX385).

Article Snippet: Sections were incubated with blocking buffer (Zymed Laboratories Inc., San Diego, USA) containing 20% normal goat serum (Gibco) and 80% PBS (0.01 M, pH 7.4) at 37°C for 30 min. After discarding the goat serum, sections were incubated in primary monoclonal antibodies against HBsAg and HBcAg (Zhongshan Golden Bridge Biotech Co. Ltd., Beijing, China) diluted in PBS, for 2 hours at 37°C.

Techniques: Immunohistochemical staining

Journal: Virology Journal

Article Title: Prevalence of a virus similar to human hepatitis B virus in swine

doi: 10.1186/1743-422x-7-60

Figure Lengend Snippet: Figure 4 Viral particles in swine sera and hepatocytes revealed by electron microscopy. A: Electron micrographs of negatively stained SHBV particles from HBsAg positive serum. Two types of particles were observed which are similar in size (20 nm and 40 nm) and morphology, like complete and empty viral particles of SHBV. B: Virus-like particles in the nucleus of hepatocytes (liver sample from DX385).

Article Snippet: Sections were incubated with blocking buffer (Zymed Laboratories Inc., San Diego, USA) containing 20% normal goat serum (Gibco) and 80% PBS (0.01 M, pH 7.4) at 37°C for 30 min. After discarding the goat serum, sections were incubated in primary monoclonal antibodies against HBsAg and HBcAg (Zhongshan Golden Bridge Biotech Co. Ltd., Beijing, China) diluted in PBS, for 2 hours at 37°C.

Techniques: Electron Microscopy, Staining, Virus