Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Barouch DH, Tomaka FL Wegmann F, et al. Evaluation of a mosaic HIV-1 vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase 1/2a clinical trial (APPROACH) and in rhesus monkeys (NHP 13-19). Lancet 218; published online July 6. http://dx.doi.org/1.116/s14-6736(18)31364-3.
SUPPLEMENTARY APPENDICES Contents: 1) Frequently used abbreviations 2) Supplementary methods 3) Supplementary results 4) List of supplementary tables and figures 5) Supplementary figure legends 6) References 7) Supplementary tables and figures
1) FREQUENTLY USED ABBREVIATIONS ADCC: antibody-dependent cell-mediated cytotoxicity ADCD: adherent cell differentiation and cytotoxicity ADCP: antibody dependent cellular phagocytosis AE: adverse event ALT: alanine aminotransferase BAMA: binding antibody multiplex assay BL: baseline CI: confidence interval BMI: body mass index ELISA: enzyme-linked immunosorbent assay ELISPOT: Enzyme-Linked ImmunoSpot ENV: Envelope : high dose HIV: human immunodeficiency virus ICS: intracellular cytokine staining IG: immunogenicity : low dose LOD: limit of detection LLOQ: lower limit of quantification PBMC: peripheral blood mononuclear cells PTE: potential T-cell epitope P95: 95th percentile SAE: serious adverse event SFU: spot forming units VNA: virus neutralization assay
2) SUPPLEMENTARY METHODS APPROACH STUDY Study design The study was reviewed and approved by the institutional ethics review board at each clinical site. All participants signed informed written consent and passed a test of understanding before enrollment. The study was sponsored by Janssen Pharmaceuticals and the protocol developed in conjunction with its partners. An independent Data Monitoring Committee (DMC) and an internal Protocol Safety Review Team (PSRT), comprising of medical and safety representatives of the sponsor and partners evaluated safety and tolerability data. To decide whether further dosing could continue, the DMC reviewed blinded safety data (2 weeks of follow-up) after 3% of participants received their first injection, and unblinded data after 1% of participants received their third injection at week 24 and after 3% of participants have received their third injection at week 24. Full details on safety monitoring are included in the online version of the clinical trial protocol. Breadth and specificity of antibody responses to gp41, gp12, gp14 and gp7v1v2 constructs were further assessed (Supplemental Figure 3). Participants Participants were recruited from centers in the USA, Rwanda, Uganda, South Africa, and Thailand. Male and non-pregnant female participants were eligible to take part in the study providing they were between 18 and 5 years old, negative for HIV following screening, and considered low risk for the infection. Key exclusion criteria for this study were chronic active hepatitis B or active hepatitis C, and sexually transmitted infections. All participants gave written informed consent and the relevant institutional boards or ethics committees approved research protocol. Human immunologic assays: ELISPOT, ADCP, BAMA, ICS, HIV neutralisation and neutralization assays were performed as previously described. 1 3 ELISA assays Antibody binding to five different gp14 antigens was determined by ELISA. The gp14 antigens that were used are, WT_A_92UG37 (clade A), WT_B_199A (clade B), WT_C_CONC (consensus clade C), VaccineTake_C_C97ZA (clade C) and VaccineTake_Mos1 (mosaic). MaxiSorp 96-wells plates (ThermoFisher Scientific, Waltham, US) were coated overnight with the subsequent antigens (Janssen Pharmaceuticals, Leiden, The Netherlands) at 1 µg/ml in PBS. After washing with Wash-Buffer (WB; PBS/.5% Tween-2), 2% Gelatin Blocking Buffer (GBB) was added to further block non-specific protein-binding sites. HIV reference standard samples, QC samples and heat-inactivated human serum samples (pre-diluted in 2%GBB) were added to the plate in a 1:1 dilution (1 µl sample and 9 ul
2%GBB). Sample plates were left for incubation (12 min at RT) to allow the specific Env protein antibodies to bind to the coating, followed by washing with WB. Horseradish peroxidase labeled mouse-anti-human IgG (Jackson Immunoresearch, Suffolk, England) was added for 6 min at RT, at a 1:2. dilution in 2% GBB followed by washing with WB. Colorimetric detection was performed using the TMB 2-Component Microwell Peroxidase Substrate Kit (Seracare, Milford, US). Color development was stopped using 1M H 2SO 4, and absorption was measured at 45 nm. To calculate the concentration, a 4-parameter logistic curve plot was fitted for the reference standard samples and the OD45 values of each individual samples was read back into this reference standard curve to obtain a concentration readout. The concentrations are reported in arbitrary ELISA Units/ml, based on the assignment of a 1. EU/mL concentration of the first dilution in the reference standard (for 1% serum).
RHESUS MONKEY STUDY ELISA assays Antibody binding to the clade C gp14 and mosaic gp14 antigens was determined by ELISA. Antigen was coated at 1µg/mL in phosphate buffered solution and serum samples were serially diluted and incubated on plates. Binding antibody was determined using horseradish peroxidase-conjugated detection antibody and SureBlue (KPL, 52-- 3) solution. Reportable values were generated by four-parameter logistic curve fitting with the lower plateau values fixed to the average plate background of the assay run using GraphPad Prism software. ELISPOT assays ELISPOT plates were coated with mouse anti-human IFN-γ monoclonal antibody (BD Pharmingen, 554699) and incubated overnight at 4 C. The plates were washed and blocked for 1 to 4 hours in 37 C. The block was removed and 5μL of peptide at a concentration of 2 μg/ml was added to the corresponding wells. 5μL of PBMCs was added at a concentration of 4x1 6 cells/ml resulting in a total of 2x1 5 cells/well at a final peptide concentration of 1 μg/ml. Media and phytohemagglutinin were used for quantitative and qualitative controls respectively. The plates were placed in a 37 C incubator for 18 to 24 hours. After washing, rabbit polyclonal anti-human IFN-γ biotin (U- Cytech, CT243) was added to each well. The plates were incubated for approximately 2 hours at room temperature. After washing, Strepavidin-AP (Southern Biotechnology, 71-4) was added to each well. The plates were incubated for approximately 2 hours at room temperature. The plates were washed with Coulter Wash, and BCIP/NBT chromagen (Pierce, 3442) was then added to each well and incubated at room temperature for 7 minutes. The chromagen was removed and the plate was rinsed thoroughly with tap water. The plates were dried upside down and removed from direct light for 24 hours. The median of duplicate well cell counts was calculated and background subtraction was performed per individual monkey by subtracting the median spot forming cells (SFC) value of medium stimulated wells to each median SFC value of the peptide stimulated sample. All values between and 1 were set to and pools of potential T-cell epitopes (PTE g) were summed per antigen. Values were multiplied by 5 to yield SFC/1 6 PBMC s, with 5 SFC/1 6 PBMC s as the detection limit threshold. Rhesus monkey correlate analysis To minimize the risk of chance findings in the correlates analysis and overfitting of a protection model, we selected, based on previously reported potential correlates of protection, 18 humoral and cellular immunologic parameters (Supplement Table 1) to generate an immune readoutbased prediction model. Parameters were ranked based on their individual (Spearman) correlation with time-to-infection. An ordinal logistic prediction model with an optimal subset of these parameters was generated with stepwise selection of the best correlating parameters. Groups that included an MVA boost were not included in the model.
The following methods/data were included in the correlates analysis but data are not shown: Assay category Env binding antibodies Env V1V2 loop binding antibodies Env-binding antibody function Env-specific cellular immune responses Assay Mosaic gp14 ELISA SF162p3 gp14 ELISA A244 V1V2-scaffold ELISA Clade B/case A2 V1V2-scaffold ELISA CN54 V1V2-scaffold ELISA Consensus C V1V2-scaffold ELISA Clade C gp14 ADCC (antibody-dependent cell-mediated cytotoxicity) Mosaic gp14 ADCC Clade C gp14 ADCD (antibody-dependent complement deposition) Mosaic gp14 ADCD Clade C gp14 ADCP (antibody-dependent cellular phagocytosis) Mosaic gp14 ADCP Env-specific IFN- producing CD4+ T-cells (flow cytometry; stimulation: PTE peptides) Env-specific IFN- producing CD8+ T-cells (flow cytometry; stimulation: PTE peptides)
3) SUPPLEMENTARY RESULTS Safety Three incidental HIV infections occurred at a single site in South Africa: A 19-year old male randomized to the / group was diagnosed with urethral chlamydia infection 14 days after having received the 3 rd vaccination. HIV-1 infection was diagnosed 1 week thereafter. A 27-year old female randomized to the / group was diagnosed with HIV-1 infectioned 85 days after having received the 1 st vaccination, reporting 2 incidents of unprotected sex in the weeks prior to diagnosis. A 22-year old male randomized to the /gp14 group was diagnosed with HIV-1 infection 81 days after having received the 2 nd vaccination. He describes having had a new sex partner around the time of having received his 2 nd vaccination. In each of the cases, confounding factors that would increase the risk of infection were identified. The site systematically increased its HIV prevention counselling and no other infections occurred afterwards.
4) LIST OF SUPPLEMENTARY TABLES AND FIGURES Supplementary tables Supplemental Table 1: Assay list that formed the basis for correlates analysis Supplemental Table 2: Participant demographic and baseline characteristics in the APPROACH study Supplemental Table 3: Solicited adverse events summary of full analysis set in the APPROACH study Supplemental Table 4: Post any dose incidence of unsolicited AEs that occurred in at least 5% of participants in any treatment group in the APPROACH study Supplemental Table 5: Unsolicited adverse events with at least Grade 3 severity and related to the study vaccine in the APPROACH study Supplemental Table 6: Incidence of unsolicited AEs related to the vaccination occurring in >1 participant (post any dose) in the APPROACH study Supplemental Table 7: Tier-2 (ID 5) actual values at week 28; per protocol immunogenicity analysis set in the APPROACH study Supplemental Table 8: Preclinical and clinical data contributed to go/no-go decision Supplementary figures Supplemental Figure 1: Solicited local and systemic adverse events in the APPROACH study Supplemental Figure 2: ELISA total IgG responses to clade A, clade B, clade C consensus and mosaic gp14 antigens in the APPROACH study Supplemental Figure 3: BAMA assay results for breadth and specificity of antibody responses to gp41, gp12, gp14 and gp7v1v2 in the APPROACH study Supplemental Figure 4: ELISA IgG subtype assays in the APPROACH study Supplemental Figure 5: Correlation between ADCP and ELISA data in humans and monkeys in the APPROACH study Supplemental Figure 6: MW965.27 neutralization antibody assay in the APPROACH study Supplemental Figure 7: IFN-γ subtype ELISPOT assays in response to vaccinematched peptide pools in the APPROACH study Supplemental Figure 8: ICS assays for the cytokine count: IFN-γ or IL2+ in the APPROACH study Supplemental Figure 9: Baseline -specific nabs by region in the APPROACH study Supplemental Figure 1: Baseline -specific nabs and ELISA/ELISPOT response correlations in the APPROACH study Supplemental Figure 11: Differences in immune response to vaccine stratified by gender, age, or region in the APPROACH study Supplemental Figure 12: Clade C gp14 ELISA antibody titers in the rhesus monkey study
Supplemental Figure 13: Serum neutralization of tier-1 viruses in the rhesus monkey study Supplemental Figure 14: PTE g peptide pool IFN-γ ELISPOT assays in the rhesus monkey study Supplemental Figure 15: ELISPOT Mosaic peptide vaccine matched assays in the rhesus monkey study Supplemental Figure 16: ICS assays in the rhesus monkey study Supplemental Figure 17: Correlation of clade C-binding antibody responses in rhesus monkey determined by human or rhesus monkey ELISA Supplemental Figure 18: Comparing distributions of positive T-cell responses to previous studies
5) SUPPLEMENTARY FIGURE LEGENDS Supplemental Figure 1 Overview of the solicited local (a) and systemic (b) adverse events reported and analysed in the 28 day reporting period of the APPROACH trial. Events are shown by worst severity grade by regimen; solicited local adverse events and most solicited systemic adverse events are considered related to vaccination by definition. Supplemental Figures 2 8 For the APPROACH study, immune responses to each vaccine regimen were measured by various assays. Vaccine response was defined based on Fisher's exact test between stimulated and unstimulated cells producing or not producing cytokines with a P value <.1. Responders are shown as solid coloured circles; non responders as open circles with coloured outlines. All panels show responder rates for each vaccine group for various timepoints beneath the graph. The dotted horizontal lines show the threshold for each assay performed. Supplemental Figure 9 Baseline -specific nabs in the APPROACH study were recorded; seropositive participants are shown as solid green circles and seronegative participants as white circles with an orange outline. Summaries of the total number of patients (n), geometric mean, and seropositivity/negativity are shown beneath the x-axis. The dotted horizontal line shows the lower limit of quantification used for the assay. Supplemental Figures 1 For the APPROACH study, immune responses to each vaccine regimen were measured by various assays. Vaccine response was defined as titer >threshold (if baseline is <threshold or is missing); otherwise, was a titer with a 3-fold increase from baseline (if baseline is threshold). Responders are shown as solid coloured dots; non responders as dots with coloured outlines. All panels show responder rates for each vaccine group for various timepoints beneath the graph. The dotted horizontal lines shows the threshold for each assay performed. Supplemental Figure 11: Differences in immune response to vaccine stratified by gender, age, or region in the APPROACH study
Supplemental Figure 12 For the rhesus monkey study, data are plotted for the following timepoints (weeks post first immunization): -1, 5, 16, 24, 28, 51, 56 and 72. Dashed lines represent timepoints of immunizations (weeks, 12, 24 and 52). Supplemental Figure 13 For the rhesus monkey study, serum neutralisation titers of tier 1A viruses (MW965.26, SF162.LS and MN-3), tier 1B viruses (DJ263.8, BaL.26, ZM19F.PB4) and murine leukemia virus (MuLV). Each vaccine regimen is shown as a different colour. Solid lines in each column represent the geometric mean titers or medians. Supplemental Figures 14 16 For the rhesus monkey study, immune responses to each vaccine regimen were measured by various assays. Each vaccine regimen is shown as a different colour. Solid lines in each column represent the geometric mean titers or medians. Supplemental Figure 17 Correlation between clade C-binding antibody responses in rhesus monkeys and humans using week 56 data. Supplemental Figure 18 (Row A) A comparison of results in the APPROACH study to the total number of T-cell responses per Step-study participant among the 72 individuals that eventually became infected with HIV-1. 1 (Row B) A comparison of number of per participant Gag responses in the Steptrial to this study. 1 (Row C) A comparison of the number of per participant Env T-cell responses using a natural Env protein for vaccine in an vector rather than the bivalent Mos1+natural protein studied here. In APPROACH we compared the distributions of numbers of positive responses across each row in parts A, B and C using a two-sided Wilcoxon rank sum test; P values are not exact due to ties in the data. Nine tests were performed, and Bonferroni corrected P values below. There was no statistical difference between the two vaccine groups
investigated in this study the APPROACH study, /+gp14 and the /, in terms of the number of total responses (A), Gag responses (B), and Env responses (C). In row (A), /+gp14 and / each had significantly more responses than Janes et al. the Step-study per participant (corrected P values,.6 and.4, respectively). In row (B), /+gp14 and / each had more Gag responses than Janes et al. per participants in the Step-study (corrected P values.1 and.5, respectively). In row (C) /+gp14 and / each had more Env responses than in the APPROACH study per participant (corrected P values.4 and.7, respectively). 6) REFERENCES: 1. Haynes BF, Gilbert PB, McElrath J, et al. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med 212;366:1275-86. 2. Yates NL, Liao HX, Fong Y, et al. Vaccine-induced Env V1-V2 IgG3 correlates with lower HIV-1 infection risk and declines soon after vaccination. Sci Transl Med 214;19;228ra39. 3. Yates et al. HIV-1 envelope glycoproteins from diverse clades differentiate antibody responses and durability among vaccines. J Virol 218; 92:e1843-17. 4. Janes H, Friedrich DP, Krambrink A, et al. Vaccine-induced gag-specific T-cells are associated with reduced viremia after HIV-1 infection. J Infect Dis 213;28:1231 9.
Supplemental Table 1: Assay list that formed the basis for correlates analysis Assay category Assay Spearman correlation p-value Clade C gp14 ELISA.3** Env binding antibodies Mosaic gp14 ELISA.32 SF162p3 gp14 ELISA.4 A244 V1V2-scaffold ELISA.7** Env V1V2 loop binding antibodies Clade B/case A2 V1V2-scaffold ELISA.795 CN54 V1V2-scaffold ELISA.99 Consensus C V1V2-scaffold ELISA.435 Clade C gp14 ADCC.291 Mosaic gp14 ADCC.726 Env-binding antibody function Clade C gp14 ADCD.777 Mosaic gp14 ADCD.433 Clade C gp14 ADCP.66 Mosaic gp14 ADCP.33 Env-specific cellular immune responses Env-specific ELISPOT (stimulation: PTE g peptide pools).1*** Env-specific IFN-γ producing CD4+ T-cells (flow cytometry; stimulation: PTE g peptide pools) Env-specific IFN-γ producing CD8+ T-cells (flow cytometry; stimulation: PTE g peptide pools).135.45
Supplemental Table 2: Participant demographic and baseline characteristics in the APPROACH study Age (years) at screening n + gp14 5 + gp14 MVA+ gp14 48 MVA+ gp14 MVA gp14 Placebo 5 All Participants 393 Mean Median Min; Max 29.8 29. (18; ) 29.6 29. (19; ) 31.3 33. (18; 48) 29.6 28. (19; 48) 3.3 28. (19; 48) 29.2 26. (18; ) 3.3 29. (19; 5) 29. 26. (19; 5) 29.9 29. (18; 5) Sex, n 5 48 5 393 Female, n (%) Male, n (%) 25 (5.) 25 (5.) 26 (53.1) 23 (46.9) 28 (57.1) 21 (42.9) 19 (39.6) 29 (6.4) 25 (51.) 24 (.) 19 (38.8) 3 (61.2) 19 (38.) 31 (62.) 2 (4.8) 29 (59.2) 181 (46.1) 212 (53.9) Race n, (%) 5 48 5 393 Asian, n (%) Black, n (%) White, n (%) Other, n (%) Region n, (%) 9 (18.) 27 (54.) 14 (28.) 5 7 (14.3) 29 (59.2) 13 (26.5) 9 (18.4) 27 (55.1) 11 (22.4) 2 (4.1) 7 (14.6) 27 (56.3) 14 (29.2) 48 7 (14.3) 26 (53.1) 15 (3.6) 8 (16.3) 26 (53.1) 13 (26.5) 2 (4.1) 8 (16.) 32 (64.) 9 (18.) 5 9 (18.4) 25 (51.) 15 (3.6) 64 (16.3) 219 (55.7) 14 (26.5) 6 (1.5) 393 Eastern Africa, n (%) South Africa, n (%) Thailand, n (%) USA, n (%) 16 (32.) 7 (14.) 8 (16.) 19 (38.) 14 (28.8) 1 (2.4) 7 (14.3) 18 (36.7) 18 (36.7) 5 (1.2) 7 (14.3) 19 (38.8) 16 (33.3) 7 (14.6) 7 (14.6) 18 (37.5) 18 (36.7) 5 (1.2) 7 (14.3) 19 (38.8) 16 (32.7) 7 (14.3) 7 (14.3) 19 (38.8) 14 (28.) 9 (18.) 8 (16.) 19 (38.) 17 (34.7) 6 (12.2) 7 (14.3) 19 (38.8) 129 (32.8) 56 (14.2) 58 (14.8) 15 (38.2) Country n, (%) 5 48 5 393 Rwanda, n (%) South Africa, n (%) Thailand, n (%) Uganda, n (%) USA, n (%) 7 (14.) 7 (14.) 8 (16.) 9 (18.) 19 (38.) 7 (14.3) 1 (2.4) 7 (14.3) 7 (14.3) 18 (36.7) 1 (2.4) 5 (1.2) 7 (14.3) 8 (16.3) 19 (38.8) 8 (16.7) 7 (14.6) 7 (14.6) 8 (16.7) 18 (37.5) 9 (18.4) 5 (1.2) 7 (14.3) 9 (18.4) 19 (38.8) 6 (12.2) 7 (14.3) 7 (14.2) 1 (2.4) 19 (38.8) 5 (1.) 9 (18.) 8 (16.) 9 (18.) 19 (38.) 6 (12.2) 6 (12.2) 7 (14.3) 11 (22.4) 19 (38.8) 58 (14.8) 56 (14.2) 58 (14.8) 71 (18.1) 15 (38.2) Ethnicity n, (%) 5 48 48 5 392 Not Reported, n (%) Hispanic or Latino, n (%) Not Hispanic or Latino, n (%) 4 (8.) 46 (92.) 6 (12.2) 43 (87.8) 1 2 (4.2) 46 (95.8) 6 (12.5) 42 (87.5) 3 (6.1) 46 (93.9) 5 (1.2) 44 (89.8) 3 (6.) 47 (94.) 6 (12.2) 43 (87.8) 1 35 (8.9) 357 (91.1)
Supplemental Table 3: Solicited adverse events in the APPROACH study summary of full analysis set (solicited AEs with unknown severity are not taken into account in this table) Post any dose + gp14 (N=5) + gp14 (N=) (N=) MVA+ gp14 (N=48) MVA+ gp14 (N=) MVA (N=) gp14 (N=5) Placebo (N=) At least one solicited AE n, (%) 46 (92.) 46 (93.9) 41 (83.7) 42 (87.5) 46 (93.9) 39 (79.6) 4 (8.) 36 (73.5) At least one solicited AE with severity Grade 3 as worst grade n, (%) At least one solicited local AE n, (%) At least one solicited local AE with severity Grade 3 as worst grade n, (%) At least one solicited local AE leading to permanent stop of the vaccine n, (%) 4 (8.) 2 (4.1) 5 (1.2) 3 (6.3) 3 (6.1) 6 (12.2) 2 (4.) 43 (86.) 4 (81.6) 34 (69.4) 36 (75.) 38 (77.6) 35 (71.4) 34 (68.) 26 (53.1) At least one solicited systemic AE n, (%) 42 (84.) 41 (83.7) 36 (73.5) 35 (72.9) 43 (87.8) 36 (73.5) 3 (6.) 27 (55.1) At least one solicited systemic AE with severity Grade 3 as worst grade n, (%) 4 (8.) 2 (4.1) 5 (1.2) 3 (6.3) 3 (6.1) 6 (12.2) 2 (4.) At least one solicited systemic AE that is related to the vaccine n, (%) At least one solicited systemic AE with severity Grade 3 that is related to the vaccine n, (%) At least one solicited systemic AE leading to permanent stop of vaccine n, (%) 42 (84.) 4 (81.6) 35 (71.4) 35 (72.9) 41 (83.7) 35 (71.4) 28 (56.) 26 (53.1) 3 (6.) 2 (4.1) 4 (8.2) 2 (4.2) 3 (6.1) 4 (8.2) 2 (4.)
Supplemental Table 4: Post any dose incidence of unsolicited AEs that occurred in at least 5% of participants in any treatment group in the APPROACH study + gp14 (N=5) + gp14 (N=) (N=) MVA+ gp14 (N=48) MVA+ gp14 (N=) MVA (N=) gp14 (N=5) Any event n, (%) 39 (78.) 34 (69.4) 4 (81.6) 38 (79.2) 31 (63.3) 39 (79.6) 42 (84.) 37 (75.5) Placebo (N=) Infections and infestations n, (%) 18 (36.) 16 (32.7) 21 (42.9) 2 (41.7) 2 (4.8) 21 (42.9) 22 (44.) 22 (44.9) Upper respiratory tract infection 4 (8.) 6 (12.2) 6 (12.5) 3 (6.1) 6 (12.2) 6 (12.) 8 (16.3) Influenza 3 (6.) 4 (8.2) 5 (1.4) 4 (8.2) 3 (6.) 3 (6.1) Urinary tract infection 5 (1.2) 3 (6.3) 3 (6.1) 2 (4.1) Malaria 2 (4.) 2 (4.1) 3 (6.1) 1 (2.1) 3 (6.1) 2 (4.) 2 (4.1) Viral infection 2 (4.) 1 (2.1) 3 (6.1) 3 (6.) Pharyngitis 1 (2.1) 3 (6.1) Investigations n, (%) 13 (26.) 9 (18.4) 13 (26.5) 11 (22.9) 6 (12.2) 14 (28.6) 8 (16.) 11 (22.4) Alanine aminotransferase increased 5 (1.) 2 (4.1) 2 (4.1) 1 (2.1) 4 (8.2) 3 (6.1) 3 (6.) Aspartate aminotransferase increased 3 (6.) 3 (6.1) 1 (2.1) 3 (6.1) 3 (6.) 2 (4.1) Electrocardiogram QT prolonged 2 (4.) 3 (6.1) 3 (6.3) 4 (8.2) 2 (4.) 2 (4.1) Haemoglobin decreased 2 (4.1) 4 (8.2) 3 (6.3) 3 (6.1) Blood creatine increased 3 (6.) 1 (2.1) 3 (6.1) Gastrointestinal disorders n, (%) 1 (2.) 4 (8.2) 1 (2.4) 9 (18.8) 4 (8.2) 1 (2.4) 6 (12.) 7 (14.3) Diarrhoea 4 (8.) 2 (4.1) 2 (4.2) 2 (4.1) 4 (8.) General disorders and administration site conditions n, (%) 5 (1.) 6 (12.2) 5 (1.2) 6 (12.5) 7 (14.3) 6 (12.2) 9 (18.) 7 (14.3) Injection site bruising 2 (4.1) 2 (4.1) 5 (1.2) 2 (4.1) 2 (4.) Influenza-like illness 2 (4.) 1 (2.1) 4 (8.2) Nervous system disorders n, (%) 5 (1.) 7 (14.3) 7 (14.3) 5 (1.4) 6 (12.2) 5 (1.2) 5 (1.) 7 (14.3)
Headaches 3 (6.1) 3 (6.1) 3 (6.3) 4 (8.2) 2 (4.) Renal and urinary disorders n, (%) 6 (12.) 5 (1.2) 5 (1.4) 8 (16.3) 8 (16.) 7 (14.3) Proteinuria 4 (8.) 4 (8.2) 3 (6.3) 5 (1.2) 6 (12.) 5 (1.2) Haematuria 3 (6.3) 2 (4.1) 2 (4.1) Musculoskeletal and connective tissue disorders n, (%) 5 (1.) 4 (8.2) 5 (1.2) 6 (12.5) 3 (6.1) 4 (8.2) 6 (12.) 2 (4.1) Back pain 3 (6.1) 3 (6.1) 4 (8.3) 2 (4.1) 2 (4.) 1 (2. Respiratory, thoracic and mediastinal disorders n, (%) 6 (12.) 3 (6.1) 6 (12.5) 4 (8.2) 4 (8.2) 7 (14.) 2 (4.1) Cough 2 (4.) 2 (4.2) 4 (8.) Oropharyngeal pain 2 (4.) 3 (6.1) Injury, poisoning and procedural complications n, (%) 5 (1.2) 4 (8.2) 2 (4.2) 4 (8.2) 5 (1.2) Skin and subcutaneous disorders n, (%) 3 (6.) 2 (4.1) 2 (4.1) 3 (6.3) 4 (8.2) 2 (4.1) 2 (4.) 2 (4.1) Blood and lymphatic system disorders n, (%) 3 (6.1) 4 (8.2) 2 (4.1) 6 (12.) Neutropenia 3 (6.1) 4 (8.) Reproductive system and breast disorders n, (%) 2 (4.1) 3 (6.3) 3 (6.1) 4 (8.) Eye disorders n, (%) 2 (4.1) 2 (4.1) 4 (8.3) 3 (6.1) Psychiatric disorders n, (%) 3 (6.3)
Supplemental Table 5: All Grade 3 unsolicited AEs occurred within the 28 days post-vaccination in the APPROACH study Treatment regimen Phase (start/stop) Preferred term Relation to vaccine Outcome Duration /+gp14 Post-dose 2 Aspartate aminotransferase increased Related Recovered 7 /+gp14 Post-dose 3 Anaphylactic reaction Not related Recovered 1 /+gp14 Post-dose 3 Alanine aminotransferase increased Not related Recovered 138 / Post-dose 3 Neutrophil count decreased Not related Recovered 15 / Post-dose 1 Procedural headache Not related Recovered 2 / Post-dose 2 Bacteraemia Not related Recovered 4 / Post-dose 2 Malaria Not related Recovered 4 / Post-dose 4 Pyrexia Not related Recovered 2 / Post-dose 3 Typhoid fever Not related Recovered 15 / Post-dose 2 Pyelonephritis Not related Recovered 7 / Post-dose 2 Alanine aminotransferase increased Not related Recovered 13 / Post-dose 1 Malaise Related Recovered 1 / Post-dose 3 Pyelonephritis Not related Unknown 174 / Post-dose 2 Upper respiratory tract infection Not related Recovered 8 /MVA+ gp14 Post-dose 1 Abdominal pain Related Recovered 1 / Post-dose 1 Diarrhoea Related Recovered 1 / Post-dose 4 Neutropenia Not related Recovered 4 /MVA Post-dose 1 Abdominal pain Not related Recovered 1 /MVA Post-dose 1 Back pain Related Recovered 8 /MVA Post-dose 1 Aspartate aminotransferase increased Not related Recovered 12 /gp14 Post-dose 1 Hypersensitivity Related Recovered 2 /gp14 Post-dose 4 Influenza Not related Recovered 15 /gp14 Post-dose 2 Electrocardiogram QT prolonged Not related Recovered 143 Placebo Post-dose 1 Dizziness postural Related Recovered 19 Placebo Post-dose 1 Stab wound Not related Recovered 13
Supplemental Table 6: Incidence of unsolicited AEs related to the vaccination occurring in >1 participant (post any dose) in the APPROACH study MedDRA System Organ Class Dictionary-derived Term + gp14 (N=5) + gp14 (N=) (N=) MVA+ gp14 (N=48) MVA+ gp14 (N=) MVA (N=) gp14 (N=5) Any event, n (%) 14 (28.%) 12 (24.5%) 15 (3.6%) 9 (18.8%) 14 (28.6%) 13 (26.5%) 13 (26.%) 14 (28.6%) General disorders and administration site conditions 4 (8.%) 5 (1.2%) 3 (6.1%) 3 (6.3%) 4 (8.2%) 3 (6.1%) 1 (2.%) 3 (6.1%) Injection site bruising 1 (2.%) 2 (4.1%) 2 (4.1%) 4 (8.2%) 2 (4.1%) 1 (2.%) Malaise 1 (2.%) 2 (4.2%) Influenza like illness 2 (4.1%) Injection site pain 1 (2.%) 1 (2.1%) Nervous system disorders 3 (6.%) 3 (6.1%) 4 (8.2%) 2 (4.2%) 4 (8.2%) 3 (6.1%) 2 (4.%) 3 (6.1%) Dizziness postural 1 (2.%) 1 (2.%) 2 (4.1%) 1 (2.1%) 1 (2.%) 2 (4.1%) 1 (2.%) 2 (4.1%) Dizziness 1 (2.%) 1 (2.%) 1 (2.1%) 1 (2.%) 1 (2.%) 1 (2.%) Paraesthesia 1 (2.%) 1 (2.%) 1 (2.%) Dizziness exertional 1 (2.%) 1 (2.%) Gastrointestinal disorders 2 (4.%) 1 (2.%) 4 (8.2%) 1 (2.1%) 4 (8.2%) 2 (4.1%) 2 (4.%) 1 (2.%) Diarrhoea 1 (2.%) 1 (2.%) 3 (6.1%) 2 (4.%) Abdominal pain 1 (2.%) 1 (2.1%) 2 (4.1%) Abdominal pain upper 1 (2.%) 1 (2.%) 1 (2.%) Dyspepsia 2 (4.1%) Investigations 2 (4.%) 1 (2.%) 1 (2.1%) 4 (8.2%) 4 (8.2%) Neutrophil count decreased 1 (2.%) 2 (4.1%) 1 (2.%) Alanine aminotransferase increased 2 (4.%) 1 (2.%) Aspartate aminotransferase increased Placebo (N=) 1 (2.%) 2 (4.1%) Blood pressure increased 1 (2.1%) 1 (2.%) Respiratory, thoracic and mediastinal disorders 2 (4.%) 1 (2.%) 1 (2.%) 3 (6.1%) 2 (4.1%) 1 (2.%) 1 (2.%) Oropharyngeal pain 1 (2.%) 3 (6.1%) 1 (2.%) Nasal congestion 2 (4.%) 1 (2.%) Sneezing 1 (2.%) 1 (2.%) Blood and lymphatic system disorders 1 (2.%) 2 (4.1%) 2 (4.1%) 2 (4.1%) 2 (4.%) Neutropenia 1 (2.%) 1 (2.%) 1 (2.%) 2 (4.%) Lymphadenopathy 1 (2.%) 1 (2.%) 2 (4.1%) Infections and infestations 2 (4.%) 1 (2.%) 1 (2.1%) 2 (4.1%) 1 (2.%) 1 (2.%) 1 (2.%) Rhinitis 1 (2.%) 1 (2.1%) Skin and subcutaneous tissue disorders 1 (2.%) 2 (4.1%) 2 (4.2%) 1 (2.%) 1 (2.%) 2 (4.1%) Hyperhidrosis 1 (2.1%) 1 (2.%) 1 (2.%) Musculoskeletal and connective tissue disorders 1 (2.%) 1 (2.%) 2 (4.1%) 1 (2.%) 1 (2.%) 1 (2.%)
Neck pain 1 (2.%) 1 (2.%) Metabolism and nutrition disorders 1 (2.%) 1 (2.%) 1 (2.%) 1 (2.%) 1 (2.%) Decreased appetite 1 (2.%) 1 (2.%) 1 (2.%) 1 (2.%) 1 (2.%) Eye disorders 2 (4.1%) 1 (2.%) Cardiac disorders 1 (2.%) 1 (2.%) Ear and labyrinth disorders 1 (2.%) 1 (2.%) Vertigo 1 (2.%) 1 (2.%) Psychiatric disorders 1 (2.1%) 1 (2.%) Renal and urinary disorders 1 (2.%) 1 (2.%)
Supplemental Table 7: Tier-2 (ID 5) actual values at week 28; per protocol immunogenicity analysis set Geometric mean (95% CI) +gp14 +gp14 MVA gp14 Placebo Clade A (398F1) nab Clade AC (246F3) nab Clade AE (CNE55) nab Clade AE (CNE8) nab Clade B (TRO11) nab Clade B (X2278) nab Clade BC (CH119) nab Clade C (Ce1176) nab Clade C (2571) nab Clade C (Ce217) nab Clade G (X1632) nab
Supplemental Table 8: Go/no go criteria and clinical results. These criteria were selected based on rhesus monkey regimen data. Criteria Endpoint Target (Lower limit of 95% CI) Post 3 rd APPROACH results Post 4 th Humoral IgG binding responses to clade C Env 9% ( 77%) 1% (93%) 1% (92%) ADCP responses to clade C Env 56% ( 4%) 72% (57%) 8% (65%) Cellular ELISPOT responses to at least one Env peptide pool 5% ( 35%) 77% (62%) 83% (68%) Env boost IgG ELISA to clade C Env of Ad/Ad+Env over / 1.5 fold 5.5 fold (3.5) 6.9 fold (4.5) Magnitude >2.15 log1 cpteg Env ELISPOT or >3.8 log1 clade C gp14 ELISA Subjects should be above BOTH response thresholds post 3 rd : post 4 th : >6% >75% post 3 rd : post 4 th : >4% >6% 94% 93% 64% 8%
Supplemental Figure 1A: Solicited Local Adverse Events
Supplemental Figure 1B: Solicited Systemic Adverse Events
Supplemental Figure 1C: Solicited Systemic Adverse Events
Supplemental Figure 2: ELISA Total IgG Responses to Clades Baseline W28 Responder W52 Responder W28 Non Responder W52 Non Responder GMT A. Env ELISA IgG-t gp14: Clade A (92UG37.1) B. Env ELISA IgG-t gp14: Clade B (199a) 1 1 6 +gp14 +gp14 MVA gp14 Placebo 1 1 6 +gp14 +gp14 MVA gp14 Placebo 1 1 5 1 1 5 ELISA titer ELISA titer LLOQ (625) LLOQ (156) 1 1 1 1 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 GMT: 2 26923 45819 11885 31722 8581 12468 3581 57937 16626 35992 1444 1737 1625 22365 3 2 Resp %: 1 1 98 1 98 1 95 98 98 1 98 1 95 97 2 5 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 GMT: 4 466 5965 3143 36571 15596 16117 4541 64253 35661 4652 2778 26191 155 27392 7 6 Resp %: 1 1 1 1 1 1 1 98 1 1 1 1 1 1 7 1 C. Env ELISA IgG-t gp14: Clade C (Con C) D. Env ELISA IgG-t gp14: Mos1 1 1 7 +gp14 +gp14 MVA gp14 Placebo 1 1 7 +gp14 +gp14 MVA gp14 Placebo 1 1 6 1 1 6 1 1 5 ELISA titer 1 1 5 ELISA titer 1 1 LLOQ (625) 1 1 LLOQ (78) BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 GMT: 13 69 7579 337 838 14532 19276 54843 96846 38426 56459 29857 32565 21766 43434 16 9 Resp %: 1 1 1 98 1 1 95 95 1 1 1 1 98 97 7 2 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 GMT: 2 34343 38336 26332 3661 1256 12359 39397 52236 34139 3574 2986 2934 1379 23574 3 2 Resp %: 1 1 1 98 1 1 95 95 1 1 1 1 98 97 4
Supplemental Figure 3A-D: BAMA assay breadth and specificity of antibody responses Baseline W28 Responder W52 Responder W28 Non Responder W52 Non Responder Median A. HIV Env gp41 (Clade C) IgG3 antibody B. HIV Env 186C_D7 gp12 (Clade C) IgG3 antibody 1 1 5 +gp14 +gp14 MVA gp14 Placebo 1 1 5 +gp14 +gp14 MVA gp14 Placebo MFI MFI Threshold (1) Threshold (1) 1 1 Median: Resp %: BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 5 822 1364 452 898 5 13 1459 1892 52 67 45 54 13 15 56 992 2985 179 189 716 1873 74 58 79 27 27 47 68 5 5 8 1 1 Median: Resp %: BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 5 223 363 8 244 5 132 244 399 67 77 5 23 16 55 68 181 446 146 179 116 21 68 72 56 6 33 29 7 5 5 3 C. HIV Env 186C gp14 (Clade C) IgG3 antibody D. HIV Env 186C_V1V2 gp7 (Clade C) IgG3 antibody 1 1 5 +gp14 +gp14 MVA gp14 Placebo 1 1 5 +gp14 +gp14 MVA gp14 Placebo MFI MFI Threshold (1) Threshold (1) 1 1 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 1 1 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 Median: Resp %: 5 5 342 197 31 134 189 514 78 67 43 56 23 16 55 68 623 819 387 337 44 464 68 72 56 6 33 29 7 5 5 3 Median: Resp %: 5 5 5 5 5 5 5 5 5 19 26 13 11 7 1 23 5 5 5 5 5 5 29 16 15 15 1 16 16 5 5 3
Supplemental Figure 3E-H: BAMA assay breadth and specificity of antibody responses Baseline W28 Responder W52 Responder W28 Non Responder W52 Non Responder Median E. HIV Env gp41 (Clade C) IgG-t antibody F. HIV Env 186C_D7 gp12 (Clade C) IgG-t antibody 1 1 5 +gp14 +gp14 MVA gp14 Placebo 1 1 5 +gp14 +gp14 MVA gp14 Placebo MFI MFI Threshold (1) Threshold (1) 1 1 Median: Resp %: BL 198 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 28864 29916 26126 2987 678 1563 27886 291 24797 96 1 89 1 79 89 88 97 28921 15988 2728 27394 3379 25 353 86 97 89 92 9 97 6 1 1 Median: Resp %: BL 5 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 13883 2386 1146 18186 5876 624 18419 24484 1425 1 1 97 1 1 1 98 97 17529 946 857 959 1953 5 89 1 1 1 95 97 5 G. HIV Env 186C gp14 (Clade C) IgG-t antibody H. HIV Env 186C_V1V2 gp7 (Clade C) IgG-t antibody 1 1 5 +gp14 +gp14 MVA gp14 Placebo 1 1 5 +gp14 +gp14 MVA gp14 Placebo MFI MFI Threshold (1) Threshold (1) 1 1 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 1 1 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 Median: Resp %: 5 13883 2386 1146 18186 5876 624 18419 24484 1425 1 1 97 1 1 1 98 97 17529 946 857 959 1953 5 89 1 1 1 95 97 5 Median: Resp %: 5 443 65 73 5 61 141 33 534 1625 5 292 81 66 56 63 6 85 41 12 523 5 131 5 5 73 5 68 36 55
Supplemental Figure 4: ELISA IgG subtype assays Baseline W28 Responder W52 Responder W28 Non Responder W52 Non Responder GMT A. HIV ELISA Env (gp14 T) clade C (ZA) IgG-1 Ab B. HIV ELISA Env (gp14 T) clade C (ZA) IgG-2 Ab +gp14 +gp14 MVA gp14 Placebo +gp14 +gp14 MVA gp14 Placebo ELISA titer ELISA titer LLOQ (29) 1 LLOQ (12) 1 1 BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 GMT: 4 36 6 34 69 12 15 38 76 17 32 2 2 38 81 4 4 Resp %: 79 82 8 95 5 6 82 83 62 72 68 73 82 89 1 GMT: Resp %: BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 4 8 1 7 9 5 6 1 1 8 1 6 6 8 1 4 4 2 2 2 5 2 8 2 8 C. HIV ELISA Env (gp14 T) clade C (ZA) IgG-3 Ab D. HIV ELISA Env (gp14 T) clade C (ZA) IgG-4 Ab +gp14 +gp14 MVA gp14 Placebo +gp14 +gp14 MVA gp14 Placebo ELISA titer ELISA titer 1 LLOQ (12) 1 LLOQ (13) 1 GMT: Resp %: BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 4 11 12 8 9 6 6 12 14 9 1 8 7 9 12 4 4 45 43 29 42 11 7 48 51 29 36 21 15 25 43 1 GMT: Resp %: BL W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 4 4 4 4 4 4 4 4 5 4 4 4 4 4 5 4 4 2 3 4 7 5
Supplemental Figure 5: ELISA and ADCP correlations Clade C ELISA titer [log] 5.5 5. 4.5 4. 3.5 3. 2.5 A. Human data: Week 28 1 2 3 4 5 Phagocytic score (PS) Spearman r: p-value:.6957 <.1 Clade C ELISA titer [log] 5.5 5. 4.5 4. 3.5 3. 2.5 B. Monkey data: Week 28 2 4 6 8 1 Phagocytic score (PS) Spearman r:.6379 p-value: <.1
Supplemental Figure 6: MW965.27 neutralization antibody assay W28 Responder W52 Responder W28 Non Responder W52 Non Responder GMT +gp14 +gp14 MVA gp14 Placebo ID5 GMT: Resp %: W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 W28 W52 28 44 2 33 14 12 27 55 54 76 41 66 22 12 51 73 2 28 16 18 16 31 1 1 43 53 37 45 3 68 2 2
Supplemental Figure 7A-B: IFNg ELISPOT to vaccine matched peptide pools Baseline W26 Responder W5 Responder W26 Non Responder W5 Non Responder A. IFNg Env Mos1 Median +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC 1 1 1 1-1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 28 345 465 177 214 22 162 389 453 413 533 424 458 18 143 28 28 91 88 79 84 75 78 95 95 98 97 92 97 86 76 4 3 P95 (73) B. IFNg Env Mos2 +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC 1 1 1 1-1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 28 138 218 57 97 41 6 152 28 125 187 125 145 8 119 28 28 72 85 41 63 34 38 73 92 72 88 72 81 53 6 2 3 P95 (7)
Supplemental Figure 7C-E: IFNg ELISPOT to vaccine matched peptide pools Baseline W26 Responder W5 Responder W26 Non Responder W5 Non Responder C. IFNg Gag PTE Median +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC P95 (181) 1 1 1 1-1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 9 13 183 9 159 91 117 179 184 22 341 195 243 17 88 28 28 38 44 31 47 39 34 48 5 56 65 5 64 3 22 6 D. IFNg Gag Mos1 +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC P95 (87) 1 1 1 1-1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 28 77 67 58 9 43 28 118 132 135 195 116 116 28 28 28 28 45 46 38 47 36 24 55 62 56 74 5 61 35 24 3 E. IFNg Gag Mos2 +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC 1 1 1 1-1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 28 28 7 28 48 28 28 73 81 68 112 93 93 28 28 28 28 59 36 47 43 35 57 71 56 79 68 67 4 43 2 P95 (55)
Supplemental Figure 7F-H: IFNg ELISPOT to vaccine matched peptide pools Baseline W26 Responder W5 Responder W26 Non Responder W5 Non Responder F. IFNg Pol PTE +gp14 +gp14 MVA gp14 Placebo Median SFU / 1 6 PBMC P95 (15) 1 1 1 1-1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 28 615 753 16 23 34 263 359 622 62 533 415 678 212 18 28 28 83 82 56 54 61 73 68 87 86 91 92 94 67 53 G. IFNg Pol Mos1 +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC P95 (112) 1 1.1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 28 453 4 122 143 22 192 325 327 423 363 311 51 16 88 28 28 81 73 54 56 57 66 7 76 81 85 89 92 58 39 4 3 H. IFNg Pol Mos2 +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC 1 1 1 1-1 Median: Resp %: BL W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 28 427 422 13 155 162 19 323 2 448 58 433 53 157 15 28 28 85 85 77 71 68 79 8 92 86 97 95 1 7 55 P95 (63)
Supplemental Figure 8A-B: ICS assays for cytokine count W26 Responder W5 Responder W26 Non Responder W5 Non Responder Median A. ICS CD4+ HIV ENV gp12 pep pool 1 (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD4+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.78.856.53.553.368.861.813 Resp %: 46 67 43 56 23 16 55 68.338.126.885.13.714.513.374.1.1 68 72 56 6 33 29 7 B. ICS CD4+ HIV ENV gp41 pep pool 1 (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD4+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.276.387.111.176.96.272.35 Resp %: 2 34 6 12 2 3 14 32.87.37.256.123.13.21.232.1.1 21 19 6 9 18 18 2
Supplemental Figure 8C-D: ICS assays for cytokine count W26 Responder W5 Responder W26 Non Responder W5 Non Responder Median C. ICS CD4+ HIV Pol RNAseInt pep pool 1 (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD4+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.147.113.6.139.13.175.14 Resp %: 5 3 9 9 5 3 5 8.33.161.148.243.141.13.1.1.1 13 9 12 6 3 4 2 D. ICS CD4+ HIV Gag pep pool (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD4+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.158.155.79.112.64.366.155 Resp %: 2 11 6 9 7 13 11.55.232.176.347.194.112.56.1.1 14 17 9 14 3 7 5
Supplemental Figure 8E-F: ICS assays for cytokine count W26 Responder W5 Responder W26 Non Responder W5 Non Responder Median E. ICS CD4+ HIV Pol RT pep pool (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD4+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.165.135.88.78.14.228.171 Resp %: 5 3 11 9 2 11 11.78.231.241.239.171.114.58.1.1 8 6 9 9 3 2 F. ICS CD4+ HIV Any (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD4+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 1 1-5 Median: Resp %: W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5.1683.1639.1118.1195.725.712.258.1698.2383.1783.2358.14.168.913.1.1 51 68 58 33 21 55 67 68 72 57 66 37 33 7 3
Supplemental Figure 8G-H: ICS assays for cytokine count W26 Responder W5 Responder W26 Non Responder W5 Non Responder Median G. ICS CD8+ HIV ENV gp12 pep pool 1 (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD8+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.155.74.56.1.97.141.255 Resp %: 16 18 15 14 22 13 19 27.58.234.295.223.256.7.26.1.1 23 36 27 34 7 3 4 H. ICS CD8+ HIV ENV gp41 pep pool 1 (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD8+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.73.56..17.9.85.14 Resp %: 18 21 18 17 11 9 17 21.14.148.195.63.81.32.11.1.1 16 19 14 19 12 9 3
Supplemental Figure 8I-J: ICS assays for cytokine count W26 Responder W5 Responder W26 Non Responder W5 Non Responder Median I. ICS CD8+ HIV Pol RNAseInt pep pool 1 (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD8+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.267.17.65.69.131.185.184 Resp %: 36 31 21 28 33 19 33 28.65.365.62.287.279.11.7.1.1 4 42 38 39 23 15 5 J. ICS CD8+ HIV Gag pep pool (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD8+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.7.72.73.7.51.182.33 Resp %: 18 21 15 17 9 11 23 32.57.214.116.79.14.79.13.1.1 26 37 24 26 19 12 2
Supplemental Figure 8K-L: ICS assays for cytokine count W26 Responder W5 Responder W26 Non Responder W5 Non Responder Median K. ICS CD8+ HIV Pol RT pep pool (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD8+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 Median:.246.321.233.15.78.198.229 Resp %: 33 34 18 22 3 26 37 39.68.29.23.17.188.8.64.1.1 4 34 27 39 21 15 2 2 L. ICS CD8+ HIV Any (Mos1) +gp14 +gp14 MVA gp14 Placebo 1 1 % of CD8+ T-cells 1 1-1 1 1-2 1 1-3 1 1-4 1 1-5 Median: W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5 W26 W5.3221.2183.153.926.741.651.259.3457.3993.432.2126.3419.64.365.38.1 Resp %: 58 64 38 51 48 41 71 77 61 77 63 74 39 31 4 5
Supplemental Figure 9: VNA at Baseline by Region GM IC9 LLOQ (17) N: GM: Eastern Africa South Africa Thailand USA 126 56 58 15 14 15 35 12 Serop %: 85 89 41 13
Supplemental Figure 1: Baseline -specific nabs and ELISA/ELISPOT response correlations Pooled Baseline Responders Responders Non Responders Non Responders A. Env ELISA IgG-t - Week 28 by Baseline VNA: Clade C (C97ZA.12) B. ELISPOT - Week 26 by Baseline VNA: Env pep pool (Clinical PTE) 1 1 6 +gp14 +gp14 MVA gp14 Placebo GM +gp14 +gp14 MVA gp14 Placebo Median 1 1 5 ELISA titer LLOQ (156) SFU / 1 6 PBMC P95 (1) 1 1 1 1-1 GM: Resp %: 2 2131 22583 22822 8235 5184 3151 28991 11823 21279 713 7242 895 17125 1146 2 3 1 1 1 1 1 1 95 1 1 1 1 1 1 1 4 Pooled Baseline 1 1 1 1-1 Median: 28 433 23 312 93 145 78 381 Resp %: 86 61 8 48 86 39 91 Pooled Baseline 181 453 25 382 189 173 12 28 28 62 1 86 84 79 81 62 4 C. Env ELISA IgG-t - Week 52 by Baseline VNA: Clade C (C97ZA.12) D. ELISPOT- Week 5 by Baseline VNA: ENV pep pool (Clinical PTE) 1 1 6 +gp14 +gp14 MVA gp14 Placebo GM +gp14 +gp14 MVA gp14 Placebo Median 1 1 5 ELISA titer LLOQ (156) SFU / 1 6 PBMC P95 (1) 1 1 1 1-1 GM: Resp %: 2 29952 3777 31128 16696 4267 5163 55347 28586 361 15125 675 7644 3463 18189 3 2 1 1 1 1 1 1 95 1 1 96 1 1 1 96 5 Pooled Baseline 1 1 1 1-1 Median: 28 514 281 335 125 179 93 457 Resp %: 88 73 73 55 75 43 95 Pooled Baseline 294 486 385 453 242 348 111 28 28 72 1 75 9 69 91 55
Supplemental Figure 11A-D: Immune response to vaccine stratified by sex and region Male Female GMT A. ELISA Week 28 Clade C97ZA.12: Central Africa B. ELISA Week 28 Clade C97ZA.12: South-Africa 1 1 6 +gp14 +gp14 MVA gp14 Placebo 1 1 6 +gp14 +gp14 MVA gp14 Placebo 1 1 5 1 1 5 ELISA titer LLOQ (156) ELISA titer LLOQ (156) 1 1 1 1 BL W28 W28 W28 W28 W28 W28 W28 W28 GMT 4 11989 7895 359 819 643 6534 648 4 GMT M: 3 1583 6256 387 6416 596 4729 4852 3 GMT F: 4 845 9639 3486 1233 689 1612 1242 7 BL W28 W28 W28 W28 W28 W28 W28 W28 GMT 1 3628 97 291 4326 16314 1886 1588 1 GMT M: 1 29757 8363 291 28154 16314 1489 15458 1 GMT F: 2 1132 1248 82398 48547 1697 C. ELISA Week 28 Clade C97ZA.12: Thailand D. ELISA Week 28 Clade C97ZA.12: USA 1 1 6 +gp14 +gp14 MVA gp14 Placebo 1 1 6 +gp14 +gp14 MVA gp14 Placebo 1 1 5 1 1 5 ELISA titer LLOQ (156) ELISA titer LLOQ (156) 1 1 1 1 BL W28 W28 W28 W28 W28 W28 W28 W28 GMT 2 19884 11451 3319 25879 1123 753 16356 14 GMT M: 3 18793 1997 6485 344 15282 782 19272 142 GMT F: 1 2743 1252 2374 6236 7952 775 14462 5 BL W28 W28 W28 W28 W28 W28 W28 W28 GMT 1 31642 24563 5623 2834 22446 7646 16453 1 GMT M: 1 33575 36454 8425 22168 16323 7715 9938 1 GMT F: 1 3717 261 4237 35256 2853 7586 27238 2
Supplemental Figure 11E-H: Immune response to vaccine stratified by sex and region Male Female Median E. ELISPOT Week 26 PTE Env: Central Africa F. ELISPOT Week 26 PTE Env: South Africa +gp14 +gp14 MVA gp14 Placebo +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC P95 (55) SFU / 1 6 PBMC P95 (55) 1 1 1 1 1 1-1 1 1-1 Median: Median M: Median F: 139 83 96 137 195.5 183 16 27.5 186 68 86 137 158 22 116 27.5 98 93 96 144.5 373 152.5 9 27.5 Median: Median M: Median F: 217 137 132 298.5 188 167 12 27.5 217 158 132 39.5 188 213.5 12 27.5 131 228 27.5 75.3 G. ELISPOT Week 26 PTE Env: Thailand H. ELISPOT Week 26 PTE Env: USA +gp14 +gp14 MVA gp14 Placebo +gp14 +gp14 MVA gp14 Placebo SFU / 1 6 PBMC P95 (55) SFU / 1 6 PBMC P95 (55) 1 1 1 1 1 1-1 1 1-1 Median: Median M: Median F: 384 174.5 141 46.5 248.5 33 18 27.5 583 31 372 513 164 281 18 117.3 234.5 164 122 263 282 8 15.5 27.5 Median: Median M: Median F: 51 312 326 374 454.5 382 211 27.5 727 2252 472 321 432.5 599 211 27.5 453.5 165.5 224 374 54 32 232.5 27.5
Supplemental Figure 11I-J: Immune response to vaccine stratified by age I. ELISA Clade C Week 28 immune response to vaccine by age 1 1 6 1 1 5 Clade C (C972A.12) 1 2 3 4 5 J. ELISPOT Clinical PTE Week 26 immune response to vaccine by age ENV Clinical PTE (SFC/1^6 PBMC) 1 1 1 2 3 4 5 Age (years) / + gp14 / + gp14 / /MVA + gp14 /MVA + gp14 /MVA /gp14
Supplemental Figure 12: Clade C gp14 ELISA antibody titers 5 [log1] ELISA titer 4 3 2 LOD 1 1 2 3 4 5 6 7 Weeks post first immunization / /gp14 /+gp14 /MVA /
Supplemental Figure 13A-D: Serum neutralization of tier-1 viruses GMT A. MW965.26 B. SF162.LS +gp14 MVA gp14 Placebo +gp14 MVA gp14 Placebo ID 5 ID 5 BL W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 GMT: 37 198 165 62 166 185 574 137 13 159 82 4 2 BL W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 GMT: 2 83 19 44 58 111 89 66 35 83 111 21 2 C. MN-3 D. DJ263.8 +gp14 MVA gp14 Placebo +gp14 MVA gp14 Placebo ID 5 ID 5 BL W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 GMT: 26 386 625 18 225 373 33 33 29 25 33 38 2 BL W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 GMT: 24 66 27 32 2 58 2 69 2 37 22 3 2
Supplemental Figure 13E-G: Serum neutralization of tier-1 viruses GMT E. BaL_26 F. ZM19F.PB4 +gp14 MVA gp14 Placebo +gp14 MVA gp14 Placebo ID 5 ID 5 BL W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 GMT: 27 44 25 24 2 38 22 35 2 34 22 25 2 BL W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 GMT: 27 42 2 21 2 24 2 25 2 24 2 25 2 G. MuLV +gp14 MVA gp14 Placebo ID 5 BL W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 W28 W56 GMT: 33 41 2 22 2 26 2 28 2 28 2 38 2
Supplemental Figure 14: PTE g peptide pool IFN-g ELISPOT assays A. ELISPOT PTE g Gag GMT +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W72 W26 W54 W72 W26 W54 W72 W26 W54 W72 W26 W54 W72 GMT: 342 25 195 118 143 161 983 1942 1184 5 124 513 128 126 126 B. ELISPOT PTE g Pol +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W72 W26 W54 W72 W26 W54 W72 W26 W54 W72 W26 W54 W72 GMT: 18 155 158 89 18 114 423 718 389 465 147 588 97 8 9
Supplemental figure 15A-C: ELISPOT mosaic peptide vaccine matched assays A. Mos1 Env GMT +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W26 W54 W26 W54 W26 W54 W26 W54 GMT: 23 28 13 173 744 154 573 1 157 33 B. Mos1 Gag +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W26 W54 W26 W54 W26 W54 W26 W54 GMT: 155 99 76 88 396 893 326 51 87 74 C. Mos1 Pol +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W26 W54 W26 W54 W26 W54 W26 W54 GMT: 163 123 118 146 282 478 436 673 76 69
Supplemental figure 15D-F: ELISPOT mosaic peptide vaccine matched assays D. Mos2 Env GMT +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W26 W54 W26 W54 W26 W54 W26 W54 GMT: 113 226 68 74 38 112 268 711 98 26 E. Mos2 Gag +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W26 W54 W26 W54 W26 W54 W26 W54 GMT: 214 143 8 9 529 1152 399 582 87 94 F. Mos2 Pol +gp14 MVA gp14 SFU / 1 6 PBMC W26 W54 W26 W54 W26 W54 W26 W54 W26 W54 GMT: 137 121 113 143 22 38 412 71 69 63
Supplemental figure 16A-C: ICS assays A. CD4+ Env Median 1 1-1 +gp14 MVA gp14 % IFNg + / IL-2 + of CD4+ T cells 1 1-2 LLOQ (.55) W3 W61 W3 W61 W3 W61 W3 W61 W3 W61 Median:.5.6.5.5.14.11.8.1.5.1 B. CD4+ Gag 1 1-1 +gp14 MVA gp14 % IFNg + / IL-2 + of CD4+ T cells 1 1-2 LLOQ (.55) Median: W3 W61 W3 W61 W3 W61 W3 W61 W3 W61.5.5.5.5.5.5.5.5.5.5 C. CD4+ Pol 1 1-1 +gp14 MVA gp14 % IFNg + / IL-2 + of CD4+ T cells 1 1-2 LLOQ (.55) Median: W3 W61 W3 W61 W3 W61 W3 W61 W3 W61.5.5.5.5.5.5.5.5.5.5
Supplemental figure 16D-F: ICS assays D. CD8+ Env Median 1 1-1 +gp14 MVA gp14 % IFNg + / IL-2 + of CD8+ T cells 1 1-2 LLOQ (.69) Median: W3 W61 W3 W61 W3 W61 W3 W61 W3 W61.6.6.6.6.1.13.6.11.6.6 E. CD8+ Gag 1 1 +gp14 MVA gp14 % IFNg + / IL-2 + of CD8+ T cells 1 1-1 1 1-2 LLOQ (.69) Median: W3 W61 W3 W61 W3 W61 W3 W61 W3 W61.6.6.6.6.37.46.11.16.6.6 F. CD8+ Pol 1 1 +gp14 MVA gp14 % IFNg + / IL-2 + of CD8+ T cells 1 1-1 1 1-2 LLOQ (.69) Median: W3 W61 W3 W61 W3 W61 W3 W61 W3 W61.6.6.6.6.12.6.14.13.6.6