Fig 1.
Design of NHP experiments with Env switching challenge viruses.
(A) Conversion of switching challenge viruses in the absence of bnAbs. All SIVdup pseudotyped particles use their respective Envs to infect the first cell but then switch to SIV Env usage for the following rounds of replication. Each challenge virus possesses a unique genetic tag that is maintained in the viral genome after switching to SIV Env usage. (B) Potential outcomes of challenge with the switching viruses in the presence of PGT121 and its LALA-PG mutant. As PGT121 only binds to HIV Env and not SIV Env, inhibition of entry should occur before the first round of infection. The inhibition of the fdEnv challenge virus would provide evidence of non-neutralizing mechanisms contributing to sterilizing immunity and different fdEnv densities could reveal whether the number of antibody binding sites affect the degree of sterilizing immunity. Created with BioRender.com.
Fig 2.
Characterization of switching challenge viruses.
(A) Western blot analyses of particles generated by co-transfection of SIVdup with the indicated Env expression plasmids. SfdEnvLow, SfdEnvInter and SfdEnvHigh correspond to SIVdup pseudotyped particles that co-incorporated SIV Env and a low, intermediate or high quantity of fusion-defective Env, respectively. (B) Infectious titers of SIVdup particles on TZMbl cells. One out of two independent experiments is displayed. Mean and SD from duplicates are shown. The dotted line corresponds to the detection limit of the assay and values below the detection level are indicated with nd. (C) Depletion of infectious pseudotyped particles by PGT121-coated beads. Dynabeads protein G previously coated with PGT121 or anti-RSV-F (Nirsevimab) antibodies were incubated in triplicates with the indicated pseudotyped particles and then depleted by magnetic separation. The loss of infectious particles compared to non-treated samples was measured by luciferase activity on TZMbl cells. Bars represent mean and SD of the experimental triplicates. The overlaid data points display the mean of triplicate measurements on TZMbl cells for each experimental triplicate. (D) Replication kinetics of the indicated SIVdup pseudotypes in CEMxSEAP reporter cells. The infectious dose was adjusted to 2x104 IU. Mock condition consisted of cells cultured in R10 medium only, while non-pseudotyped SIVdup particles were used as undiluted supernatant of transfected HEK293T/17 cells. Mean and SD of SEAP activity from the infected cells was measured in duplicates for each time point. One representative experiment out of two is shown.
Table 1.
Determination of the mean of fdEnv trimers on SIVdup challenge viruses.
Fig 3.
In vitro binding and neutralization activities of PGT121 and PGT121LALA-PG.
(A) Binding of PGT121 and PGT121LALA-PG to HEK293T/17 cells transfected with either an empty vector (= Mock), HIV Env, fdEnv or SIV Env expression plasmids. The mean fluorescence intensity of the cell population is displayed on the top right corner of each panel. One representative experiment out of two is shown. (B) Neutralization of SIVdup particles pseudotyped with either SIV Env, HIV Env or SfdEnvHigh by PGT121 on TZMbl cells. Mean and SD of two independent experiments each done in duplicates are shown. (C) Dose-response curve of binding of PGT121 and PGT121LALA-PG to fdEnv-expressing cells. Mean of duplicates and SD of one representative experiment out of two is shown. (D) Dose-response curve of neutralization of HIV Env pseudotyped SIVdup particles by PGT121 and PGT121LALA-PG. The IC50s of the antibodies are displayed on the top right corner of the graph. Mean and SD of two independent experiments each performed in triplicates are shown.
Fig 4.
Fc-γ receptor binding and C1q recruitment by PGT121 and PGT121LALA-PG.
(A) PGT121 and PGT121LALA-PG binding to HEK293T/17 cells transiently transfected with the indicated FcγRs of macaques. Mean of duplicates and SD of one representative experiment out of two are shown. (B) Deposition of C1q of rhesus macaques on PGT121 and PGT121LALA-PG. ELISA plates were coated with serial dilutions of the two antibodies and subsequently incubated with 10% rhesus macaque serum. After washing, C1q deposition was detected using a C1q antiserum. Mean of duplicates and SD of one representative experiment out of two is displayed.
Fig 5.
Simultaneous challenge experiment in non-human primates.
(A) Scheme of experimental schedule. Six rhesus macaques per group were infused intravenously (i.v.) either with a saline solution (mock), 5 mg/kg PGT121 (PGT121), 5 mg/kg PGT121LALA-PG (PGT121LALA-PG) or a low-dose of 1 mg/kg PGT121 (PGT121-LD) seven days prior to intrarectal (i.r.) simultaneous challenge with HIV Env, SIV Env, SfdEnvLow, SfdEnvInter and SfdEnvHigh pseudotypes. Blood samples were collected on day 7 post-challenge and lymphoid organs were sampled after euthanasia of the animals on day 10 to 11 post-challenge. Created with BioRender.com. (B) PGT121 serum concentrations on the day of challenge. Bars represent mean and SD of the groups. The individual data points correspond to the mean of PGT121 serum concentrations measured for each animal from two independent experiments each performed in duplicates. The dotted line represents the cut-off value of the ELISA. Statistically significant differences were determined by a two-tailed Mann-Whitney test between each respective group (** p<0.005). (C) Viral RNA load in plasma on days 7 and 10/11 post-challenge. Mean of duplicates are shown for each animal. The animal designation numbers are displayed on the top left corner of each graph. Viral loads of the experimental groups did not differ significantly from the viral load of the mock control group (two-tailed Mann-Whitney test, p>0.05). Values for animal #16995 are highlighted in purple.
Fig 6.
Ratio of switching challenge viruses.
Mean of ratio of switched challenge viruses derived from pseudotypes with (A) HIV Env, (B) SfdEnvLow, (C) SfdEnvInter, or (D) SfdEnvHigh to SIV Env pseudotypes. For each animal (n = 6 per group) the mean ratios of the challenge viruses in plasma and lymph nodes from three different locations (mesenteric, inguinal and submandibular) collected on the day of necropsy are shown as individual data points. Bars represent the median of each group. The ratios for the animal #16995, largely deviating in the HIV Env/SIV Env ratio are marked by a purple inverted triangle symbol. Statistically significant differences between groups as determined by Kruskal-Wallis tests followed by Dunn’s multiple comparison tests are indicated (* p<0.05).