Journal: mBio

Article Title: Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and Pathogenesis In Vivo

doi: 10.1128/mBio.00981-14

Figure Lengend Snippet: Expression kinetics of MHV68-encoded miRNAs during lytic infection. Stem-loop qRT-PCR was used to determine the level of expression of MHV68-encoded mature miRNAs during lytic replication. NIH 3T12 fibroblasts were infected with MHV68 at MOI 5 and then harvested at the indicated time points. Stem-loop qRT-PCR was performed using TaqMan primers and probe sets (see Table S2 in the supplemental material). (A) Expression time course for all tested viral miRNAs relative (10 2 ) to the endogenous noncoding RNA control snoRNA234. (B) Time course for all tested viral miRNAs expressed as approximate miRNA copy number per cell, based on the synthetic miR-2-5p standard curve. (C) Viral miRNAs that peak between 16 and 48 hpi, expressed as miRNA copy number per cell. (D) Viral miRNAs that peak at 48 hpi. (E) Viral miRNAs with consistent low-level expression throughout the time course, depicted on an enhanced scale. All data are means ± standard deviations (SD) from 3 experiments.

Article Snippet: NIH 3T12 murine fibroblasts (ATCC CCL-164) and murine embryonic fibroblasts (MEFs) from C57BL/6J mice (Global Stem GSC-6002) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin, and 2 mM l -glutamine.

Techniques: Expressing, Infection, Quantitative RT-PCR, Control

Journal: mBio

Article Title: Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and Pathogenesis In Vivo

doi: 10.1128/mBio.00981-14

Figure Lengend Snippet: miRNA mutations incorporated in MHV68.Zt6 and corresponding expression of miRNAs and surrounding genes. (A) Depiction of MHV68.Zt6 mutations, including deletion of TMER1 to TMER5, TMER7, and TMER8 miRNA stem-loops (red X) and insertion of the PolIII stop site (red line) following vtRNA6 in TMER6. (B) Expression of viral miRNAs from wild-type MHV68 marker virus or MHV68.Zt6, relative (10 2 ) to the endogenous noncoding RNA control snoRNA202. NIH 3T12 fibroblasts were infected with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 and then harvested at 48 hpi. Stem-loop qRT-PCR was performed as described for Fig. 1 . Values are means ± SD from 2 experiments. (C) Expression of viral coding genes surrounding miRNA mutations. NIH 3T12 fibroblasts were infected with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 for 24 h. M1, M2, and M3 expression was quantified using qRT-PCR. Values are relative (10 3 ) to endogenous GAPDH expression and are means ± SD from 3 experiments.

Article Snippet: NIH 3T12 murine fibroblasts (ATCC CCL-164) and murine embryonic fibroblasts (MEFs) from C57BL/6J mice (Global Stem GSC-6002) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin, and 2 mM l -glutamine.

Techniques: Expressing, Marker, Virus, Control, Infection, Quantitative RT-PCR

Journal: mBio

Article Title: Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and Pathogenesis In Vivo

doi: 10.1128/mBio.00981-14

Figure Lengend Snippet: MHV68 miRNAs are dispensable for lytic replication. Plaque assays were used to determine the titer of wild-type or miRNA deletion mutant viruses during acute replication in vitro and in vivo . (A) Lytic replication in fibroblasts in vitro . Single-step and multistep growth curves were generated following infection of NIH 3T12 fibroblasts with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 or 0.05, respectively. At the specified time points, cells and supernatant fluid were collected, and titers were determined by plaque assay. Data are means ± SD of 3 experiments. (B) Lytic replication in lungs in vivo . C57BL6/J mice were infected i.n. with 10 4 PFU MHV68.ORF73βla or MHV68.Zt6. At 5 or 8 dpi, lungs were harvested and viral titers were determined by plaque assay. Lines represent the mean titers for eight individual mice. For all experiments, statistical significance was determined by Student’s t test. *, P < 0.05; **, P < 0.01.

Article Snippet: NIH 3T12 murine fibroblasts (ATCC CCL-164) and murine embryonic fibroblasts (MEFs) from C57BL/6J mice (Global Stem GSC-6002) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin, and 2 mM l -glutamine.

Techniques: Mutagenesis, In Vitro, In Vivo, Generated, Infection, Plaque Assay

Journal: NPJ Vaccines

Article Title: Heterologous immunization modulates B-cell epitope competition between helper peptides and the MPER segment in MPER/liposome vaccines

doi: 10.1038/s41541-026-01371-6

Figure Lengend Snippet: A Schematics of immunization schedule and heterologous immunization regimens. Balb/c mice were immunized subcutaneously with various MPER/liposome vaccines formulated with helper peptides and adjuvant at the specified time points. B MPER-specific IgG endpoint titers (log10) from ELISA of immune sera against NpMPER/liposome collected 30 days after the third immunization ( n = 5). Data are representative of two independent experiments. Error bars represent mean ± SEM. Statistical differences were assessed by the Kruskal–Wallis test with Dunn’s comparison test denoted by p values * p < 0.05, ** p < 0.01. C Relative binding activity of the purified polyclonal IgG antibodies elicited by each group for high-density MPER/liposome (1:50) assessed by SPR analysis. The NpMPER/liposome was captured on the L1 chip surface, and sensorgrams illustrate the specific binding signal, measured in response units (RUs), on the y -axis as a function of time during the association and dissociation phases on the x -axis for each antibody at a concentration of 30 μg/ml. Data shown are representative of three independent experiments, with non-specific antibody binding to NP366/liposome subtracted. D Frequency of MPER-specific ASCs in bone marrow 30 days after the final immunization with each group indicated in ( A ). Values represent background-subtracted data, with baseline ASC frequencies from naïve mice subtracted from each experimental group. Numbers of MPER-binding ASCs were determined by ELISPOT assay using high density NpMPER/liposome (1:50) as the capture antigen. Data are represented as mean ± SEM from one of three independent experiments ( n = 5). Statistically significant differences between different groups were determined by Kruskal-Wallis test with Dunn’s comparison test denoted by p values: * p < 0.05, *** p < 0.001. E % relative affinities of antibodies secreted from ASCs measured by ELISPOT assay. The % relative affinities were calculated by determining the ratio of ASCs detected on low density MPER/Liposome (1:500) and high density (1:50) MPER/liposome from bone marrow cells. Data is represented as mean ± SEM from one of the three independent experiments ( n = 5). No statistically significant differences were observed. F % relative affinities of MPER-specific IgGs in serum collected 30 days after the third immunization were assessed by calculating the ratio of MPER/liposome binding at low density (1:500) to high density (1:50) using ELISA (detailed in “Methods” section). The data are presented as mean ± SEM from one of two independent experiments, each with n = 5. No statistically significant differences were observed.

Article Snippet: Six-to eight-week-old Balb/c mice were purchased from Taconic Bioscience (Germantown, NY) and were housed and maintained in a specific pathogen-free animal research facility at DFCI.

Techniques: Vaccines, Adjuvant, Enzyme-linked Immunosorbent Assay, Comparison, Binding Assay, Activity Assay, Purification, Concentration Assay, Enzyme-linked Immunospot

Journal: NPJ Vaccines

Article Title: Heterologous immunization modulates B-cell epitope competition between helper peptides and the MPER segment in MPER/liposome vaccines

doi: 10.1038/s41541-026-01371-6

Figure Lengend Snippet: A Schematics of immunization schedule for Tfh and GC B cell analysis. Frequencies and numbers of Tfh (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) cells following booster immunization ( B ) (also see Supplementary Fig. for gating strategy), GC B cells (B220 + IgD − CD38 − GL7 + ) ( C ), and antigen-specific GC B cells ( D ). MPER- (B220 + IgD − CD38 − GL7 + MPER-PE + ) and LACK- (B220 + IgD − CD38 − GL7 + LACK-FITC + ) specific GC B cells or MPER- (B220 + IgD − CD38 − GL7 + MPER-PE + ) and HIV30- (B220 + IgD − CD38 − GL7 + HIV-FITC + ) specific GC B cells quantified by FACS and presented in bar graphs as percent and total cell number. For ( B – D ), Balb/c mice primed with pMPERTM/sLACK were boosted with NpMPER/sLACK (G3) or NpMPER/sHIV30 (G4). The Tfh and GC responses induced by G3 and G4 were assessed by flow cytometry from the spleen on day 12 after the boost. Symbols represent individual animals, and error bars indicate mean ± SEM. The data are cumulative from two independent experiments. Unimmunized mice were used as controls. Statistical significance for the A , B was determined using Kruskal–Wallis test with Dunn’s comparison test with p -values indicated as, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. E MPER-specific IgG titers from immune sera collected at the indicated timepoints. The broken lines indicate immune sera collected on day 29 and day 52 after primary immunization, respectively. The data are represented as binding curves, with log serial dilutions of serum on the x -axis and absorbance values on the y -axis. F ELISA to measure serum IgG titers for MPER/liposome, LACK/liposome, HIV30/liposome and OVA/liposome. The immune sera were collected 30 days after the third immunization and LACK- vs MPER-specific antibody titers induced by G3 regimen (left) are compared with MPER-, LACK-, HIV30- and OVA-specific antibody titers induced by G4 regimen (right). All peptides were N-terminally palmitoylated and arrayed on the surface of liposome for ELISA assay.

Article Snippet: Six-to eight-week-old Balb/c mice were purchased from Taconic Bioscience (Germantown, NY) and were housed and maintained in a specific pathogen-free animal research facility at DFCI.

Techniques: Cell Analysis, Flow Cytometry, Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: NPJ Vaccines

Article Title: Heterologous immunization modulates B-cell epitope competition between helper peptides and the MPER segment in MPER/liposome vaccines

doi: 10.1038/s41541-026-01371-6

Figure Lengend Snippet: Flow cytometric analysis and quantification of the percentage of Tfh cells ( A ), GC B cells ( B ) and percentage and total cell number of antigen-specific GC B cells ( C ) in the spleens of Balb/c mice on day 12 post primary immunization. Statistical significance was determined using Kruskal–Wallis test with Dunn’s comparison test, with p-values indicated as, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Mice were immunized with NpMPER/liposome with sLACK (sG1), sHIV30 (sG2) or sOVA helper peptides (sG3) encapsulated inside the liposome vaccine, respectively. Naïve mice ( n = 5), served as controls. Error bars represent mean ± SEM from n = 9 animals, based on one of the two independent experiments. D Comparison of antigen-specific serum IgG titers measured by ELISA after homologous boosting. MPER-specific IgG titers from immune sera of mice in each group collected 10 days after a single homologous boost were compared with anti-LACK (sG1), anti-HIV30 (sG2) or anti-OVA antibody titers (sG3). GC responses induced by co-delivery of helper peptides pLACK, pHIV30 or pOVA with NpMPER/liposome at primary response. Frequency of Tfh cells ( E ), frequency of GC B cells ( F ) and frequency and number of antigen-specific GC B cells ( G ) in the spleen of Balb/c mice on day 12 after primary immunization with NpMPER/pLACK (pG1), NpMPER/pHIV30 (pG2) and NpMPER/pOVA (pG3). Responses in naïve mice ( n = 4) were assessed as controls. Data shown are representative of two independent experiments ( n = 5). Statistical significance was determined using Kruskal–Wallis test with Dunn’s comparison test, with p -values indicated as, * p < 0.05, ** p < 0.01. H Immunogenicity of three different helper peptide antigens relative to the MPER in indicated formulations after homologous boosting. MPER-specific IgG titers by ELISA are compared across the three groups, each corresponding to the specific CD4 T cell helper peptide pLACK (pG1), pHIV30 (pG2), and pOVA (pG3). The data are presented as binding curves, with log serial dilutions of serum on the x -axis and absorbance values on the y -axis. I A relative binding affinity of the purified polyclonal IgG antibodies for MPER/liposome. The MPER/liposomes at low density (peptide to lipid ratio of 1:500) were captured on the L1 chip surface and the polyclonal IgG antibodies elicited by each group were passed over the chip surface for 3 min. The polyclonal IgG antibodies from each group were purified from immune sera collected 10 days after boost. Data shown are representative of three independent experiments, with non-specific antibody binding to NP366/liposome subtracted.

Article Snippet: Six-to eight-week-old Balb/c mice were purchased from Taconic Bioscience (Germantown, NY) and were housed and maintained in a specific pathogen-free animal research facility at DFCI.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Immunopeptidomics, Binding Assay, Purification, Liposomes

Journal: NPJ Vaccines

Article Title: Heterologous immunization modulates B-cell epitope competition between helper peptides and the MPER segment in MPER/liposome vaccines

doi: 10.1038/s41541-026-01371-6

Figure Lengend Snippet: A Schematic of heterologous immunization strategies. B Anti-MPER polyclonal IgG titers determined against high density NpMPER/liposome (1:50) by ELISA. Immune sera were collected 30 days after the third immunization, combined from 5 mice for each group and purified using gamma-bind plus affinity column for the assay. Data shown is representative of two independent experiments. C Frequency of MPER-specific ASCs in spleen and bone marrow. The MPER-specific ASCs from spleen (left) and bone marrow cells (right) at 30 days after the third immunization were determined from 8 mice by ELISPOT assay. Quantification of ASCs calculated from 10 6 cells from each group is indicated. Data are background-subtracted using ASC frequencies from naïve mice as baseline controls. Error bars indicate mean ± SEM. D % relative affinities of MPER/liposome binding IgG ASCs in bone marrow as assessed by ELISPOT assay. The frequencies of low-density MPER/liposome (1:500) and high-density MPER/liposome (1:50) binding IgG ASCs were determinedand the ratios of low-density MPER to high-density MPER binding plotted as % relative affinity. Higher percentage is indicative of greater affinity antibodies. Data are represented as mean ± SEM. Statistically significant differences between different groups were determined using Kruskal–Wallis test with Dunn’s comparison test, denoted by p values: * p < 0.05. E % relative affinities of MPER-specific purified polyclonal IgG antibodies measured by ELISA against low density (1:500) vs. high density (1:50 peptide to lipid ratio) MPER/liposome and two dilutions below the saturation point were considered as explained in detail in Methods section. The polyclonal antibodies were purified from immune sera combined from 5 mice for each group collected on day 30 after the third immunization. No statistically significant differences were observed. F Vaccine-elicited antibody binding to ADA gp145 expressed on the surface of 293T cells by flow cytometry. 293T cells were transfected to express ADA gp145 with MPER sequence mutated to the MPER immunogen (HxB2 MPER sequence), and binding was measured using anti-mouse IgG-PE. Flow cytometry plots show side scatter area (SSC-A) on the y -axis and PE fluorescence (ADA gp145 binding) on the x -axis. The data are presented as a representative example from one of two independent experiments. G Frequency and total cell number of MPER-specific memory B cells. Balb/c mice were immunized with pMPERTM/sLACK liposome (mG1) or pMPERTM/pHIV30 liposome (mG2), and the MPER-specific IgG memory B cells from each group was assessed in spleen on day 30 post-primary immunization. Data shown are represented as the aggregate of two independent experiments ( n = 12). Statistically significant differences between the groups were determined by two-way ANOVA (or mixed model) with Tukey’s multiple comparison test denoted by p values: **** p < 0.0001. H Difference in quantity of anti-MPER IgG responses elicited by mG1 and mG2. Immune sera were collected 10 days after the homologous booster immunization. I Comparison of MPER and helper peptide-specific antibody titers elicited by pMPERTM/sLACK (mG1) and by pMPERTM/pHIV30 (mG2). Data in H , I are represented as one of the two independent experiments with n = 6.

Article Snippet: Six-to eight-week-old Balb/c mice were purchased from Taconic Bioscience (Germantown, NY) and were housed and maintained in a specific pathogen-free animal research facility at DFCI.

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Affinity Column, Enzyme-linked Immunospot, Binding Assay, Comparison, Flow Cytometry, Transfection, Sequencing, Fluorescence