Supporting Information Fouts et al. 10.1073/pnas.1423669112 b. a. Naïve rhFLSC Gp120 rhFLSC RC529 Gp120 Iscomatrix c. d. IFN-γ SFC </= Mean (n=14) Challenges to Infection 5 11-12 IFN-γ SFC >/= Mean (n=10) 0.8 7-8 Log ADCC Fraction Uninfected 1.0 0.6 0.4 p = 0.325 6-7 4-5 4 3-4 1-2 0.2 3 0.0 0 2 4 6 8 10 12 14 Number of Intrarectal Challenges 2 1000 800 600 400 200 0 IL-2 ELISPOT e. Immunogen Adjuvant Animal # Naïve Naïve Naïve Naïve Naïve Naïve gp120 gp120 gp120 gp120 gp120 gp120 rhFLSC rhFLSC rhFLSC rhFLSC rhFLSC rhFLSC gp120 gp120 gp120 gp120 gp120 gp120 rhFLSC rhFLSC rhFLSC rhFLSC rhFLSC rhFLSC None None None None None None RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE RC529-SE Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix Iscomatrix A1N099 A3N049 A3N058 A3N100 A3N107 A4N053 A2N041 A3N057 A3N061 A3N104 A4N037 A4N078 A3N052 A3N065 A3N078 A4N030 A4N051 A4N079 A3N064 A3N068 A3N072 A3N085 A4N036 A4N046 A3N071 A3N080 A3N086 A3N109 A4N042 A4N049 Clade B Tier 1 isolates MN SF162.LS BaL.26 <20 <20 <20 <20 <20 <20 500 155 200 600 1188 866 7010 183 1992 717 947 239 370 5599 2641 2432 3499 843 43740 4751 5198 5198 1956 1225 <20 <20 <20 <20 <20 <20 226 39 115 191 366 108 5668 73 597 405 188 129 129 1055 1100 87 813 372 9566 712 1364 1364 410 567 <20 <20 <20 <20 <20 <20 41 21 30 42 56 62 176 <20 93 44 <20 34 27 226 116 33 89 175 348 130 134 134 48 89 >20-100 >100-1000 >1000-10,000 >10,000 Fig. S1. (A) Longitudinal IFN-γ and IL-2 ELISPOT responses (spot-forming cells; SFC) to HIV-1Ba-L gp120 peptides. The asterisk indicates a significantly higher ELISPOT response with Iscomatrix versus RC529 adjuvants for the same antigen (P < 0.05, t test). Week 63 is the day of challenge. (B) Polyfunctional FACS analysis of PBMCs harvested at week 26, 2 wk after the third inoculation. Percentages inside the pie represent the total number of T cells responding to the HIV-1Ba-L gp120 peptide stimulation. Polyfunctionality was assessed against IFN-γ, IL-2, TNF-α, MIP-1β, and CD107a as described (1, 2). The colored portions of the pie represent the portion of the T-cell response presenting a single function (red), two functions (green), or three functions (blue). Data shown are those where the total T-cell response was greater than background measured with vaccine-naive control macaques. (C) Comparison of the rate of acquisition between animals dichotomized by the mean IFN-γ ELISPOT responses (SFC) measured at week 61 (2 wk after the final boost). Data were analyzed by log-rank test. (D) Comparisons of IL-2 versus log ADCC responses (measured at the time of first challenge) versus acquisition for all study 1 animals. The two measures are plotted for each animal (circles). The circles are color-coded according to the number of challenges needed to infect the animal. (E) Neutralizing of tier 1 pseudoviruses by sera collected at week 26 (2 wk after the third inoculation) in a TZM-bl–based assay (main text). Values presented are the maximum serum dilution at which relative luminescence units were reduced by 50% compared with virus control wells without serum. The titer values are shown and colorcoded (right box). An MuLV pseudotyped virus served as control for nonspecific effects and was not neutralized (not shown). 1. Betts MR, et al. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281(1-2):65–78. 2. Xu R, et al. (2008) Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines. Vaccine 26(37):4819–4829. Fouts et al. www.pnas.org/cgi/content/short/1423669112 1 of 4 a. Half-maximal Competition Binding Titer b. Fig. S2. Dose escalation study. (A) Comparison of half-maximal serum-binding titers to rhFLSC or HIV-1Ba-L gp120 in studies 1 and 2, compared with an unpublished dose escalation study (with 300, 600, or 1,200 μg rhFLSC) and a previously reported study that used QS21 adjuvant (1). Mean group titers are shown; bars indicate SD. In all studies, titers were measured by antigen capture ELISA 2 wk after the final immunization as previously described (1). The amount of antigen and/or adjuvant used is shown. (B) Comparison of cumulative competition binding titers against CD4i epitopes (17b, 19e, A32) measured as previously described (1). Mean group titers are shown; bars indicate SD.The immunization groups are the same as in A. Pairwise group comparisons were made by Mann–Whitney–Wilcoxon test. In the dose escalation study, the group that received 1,200 μg of rhFLSC exhibited the highest cumulative titer compared with the other two groups (P = 0.013). 1. DeVico A, et al. (2007) Antibodies to CD4-induced sites in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit immunogens. Proc Natl Acad Sci USA 104(44):17477–17482. Fouts et al. www.pnas.org/cgi/content/short/1423669112 2 of 4 Fig. S3. Tat and Tat toxoid binds to rhFLSC. Tat toxoid was mixed with rhFLSC at the same concentrations used to formulate the vaccine (Fig. 1) and then crosslinked with BS3 as previously described (1). RhFLSC and HIV-1 Tat alone were treated with cross-linker as controls. Cross-linked preparations were analyzed by Coomassie-stained SDS/PAGE (A) and anti-rhFLSC (B) or anti-Tat (C) Western blots. RhFLSC, Tat, and Tat toxoid were run as controls for comparison under reducing and nonreducing conditions. Recognition of the same higher molecular weight bands (arrows) in cross-linked material by both the anti-rhFLSC and anti-Tat antibodies in lanes 3 and 4 indicates that Tat and Tat toxoid associates with rhFLSC. 1. DeVico A, Silver A, Thronton AM, Sarngadharan MG, Pal R (1996) Covalently crosslinked complexes of human immunodeficiency virus type 1 (HIV-1) gp120 and CD4 receptor elicit a neutralizing immune response that includes antibodies selective for primary virus isolates. Virology 218(1):258–263. Fouts et al. www.pnas.org/cgi/content/short/1423669112 3 of 4 a. Total SIVmac239 Env-Specific Responses Total SIVmac239 Gag-Specific Responses b. Fig. S4. (A) Polyfunctional FACS analysis of animals in study 3 using samples collected at week 12, 2 wk after the final DNA immunization. Responses were measured with SIVmac239 Env or Gag peptides. Polyfunctionality was assessed against IFN-γ, IL-2, TNF-α, MIP-1β, and CD107a (1, 2). The colored portions of the pie represent the portion of the T-cell response presenting a single function (red), two functions (green), three functions (blue), or four functions (orange). The number outside the pie represents the percentage of the total response that has two or more functions. Data shown are those where the total T-cell response was greater than background measured with vaccine-naive control macaques. (B) Neutralization titers (EC50) measured in the TZM-bl assay using pseudoviruses with tier 1A SIVmac251.6, tier 1B SIVsmE660/BR-CG7V, and tier 2 SIVmac251.3. The data shown were generated from samples taken 2 wk after the subunit or sham boost (Fig. 1). Values presented are the maximum serum dilutions at which infection was reduced by 50% (ID50) compared with virus control wells without serum. Mean group values are shown; bars indicate SD. An MuLV pseudotyped virus served as control for nonspecific effects and was not neutralized (not shown). 1. Betts MR, et al. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281(1-2):65–78. 2. Xu R, et al. (2008) Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines. Vaccine 26(37):4819–4829. Fouts et al. www.pnas.org/cgi/content/short/1423669112 4 of 4
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