Fig 1.
Vaccines targeting SIV protease cleavage sites (PCSs) or full Gag and Env proteins.
(A) Diagram of the twelve protease cleavage sites (PCS1 through PCS12), located on three SIV polyproteins (Pr55Gag, Pr160Gag-Pol and Nef precursor), not drawn to scale. MA: matrix; CA: capsid; NC: nucleocapsid; TFP: transframe protein; PR: protease; RT: reverse transcriptase; and IN: integrase. (B) Peptide sequences of SIV immunogens in a conserved element vaccine targeting the PCSs (the PCS vaccine). Each sequence corresponds to -10 through +10 amino acid positions flanking each cleavage site. Slash (/) indicates the site of protease cleavage. These sequences were confirmed to be specific for SIV by NCBI protein BLAST and conserved among multiple SIV strains. The peptide immunogens were delivered as recombinant vesicular stomatitis viruses (rVSVpcs) and nanoparticles (NANOpcs). Peptide antigens with these sequences were also used in a Bio-Plex multiplexed assay to detect anti-PCS antibodies. (C) Sequences of three Gag or Env (non-PCS) peptides used in Bio-Plex to detect anti-Gag or Env antibodies, including one Gag peptide, named SIVgag, and two Env peptides, named SIVenv1 and SIVenv2. (D) Western blot analyses of protein expression from a full-length Gag and Env-based vaccine (the Gag/Env vaccine). VeroE6 cells were infected with recombinant vesicular stomatitis viruses (rVSVs) carrying full Gag or Env gene of SIVmac239 (rVSVgag/env) and the culture supernatants were analyzed by Western blot to detect Gag or Env protein expression using standard monoclonal antibodies (mAb, NIH AIDS Reagent Program) to Gag or Env. The full Gag and Env genes were also cloned into pVAX1 (a DNA vaccine vector), respectively, followed by NANO packaging (NANOgag/env). HEK293T cells were transfected with these DNA vaccines and analyzed by Western blot. (E) Vaccination scheme. Three groups (Control, PCS vaccine and Gag/Env vaccine) of eight female MCMs per group were primed and boosted on indicated weeks (wk). The Control group received empty rVSV virus and sterile water. One animal from the Gag/Env vaccine group was euthanized early due to severe health issues unrelated to vaccination, leaving seven animals in this group to complete the study. rVSV control vector (rVSV), rVSVpcs or rVSVgag/env was administered intramuscularly. NANO control vector (sterile water), NANOpcs or NANOgag/env was administered intranasally.
Fig 2.
Dynamics of PCS-specific IgG antibodies in cervicovaginal secretions.
Cervicovaginal lavage (CVL) samples from the vaccination experiments illustrated in Fig 1E were quantified for levels of IgG antibodies to each PCS peptide (y axis, expressed as ratios of anti-PCS IgG concentration to total protein concentration ×109) by a Bio-Plex multiplexed antibody assay, at indicated time points (x axis, weeks post prime). The Control group received empty rVSV virus and sterile water. Data are shown as each value from individual animals with median line, following subtraction of pre-vaccination values. Total animal numbers per group examined are n = 8 (Control or PCS group) or n = 7 (Gag/Env group). However, for technical stringency, animal samples collected on menstruation dates were excluded from analysis to rule out any potential menses blood contamination. As a result, for some of the data points in the graph, fewer individual values than the total animal number (n = 8 or n = 7) were available. Grey areas indicate a week interval post prime, 1st boost or 4th boost, with trends of antibody increase in the PCS vaccine group in response to vaccination. The trends did not reach statistical significance.
Fig 3.
Dynamics of Gag/Env-specific IgG antibodies in cervicovaginal secretions.
Cervicovaginal lavage (CVL) samples from the vaccination experiments illustrated in Fig 1E were quantified for IgG antibodies to Gag/Env (non-PCS) peptides, SIVgag, SIVenv1 and SIVenv2 (y axis, expressed as ratios of anti-non-PCS IgG concentration to total protein concentration ×109) by a Bio-Plex multiplexed antibody assay, at indicated time points (x axis, weeks post prime). The Control group received empty rVSV virus and sterile water. Data are shown as each value from individual animals with median line, following subtraction of pre-vaccination values. Total animal numbers per group examined are n = 8 (Control or PCS group) or n = 7 (Gag/Env group). However, for technical stringency, animal samples collected on menstruation dates were excluded from analysis to rule out any potential menses blood contamination. As a result, for some of the data points in the graph, fewer individual values than the total animal number (n = 8 or n = 7) were available. Grey areas indicate a week interval post prime, 1st boost or 4th boost, with trends of antibody increase in the PCS vaccine group in response to vaccination. The trends did not reach statistical significance.
Fig 4.
Fold changes of mucosal antibodies to each PCS peptide in response to vaccinations.
Graphs show fold changes of mucosal IgG antibodies to each PCS peptide (1 through 12) between the time of a prime/boost and one week after that single prime/boost (Prime, 1st, 2nd, 3rd or 4th), or between the baseline (the start of the full vaccination procedure) and one week after the last boost (the end of the full vaccination procedure) (Full). The Control group received empty rVSV virus and sterile water. Data are shown as each value from individual animals with interquartile range and median line. Total animal numbers per group examined are n = 8 (Control or PCS group) or n = 7 (Gag/Env group). However, for technical stringency, animal samples collected on menstruation dates were excluded from analysis to rule out any potential menses blood contamination. As a result, for some of the data points in the graph, fewer individual values than the total animal number (n = 8 or n = 7) were available. Fold changes of antibodies in response to prime, the 1st boost, the 4th boost and the full vaccination procedure showed trends of increase in the PCS vaccine group, compared to the other groups. The trends did not reach statistical significance.
Fig 5.
Fold changes of collective anti-PCS antibodies in response to vaccinations.
Mucosal IgG antibodies to all individual PCS types (PCS1 through PCS12) were collectively treated as anti-PCS antibodies for calculation. Graph shows fold changes between the time of a prime/boost and one week after that single prime/boost (Prime, 1st, 2nd, 3rd or 4th), or between the baseline (the start of the full vaccination procedure) and one week after the last boost (the end of the full vaccination procedure) (Full). The Control group received empty rVSV virus and sterile water. Total animal numbers per group examined are n = 8 (Control or PCS group) or n = 7 (Gag/Env group). However, for technical stringency, animal samples collected on menstruation dates were excluded from analysis to rule out any potential menses blood contamination. Bars represent mean ± SEM. The PCS vaccine group demonstrated significantly higher fold induction of anti-PCS antibodies than the Gag/Env vaccine and Control groups, in response to prime, the 1st boost, the 4th boost and the full vaccination procedure, as determined by Mann Whitney's test: **** p < 0.0001.
Fig 6.
Induction of antibodies to Gag/Env antigens.
(A) Graphs show fold changes of mucosal IgG antibodies to Gag/Env (non-PCS) peptides (SIVgag, SIVenv1 and SIVenv2) between the time of a prime/boost and one week after that single prime/boost (Prime, 1st, 2nd, 3rd or 4th), or between the baseline (the start of the full vaccination procedure) and one week after the last boost (the end of the full vaccination procedure) (Full). The Control group received empty rVSV virus and sterile water. Data are shown as each value from individual animals with interquartile range and median line. Total animal numbers per group examined are n = 8 (Control or PCS group) or n = 7 (Gag/Env group). However, for technical stringency, animal samples collected on menstruation dates were excluded from analysis to rule out any potential menses blood contamination. As a result, for some of the data points in the graph, fewer individual values than the total animal number (n = 8 or n = 7) were available. Fold changes of antibodies to SIVgag and SIVenv1 in response to prime, the 1st boost, the 4th boost and the full vaccination procedure showed trends of increase in the PCS vaccine group, compared to the other groups. The trends did not reach statistical significance. (B) Reactivity of mucosal IgG antibodies to Gag and Env proteins. Western blot membranes containing purified recombinant Gag (rGag) or Env (rEnv) protein (NIH AIDS Reagent Program) were used to probe anti-Gag/Env IgG antibodies in CVL samples, collected at one week after the last boost from animals of the PCS vaccine (animal ID: cy0759), Gag/Env vaccine (animal ID: cy0784) and Control (animal ID: cy0779) groups, respectively.