Journal: PLoS ONE

Article Title: A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice

doi: 10.1371/journal.pone.0106597

Figure Lengend Snippet: (a) After a 24 hr infection, total infected Vero cell lysates were collected and proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Blot was probed with anti-VSV-N and anti-VSV-G IN CT, which does not recognize G NJ . IN: rVSV-EnvG 4 -G 6 IN ; NJ: rVSV-EnvG 4 -G 6 NJ . (b-d) Sucrose-gradient purified rVSV-EnvG 4 -G 6 particles were separated by SDS-PAGE. (b) Blots of 10 6 pfu of rVSV-EnvG 4 -G 6 IN and 2.5×10 6 pfu rVSV-EnvG 4 -G 6 NJ vectors were probed separately for EnvG using anti-gp120. (c) Blot of 10 6 pfu rVSV-EnvG 4 -G 6 IN was probed with anti-VSV-G IN CT. (d) Blot of 10 6 pfu rVSV-EnvG 4 -G 6 IN and rVSV-EnvG 4 -G 6 NJ was probed with anti-VSV-G. (e) Replication kinetics of rVSV-EnvG 4 -G 6 and rVSV-G 4 viruses in Vero cells. Vero cells were infected in 6-well plates at an MOI of 0.1. At various intervals post infection, supernatant was collected from duplicate wells and virus was titrated. Plaques were counted manually after cell fixation.

Article Snippet: The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5′terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG.

Techniques: Infection, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Virus

Journal: PLoS ONE

Article Title: A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice

doi: 10.1371/journal.pone.0106597

Figure Lengend Snippet: Murine Immunization Regimens.

Article Snippet: The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5′terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG.

Techniques:

Journal: PLoS ONE

Article Title: A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice

doi: 10.1371/journal.pone.0106597

Figure Lengend Snippet: (a) After a 22 hr infection, Vero cells were detached from plate by gentle trypsin treatment and resuspended in PBS. 5×10 6 cells were analyzed for VSV G and HIV-1 EnvG surface expression. All cells analyzed for Env staining were first gated as positive for G staining. (b) For rVSV staining, 10 9 pfu of virus was bound to alum at 37°C with agitation. rVSV/alum conjugates were stained with titrated anti-VSV-G (Vi10) or anti-HIV-1 Env Ab followed by anti-human IgG or anti-mouse IgG2a Alexa555 and acquired on a modified LSRII flow cytometer. Median fluorescent intensity (MFI) was determined for each Ab dilution. (c) 10 9 pfu of virus was incubated with SYTO 63 nucleotide stain in PBS for 30 min at RT followed by incubation with anti-VSV-G (Vi10) and then anti-mouse IgG2a Alexa555. Virus was analyzed as described above. Minimum threshold settings on SSC were used to increase sensitivity for small particles and FSC and SSC parameters were set to log scale. Deionized water was run for 15 min to equilibrate for low threshold noise. ∼50,000 events were acquired for the PBS control, ∼10 6 events were acquired for virus samples. Particles staining positive for nucleic acid that were above the noise threshold were gated on, and the amount of anti-G staining for those populations were compared.

Article Snippet: The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5′terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG.

Techniques: Infection, Gentle, Expressing, Staining, Virus, Modification, Flow Cytometry, Incubation, Control

Journal: PLoS ONE

Article Title: A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice

doi: 10.1371/journal.pone.0106597

Figure Lengend Snippet: (a) Mean body weights and temperatures (b) of inoculated mice. (c) Mean copy numbers of VSV N genomic RNA or (d) VSV N mRNA per mg of indicated tissue or mL of blood. N = 4–12, dependent on study day. Fresh tissue specimens were homogenized, clarified by centrifugation and supernatants were subjected to RNA extraction and qPCR. All samples were tested in duplicate. Dotted lines indicate limits of detection. SEM is shown. * p <0.05 for comparison of rVSV-EnvG 4 -G 6 to rVSV-G 4 . All PBS and rVSV-EnvG 4 -ΔG values were found to be significantly lower than rVSV-EnvG 4 -G 6 and rVSV-G 4 values.

Article Snippet: The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5′terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG.

Techniques: Centrifugation, RNA Extraction, Comparison

Journal: PLoS ONE

Article Title: A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice

doi: 10.1371/journal.pone.0106597

Figure Lengend Snippet: (a) Mean anti-Env endpoint titers (4 animals/group) against JR-FL foldon trimer were determined over the course of the 11 week experiment as described previously.(b) Week 6 endpoint titers for mice primed with pEnvG/pIL-12 (group 1/2/3, N = 12), IN rVSV-EnvG 4 -G 6 (group 4/5, N = 8), or IM rVSV-EnvG 4 -G 6 (group 6/7, N = 8). *p<0.05 compared to group 1/2/3. (c) Week 8 endpoint titers for all groups. (d) Week 11 endpoint titers for all groups. *p<0.05 compared to group 1. # p<0.05 compared to group 2. + p<0.05 compared to group 3. ∧p<0.05 compared to group 4. ∇ p<0.05 compared to group 5. (e) Ratio of IgG2a to IgG1endpoint titers at week 11. (f) Neutralization of HIV-1 virus pseudo-typed with SF162.LS Env as measured in a standard TZM-bl neutralization assay using IgG purified from week 11 sera of selected groups. SEM is shown.

Article Snippet: The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5′terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG.

Techniques: Neutralization, Virus, Purification