* Corresponding author: Tel.: ; Fax:

JVI Accepted Manuscript Posted Online 8 February 2017 J. Virol. doi:10.1128/jvi.02182-16 Copyright 2017 American Society for Microbiology. All Rights Reserved. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 HIV/AIDS vaccine candidates based on replication-competent recombinant poxvirus NYVAC-C-KC expressing trimeric gp140 and Gagderived VLPs or lacking the viral molecule B19 that inhibits type I interferon activate relevant HIV-1-specific B and T cell immune functions in non-human primates Juan García-Arriaza a, Beatriz Perdiguero a, Jonathan L. Heeney b, Michael S. Seaman c, David C. Montefiori d, Nicole L. Yates d, Georgia D. Tomaras d, Guido Ferrari d, Kathryn E. Foulds e, Mario Roederer e, Steven G. Self f, Bhavesh Borate f Raphael Gottardo f, Sanjay Phogat g, Jim Tartaglia g, Susan W. Barnett h, Brian Burke h, Anthony D. Cristillo i, Deborah E. Weiss i, Carter Lee j, Karen V. Kibler k, Bertram L. Jacobs k, Ralf Wagner l, Song Ding m, Giuseppe Pantaleo n, and Mariano Esteban a * a Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. b Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom. c Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA. d Duke University Medical Center, Durham, North Carolina, USA. e Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA. f Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. g Sanofi Pasteur, Swiftwater, Pennsylvania, USA. h Novartis Vaccines and Diagnostics, Inc., Cambridge, Massachusetts, USA. i Advanced BioScience Laboratories, Inc., Rockville, Maryland, USA. j Global Solutions on Infectious Diseases, South San Francisco, California, USA. k Biodesign Institute, Arizona State University, Tempe, Arizona, USA. l University of Regensburg, Regensburg, Germany. m EuroVacc Foundation, Lausanne, Switzerland. n Division of Immunology and Allergy, Department of Medicine, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. 1

34 35 36 37 38 39 40 41 42 * Corresponding author: Tel.: +34 91 5854553; Fax: +34 91 5854506. E-mail: mesteban@cnb.csic.es Running title: HIV immunogenicity in macaques of replicating NYVACs Key words: HIV-1, poxvirus, NYVAC, B19R, non-human primates, immunogenicity, cellular responses, humoral responses. Abstract word count: 250 Manuscript word count: 6349 43 44 45 2

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 ABSTRACT The non-replicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV- 1 Env(gp120), Gag-Pol-Nef antigens (NYVAC-C) showed in phase I clinical trials limited immunogenicity. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4 + and CD8 + T cells, here we compared the HIV-1-specific immunogenicity elicited in non-human primates immunized with two replicating NYVAC vectors that have been modified by the insertion of K1L and C7L vaccinia viral host-range genes and express clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gagderived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4), and booster doses with NYVAC-C-KC vectors plus clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced an enhanced and similar HIV-1-specific CD4 + and CD8 + T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4 + and CD8 + T cells, induction of some cytokines and in the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions. 3

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia B19 protein, an inhibitor of type I interferon response. Immunization of nonhuman primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited a high level of T cell and humoral immune responses, with the vector containing the deletion in B19R inducing a trend to higher magnitude of CD4 + and CD8 + T cells and neutralization of some HIV-1 strains. These poxvirus vectors could be considered as HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection. INTRODUCTION According to the UNAIDS, about 36.7 million of adults and children were living with HIV worldwide at the end of 2015; however, the number of people newly infected continues to fall, being in 2015 38% lower than in 2001 (www.unaids.org). These data are the result of the global implementation of preventive and therapeutic strategies. Nevertheless, the development of a vaccine remains among the best hopes for controlling the HIV/AIDS pandemic. To date, the phase III clinical trial RV144 is the only HIV-1 vaccine efficacy trial that has demonstrated a modest level of protection (31.2%) against HIV-1 infection in humans (1). The RV144 study combined a recombinant canarypox virus vector (ALVAC) expressing HIV-1 antigens from clades B and E as a prime with recombinant HIV-1 gp120 proteins from clades B and CRF01_AE as a boost. Further studies evaluating potential immune correlates of protection have shown that CD4 + T cells, IgG antibodies to the V1/V2 and V3 loops of HIV-1 gp120, IgG3 antibodies to gp120, together with ADCC responses, correlated with decreased risk of HIV-1 infection, whereas IgA antibodies to the envelope correlated with a decreased vaccine efficacy in the vaccine group (2-7). These clinical findings provided for the first time evidence that an HIV/AIDS vaccine can prevent HIV-1 infection and highlighted that poxvirus vectors should be considered as one of the future HIV/AIDS vaccine candidate vectors. Among the poxviruses, the highly attenuated vaccinia virus (VACV) strain NYVAC has emerged as a potential HIV/AIDS vaccine candidate (8, 9). Importantly, several NYVAC-based recombinants expressing HIV-1 antigens from clades B or C have been evaluated as HIV/AIDS vaccine candidates in preclinical (10-28) and clinical (20, 29-34) studies with encouraging results of HIV-1-specific T cell and humoral 4

107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 immune responses. However, new strategies have been implemented to improve poxvirus vector immunogenicity (35). Among these strategies, we have previously reported that deletion of immunomodulatory VACV genes such as B19R and/or B8R, that block interferon (IFN) type I and type II pathways (17) or insertion of the host range VACV C7L gene into NYVAC recombinant vectors expressing clade C (CN54) HIV-1 Env (gp120), and Gag-Pol-Nef antigens as a polyprotein (NYVAC-C) significantly improved the magnitude and quality of the HIV-1-specific immune responses in mice (23). Furthermore, deletion of B19R and/or B8R genes in NYVAC-C triggered an upregulation of innate immune pathways in infected human monocytes with a robust expression of type I IFNs and IFN-stimulated genes (ISGs), a strong activation of the inflammasome, and upregulation of the expression of IL-1β and proinflammatory cytokines (12). Moreover, restoration of replication competence in human cells of NYVAC-C by re-incorporation of the K1L and C7L VACV host range genes (NYVAC-C- KC) with or without removal of the immunomodulatory viral molecule B19, enhanced cross-presentation and proliferation of HIV-1-specific memory CD8 + T cells in vitro (26). These recombinants selectively activated IFN-induced genes and genes involved in antigen processing and presentation as determined by microarray analysis of infected human dendritic cells (DCs) (19, 26). At the same time, these constructs maintained limited virus spread in tissues and an attenuated phenotype (26). Additionally, further improved NYVAC recombinants expressing HIV-1 immunogens, such as HIV-1 clade C (ZM96) trimeric soluble gp140 or Gag(ZM96)-Pol-Nef(CN54) as Gag-derived VLPs have been shown an enhanced HIV-1-specific immunogenicity profile in mice (24) and non-human primates (NHPs) (10, 13). Clinical trials with homologous NYVAC vectors expressing HIV-1 antigens (gp120 Env, and the polyprotein Gag-Pol-Nef) have shown a limited immunogenicity profile with preference for CD4 + T cell activation, markedly enhanced when priming was performed with a DNA vector expressing the same HIV-1 antigens (29-33). Thus, in order to optimize the immunization protocol with NYVAC vectors expressing HIV-1 antigens, various approaches have been evaluated in NHPs,either comparing NYVAC versus ALVAC (13), or combining NYVAC with DNA vectors (10), peptides (22) and with dendritic cell targets (28), and have all demonstrated promising results. Here, as part of the Poxvirus T cell Vaccine Discovery Consortium (PTVDC) from the Collaboration for AIDS Vaccine Discovery (CAVD) of Bill and Melinda Gates Foundation, we extended our previous studies with NYVAC recombinants (19, 26) and evaluated in NHP novel NYVAC recombinants. Hence, we combined in a single recombinant NYVAC vector a set of strategies: restoration of replication competence, expression of novel HIV-1 immunogens (trimeric gp140 and Gag-Pol-Nef as Gag- 5

144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 derived VLPs), and deletion of B19R immunomodulatory gene (NYVAC recombinants termed NYVAC-C-KC and NYVAC-C-KC-ΔB19R). Thus, NYVAC-C-KC and NYVAC-C- KC-ΔB19R were compared in immunized NHP to evaluate the HIV-1-specific immunogenicity profile induced by these novel NYVAC recombinants, when applied in a prime/boost approach following similar delivery of the immunogens, poxvirus and protein, as for the protocol used in the RV144 phase III clinical trial. The aim was to define the type of HIV-1-specific T cell and humoral immune responses induced by these vectors as a function of immunological markers that have been correlated with HIV-1 immune efficacy. The results showed that replicating NYVAC-C-KC vectors together with booster of the purified gp120 protein component induced enhanced activation of HIV-1-specific CD4 + and CD8 + T cell immune responses, together with a strong induction of HIV-1-specific humoral immune responses. These results demonstrate that replicating NYVAC-C-KC vectors triggered relevant HIV-1-specific immunological properties as potential correlates of protection, with VACV B19 protein exerting some control of immune functions, and supporting the use of these novel NYVAC-C-KC recombinants as HIV/AIDS vaccine candidates. 6

161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 MATERIALS AND METHODS Replication-competent recombinants NYVAC-C-KC and NYVAC-C-KC-ΔB19R expressing clade C HIV-1 antigens. VACV K1L and C7L genes were inserted into poxvirus recombinants NYVAC-gp140 and NYVAC-Gag-Pol-Nef, previously described in (24), to generate respectively the replication-competent recombinants NYVAC-KCgp140 and NYVAC-KC-Gag-Pol-Nef, following the same methodology previously described (19). Thus, the replication-competent NYVAC-C-KC vector consists of two NYVAC-KC viruses that express different clade C HIV-1 antigens under the same synthetic early/late poxvirus promoter (36), one (NYVAC-KC-gp140) expressing Env gp140 from strain 96ZM651 and the other (NYVAC-KC-Gag-Pol-Nef) expressing Gag from strain 96ZM651 and Pol and Nef from strain CN54. A description of the HIV-1 gp140 and Gag-Pol-Nef sequences included in the NYVAC-KC recombinants was previously reported (10, 24). Next, the VACV B19R gene (B18R in WR strain) was deleted from the replication-competent recombinants NYVAC-KC-gp140 and NYVAC- KC-Gag-Pol-Nef, to generate respectively the replication-competent recombinants NYVAC-KC-gp140-ΔB19R and NYVAC-KC-Gag-Pol-Nef-ΔB19R, following the same methodology previously described (19). Once generated, the resulting replicationcompetent NYVAC-KC-gp140, NYVAC-KC-Gag-Pol-Nef, NYVAC-KC-gp140-ΔB19R and NYVAC-KC-Gag-Pol-Nef-ΔB19R recombinant viruses were expanded in large cultures of primary chicken embryo fibroblast (CEF) cells followed by virus purification through two 36% (wt/vol) sucrose cushions. Titers were determined by plaque immunostaining in BSC-40 cells. Expression of HIV-1 antigens by Western blotting and analysis of plaque size were done as previously described (24). Infection of human macrophages (THP-1 cells) and RNA analysis of IFN-β, MIP-1α, IL-8, and IL-1β by quantitative real-time PCR was done as previously described (37, 38). For simplicity of terminology, we refer to the combined mixed inoculation of NYVAC-KC-gp140 plus NYVAC-KC-Gag-Pol-Nef as NYVAC-C-KC and that of NYVAC-KC-gp140-ΔB19R plus NYVAC-KC-Gag-Pol-Nef-ΔB19R as NYVAC-C-KC-ΔB19R. HIV-1 proteins. For the immunizations performed in this study we have used a bivalent HIV-1 gp120 protein containing a mixture of TV1 gp120 and 1086 gp120, both from clade C. These proteins were expressed from stably transfected Chinese hamster ovary (CHO) cell lines, purified and characterized as previously described (39). Non-human primates. Animals used in this study (designated AUP513) were outbred adult male Indian rhesus macaques (Macaca mulatta) which were housed, fed, given environmental enrichment, and handled at the animal facility of Advanced BioScience 7

198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 Laboratories (ABL), Inc. (Rockville, MD). The study protocol and animal care are in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The study was approved by the ABL, Inc., Institutional Animal Care and Use Committee in accordance with international guidelines. All procedures were carried out under anesthesia (ketamine administered at 10 mg/kg of body weight) by trained personnel under the supervision of veterinary staff, and all efforts were made to ameliorate animal welfare and to minimize animal suffering. During the study, macaques were observed for general behavior, clinical symptoms, and local reactions at the injection sites twice daily the week after immunizations. When animals were sedated for immunizations or sample collections, body weight and temperature were measured. The age of the animals ranged between 2.5 and 2.8 years, with a mean of 2.6 years and the weight range was between 3.1 to 5.7 kg, with a mean of 3.8 kg. At selected time points, a physical examination was performed by a veterinarian, and clinical chemistry and hematology parameters were measured. All rhesus macaques were negative for tuberculosis, simian retrovirus (SRV), simian T cell leukemia virus (STLV-1), herpesvirus B, simian immunodeficiency virus (SIV), measles and poxvirus immunogens prior to the study, and have also negative fecal culture for salmonella, shigella, campylobacter and yersinia. Furthermore, animals were immunologically naïve for the vaccine components. A total of 16 animals were divided into two groups of eight animals each. Vaccines and immunization schedule. Two immunizations groups of eight rhesus macaques were included in the study protocol (termed AUP513), which was also previously reported (13). Group 1 received two immunizations with NYVAC-C-KC (containing a 1:1 mixture of NYVAC-KC-gp140 and NYVAC-KC-Gag-Pol-Nef) at weeks 0 and 4 and was boosted with two immunizations of NYVAC-C-KC plus bivalent HIV-1 gp120 proteins from clade C (containing a 1:1 mixture of TV1 gp120 and 1086 gp120) at weeks 12 and 24. Group 2 received two immunizations with NYVAC-C-KC-ΔB19R (containing a 1:1 mixture of NYVAC-KC-gp140-ΔB19R and NYVAC-KC-Gag-Pol-Nef- ΔB19R) at weeks 0 and 4 and was boosted with two immunizations of NYVAC-C-KC- ΔB19R plus bivalent HIV-1 gp120 proteins from clade C (TV1 and 1086 gp120) at weeks 12 and 24. All immunizations with the replication-competent NYVAC-C-KC vectors and proteins were given intramuscularly (i.m.) in the deltoid muscle of the upper right arm for the replication-competent NYVAC-C-KC vectors and in the opposite site, the upper left arm, for the gp120 proteins. A dose of 1 x 10 8 PFU of each replication-competent NYVAC-C-KC vector (NYVAC-KC-gp140 plus NYVAC-KC-Gag- 8

235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 Pol-Nef in the case of NYVAC-C-KC, and NYVAC-KC-gp140-ΔB19R plus NYVAC-KC- Gag-Pol-Nef-ΔB19R for NYVAC-C-KC-ΔB19R; 2 x 10 8 PFU of total virus in 1.0 ml of Tris-buffered saline) and 50 µg of each of the TV1 and 1086 clade C HIV-1 gp120 proteins (with adjuvant MF59; 100 µg of total protein in 1.0 ml) was used for each immunization. At weeks 0, 6, 14, 26 and 36 (at the beginning of the study, two weeks after the second, third, and fourth immunizations, and at the end of the study, respectively), peripheral blood mononuclear cells (PBMCs) and serum samples were obtained from each immunized animal, and HIV-1-specific T cell and humoral immune responses were analyzed. Blood samples were processed following current procedures (40). Peptides. Overlapping peptides (15-mers with 11 amino acids overlapping) spanning the Env, Gag, Pol, and Nef HIV-1 clade C regions were matched to the inserts expressed by NYVAC-C-KC and NYVAC-C-KC-ΔB19R. Peptides used in the IFN-y ELISpot and intracellular cytokine staining (ICS) assays for T cell stimulation were grouped in nine different peptide pools (Env-1, Env-2, Env-3, Pol-1, Pol-2, Gag-1, Gag/Pol, Gag-2/Pol and Nef), with about 60 peptides per pool. IFN-γ ELISpot assay. The overall strength of HIV-1-specific T cell immune responses induced was analyzed by IFN-γ ELISpot analysis from freshly isolated PBMCs obtained from each immunized rhesus macaque, as previously described (10). Briefly, PBMCs were stimulated in triplicate with HIV-1 Env, Gag, Pol, and Nef peptide pools at 1 µg/ml, or with phytohemagglutinin (PHA) (2.5 µg/ml) as a positive control, while addition of medium only served as a negative control, following the protocol described previously (10). The spot forming units (SFUs) were counted as a measure of the magnitude of HIV-1-specific T cells. ICS assay. The HIV-1-specific CD4 + and CD8 + T cell immune responses induced were analyzed by polychromatic ICS from PBMCs obtained from each immunized rhesus macaque, as previously described (13, 40). In short, cryopreserved PBMCs were thawed and rested overnight in R10 medium (RPMI 1640 [BioWhittaker, Walkersville, MD], 10% FBS, 2 mm L-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin) with 50 U/ml Benzonase (Novagen, Madison, WI) in a 37ºC, 5% CO 2 incubator. Next day, 1 x 10 6 to 3 x 10 6 cells were stimulated in 96-well plates with the corresponding HIV-1 Env, Gag, Pol, and Nef peptide pools (2 μg/ml) in the presence of brefeldin A (10 μg/ml; BD Biosciences, San Jose, CA) for 6 h. Negative controls received an equal concentration of dimethyl sulfoxide (DMSO) instead of peptides. For staining, cells 9

272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 were fixed, permeabilized, and stained with different antibodies. The following fluorescence-labeled antibodies were used: CD3-Cy7-APC (clone SP34.2; BD Biosciences), CD4-BV421 (clone OKT4; BioLegend), CD8-BV570 (clone RPA-T8; BioLegend), IFN-γ-APC (clone B27; BD Biosciences), IL-2-PE (clone MQ1-17H12; BD Biosciences) and TNF-α-FITC (clone Mab11; BD Biosciences). An Aqua LIVE/DEAD kit (Invitrogen, Carlsbad, CA) was used to exclude dead cells. All antibodies were previously titrated to determine the optimal concentration. Samples were acquired on an LSR II flow cytometer and analyzed using FlowJo version 9.8 (Treestar, Inc., Ashland, OR). HIV-1-specific binding antibody assay. HIV-1-specific binding IgG and IgA antibodies to HIV-1 gp120/gp140 proteins, and murine leukemia virus (MuLV) gp70- scaffolded V1/V2 were measured using a binding antibody multiplex assay (BAMA) in serum and rectal weck elutions from each immunized rhesus macaque, as previously described (6, 7, 41). HIV-1 antigens used to analyze the levels of IgG or IgA binding antibodies included: (i) consensus gp140 proteins of clade A (A1.con.env03 140 CF), clade B (B.con.env03 140 CF), clade C (C.con.env03 140 CF); and group M (Con S gp140 CF), (ii) primary Env variants 1086 gp120 (clade C), TV1 gp120 (clade C), JRFL gp140 (clade B), and MSA4076 gp140 (clade A1; also known as OOMSA); and (iii) MuLV gp70-scaffolded V1/V2 (from clade B; gp70_b.casea2 V1/V2). Different plasma serial dilutions were made, and the primary readout was the mean fluorescence intensity (MFI). For IgA, results are expressed as the MFI minus the background MFI at dilution 1/80. For IgG, MFI data were transformed to values defining the area under the curve (AUC), which resemble the integral of the curve for MFI plotted against serial dilutions. Moreover, the specific activity of rectal mucosal IgG binding antibodies was calculated by dividing the antibody titer by the total IgG concentration. Values 3-fold above the baseline visit were considered positive, and the cutoff was established using serum-negative samples. All assays were performed under Good Clinical Laboratory Practices (GCLP)-compliant conditions. Antibody-dependent cellular cytotoxicity (ADCC) assay. The ADCC activity against recombinant gp120 proteins from isolates 1086 or TV1 (clade C) was analyzed in serum from each immunized rhesus macaque according to the ADCC-GranToxiLux (GTL) procedure, as previously described (42, 43). The results of the GTL assay were considered positive if the percentage of Granzyme B activity after background subtraction was 8% for the infected target cells (44). The log 10 titer of the ADCC antibodies present in the plasma was calculated by interpolating the log reciprocal of 10

309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 the last plasma dilution that yielded positive Granzyme B activity ( 8%). The GTL- ADCC assay was performed under GCLP-compliant guidelines. Neutralizing antibodies against HIV-1. Neutralizing antibodies (NAb) against HIV-1 were analyzed in serum from each immunized rhesus macaque using the TZM-bl cell assay and the more sensitive A3R5 cell assay, as previously described (45-48). For the TZM-bl assay pseudoviruses carrying the following Envs were used: BaL.26 (tier 1B, clade B), Ce1086 (tier 2, clade C), Ce1176 (tier 2, clade C), Ce2010 (tier 2, clade C), Du151.2 (tier 2, clade C), MN.3 (tier 1A, clade B), MW965.26 (tier 1A, clade C), SF162.LS (tier 1A, clade B), TH023.6 (tier 1 Clade AE), TV1.21 (tier 2, clade C), and MuLV (as a negative control). For the A3R5 cell assay, we used viral infectious molecular clones (IMCs) which encoded luciferase on the genome (and expressed in the cells only upon infection) and carrying the ectodomains of env genes of the following isolates: Ce1086 (tier 2, clade C), Ce1176 (tier 2, clade C), Ce2010 (tier 2, clade C), Du151.2 (tier 2, clade C), and TV1.21 (tier 2, clade C). For both assays, the neutralization titers are represented as the log 10 of the serum dilution at which relative luminescence units (RLU) were reduced by 50% (ID50) compared to virus control wells after subtraction of background RLU in cell control wells. Both assays were done under GCLP-compliant conditions. Statistical procedures. The Wilcoxon rank sum test was used to compare differences between groups. P values of <0.05 were considered significant. All values used for analyzing proportionate representation of responses were background subtracted. Box plots were used to summarize the distribution of various immune responses, where the midline of the box indicates the median, the ends of the box denote the 25th and 75th percentiles, and whiskers extended to the extreme data points that are no more than 1.5 times the interquartile range (IQR) or, if no values meet this criterion, to the data extremes. Spearman s rank-based correlations were used to compare association of CD4 + and CD8 + T cell responses with antibody responses from BAMA, GTL ADCC and NAb assays at week 26 between NYVAC-C-KC and NYVAC-C-KC-ΔB19R vectors. Specifically, DMSO-adjusted percent positivity for each cytokine and pooled antigen combination from the CD4 + and CD8 + T cell responses was assessed for correlation with: 1) AUC against gp120.tv1 antigen for the IgG subclass in the BAMA assay, 2) titer against the gp120.tv1 antigen in the ADCC GTL assay, and 3) titer against the MW965.26 isolate in the TZM.bl NAb assay. Significance of the correlations was derived using a one-sided p-value with the alternative that true correlation is greater than 0. 11

346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 RESULTS Enhanced expression, plaque size and innate immune profile of NYVAC-C-KC and NYVAC-C-KC-ΔB19R vectors. We have previously described the generation and characterization of non-replicating NYVAC vectors expressing clade C HIV-1 trimeric gp140 or Gag-Pol-Nef as a polyprotein processed into Gag-derived VLPs and their immune behavior in mice (24) and in NHPs (13). Since these vectors do not replicate in human cells, it was important to define if novel replication-competent NYVAC-KC vectors could be more immunogenic as a function of higher levels of antigen expression during infection. Analysis by Western blot of the expression of HIV-1 gp140 in human HeLa cells is shown in Fig. 1A. Clearly, higher levels of expression at late times post infection were observed in the NYVAC-C-KC vectors as opposed to parental NYVAC-C. These differences were also noticeable after analysis of the virus plaque size phenotype in cultured BSC-40 cells. The NYVAC-C-KC vectors have larger plaque size phenotype, with or without B19R, than parental NYVAC-WT or NYVAC-gp140, consistent with a higher replication capacity of NYVAC-KC vectors over parental NYVAC (Fig. 1B). Analysis by real time PCR of the innate immune response elicited in human macrophages (THP-1) infected with the NYVAC-C-KC vectors, showed that compared to NYVAC-KC-gp140, NYVAC-KC-gp140-ΔB19R triggered a significant upregulation of the mrna levels of type I IFN (IFN-β), MIP-1α, IL-8, and IL-1β (Fig. 1C), indicating some differential innate immune responses between these vaccines. Immunological responses induced in NHP after prime/boost immunization with NYVAC-C-KC or NYVAC-C-KC-ΔB19R vectors. To define the HIV-1-specific immune responses induced by these two novel optimized replication-competent NYVAC vectors expressing HIV-1 clade C immunogens and containing a deletion in VACV B19R gene, and to further evaluate whether these responses could be relevant in the control of HIV-1 replication, we analyzed a number of immune parameters that have been previously described as potential indicators for correlates of protection (2-7). The immunization protocol in NHPs is depicted in Figure 2 and follows a similar immunization scheme as described in the RV144 efficacy clinical trial (1), allowing a head-to-head comparison of the immunogenicity induced by both NYVAC-C-KC vectors. Figure 2A summarizes the 2 immunization groups included in the study (see Materials and Methods for details). Rhesus macaques were divided into two groups of eight animals each: group 1 received in the upper right arm an intramuscular (i.m.) administration of 2x10 8 pfu of NYVAC-C-KC (1:1 mixture of 1x10 8 pfu of NYVAC-KCgp140 and 1x10 8 pfu of NYVAC-KC-Gag-Pol-Nef); group 2 received a similar 12

382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 administration protocol but with NYVAC-C-KC-ΔB19R (1:1 mixture of 1x10 8 pfu of NYVAC-KC-gp140-ΔB19R and 1x10 8 pfu of NYVAC-KC-Gag-Pol-Nef-ΔB19R). The first two administrations with the NYVAC-C-KC vectors were given at weeks 0 and 4; thereafter animals received at weeks 12 and 24 i.m booster doses of the corresponding NYVAC-C-KC vectors in the upper right arm, while in the left arm animals received booster immunization with a bivalent clade C gp120 protein (1:1 mixture of 50 µg of 1086 gp120 and 50 µg of TV1 gp120 proteins adjuvanted with MF59). Immunological monitoring of HIV-1-specific T and B cell immune responses induced by both groups was performed from PBMCs, serum or rectal mucosal samples isolated at weeks 0, 6, 14, 26, and 36 (at the beginning of the study, 2 weeks after the second, third, and fourth immunizations, and at the end of the study, respectively), following similar analytical approaches as previously described for NHP (13) (Fig. 2B). HIV-1-specific T cell immune responses. The total magnitude of HIV-1-specific T cell immune responses induced by NYVAC-C-KC and NYVAC-C-KC-ΔB19R vectors was measured at weeks 0, 6, 14, 26, and 36 by IFN-γ ELISpot assay. The results showed that the mean SFU values induced by both immunization groups were low until week 14, and clearly peaked at week 26 (two weeks after the completion of the prime/boost immunization protocol) (Fig. 3A). As expected, the SFU values decline at week 36. There were no significant statistical differences in the levels of IFN-γ positive T cells between the two groups at any timepoint. However, at week 26 of immunization NYVAC-C-KC elicited a trend to higher numbers of SFUs; while at week 36 of immunization NYVAC-C-KC-ΔB19R induced a trend to higher responses. Furthermore, we also analyzed the magnitude of HIV-1-specific T cell immune responses induced by NYVAC-C-KC and NYVAC-C-KC-ΔB19R vectors by ICS assay. Thus, we measured at weeks 6, 14, 26, and 36 the percentage of HIV-1-specific CD4 + and CD8 + T cells secreting IFN-γ, IL-2 and/or TNF-α after stimulation of PBMCs obtained from each immunized rhesus macaque with peptide pools that spanned the HIV-1 Env, Gag, Pol, and Nef antigens present in the inserts. As observed in Fig. 3B and 3C, the total magnitude of HIV-1-specific CD4 + and CD8 + T cell responses peaked at week 26, with a decline at week 36, similar to the results obtained with the IFN-γ ELISpot assay. There were no statistically significant differences in the magnitude of HIV-1-specific CD4 + T cell responses induced by both immunization groups at any timepoint (Fig. 3B). However, at weeks 14 and 36 of immunization NYVAC-C-KC- ΔB19R induced a trend to higher HIV-1-specific CD4 + T cell responses; while at week 26 of immunization NYVAC-C-KC induced a trend to higher responses. On the other 13

418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 hand, the analysis of the magnitude of the total HIV-1-specific CD8 + T cell responses (Fig. 3C), showed that immunization with NYVAC-C-KC-ΔB19R induced a trend toward increased levels over NYVAC-C-KC at all time points, but the differences were not significant. Moreover, the analysis of the cytokine responses generated by both immunization groups revealed that in agreement with the total magnitude of HIV-1- specific CD4 + T cell responses, immunization with NYVAC-C-KC-ΔB19R induced at week 36 a trend to higher magnitude of HIV-1-specific CD4 + T cells producing TNF-α (Fig. 4B), or IL-2 (Fig. 4C), but the differences were not significant. However, at week 26 of immunization with NYVAC-C-KC a trend to higher magnitude of HIV-1-specific CD4 + T cells producing IFN-γ (Fig. 4A), TNF-α (Fig. 4B), or IL-2 (Fig. 4C) was observed. In the case of HIV-1-specific CD8 + T cells, and in agreement with the total values of CD8 + T cells in Figure 3C, immunization with NYVAC-C-KC-ΔB19R induced at most of the time points a trend toward higher magnitude of HIV-1-specific CD8 + T cells producing IFN-γ (Fig. 4D), TNF-α (Fig. 4E), or IL-2 (Fig. 4F), although there were no statistical differences between groups. Binding antibody responses. Since the RV144 phase III clinical trial showed that IgG antibodies against the V1/V2 and V3 regions of HIV-1 gp120 correlated with decreased risk of HIV-1 infection (2-7), we next analyzed the HIV-1 specific humoral immune responses elicited after immunization with the two NYVAC-C-KC vectors. Thus, we quantified in individual serum samples obtained from each immunized rhesus macaque the total binding of IgG antibody levels against clade C HIV-1 gp140, gp120, and MuLVgp70-scaffolded V1/V2 proteins at weeks 6, 14, 26, and 36 (Fig. 5). Results showed that there was strong IgG binding antibody levels to the different HIV-1 envelopes tested in all animals when the NYVAC-C-KC vectors were immunized together with the gp120 protein component, although there were no statistical significant differences between the two groups. In these immunization groups, the maximum IgG antibody levels were detected at week 26 (two weeks after the second booster dose with NYVAC-C-KC/gp120 protein), and by week 36 IgG antibody levels declined but still were maintained at levels superior to two doses of NYVAC-C-KC vectors (week 6). Immunization with NYVAC-C-KC elicited a trend to higher levels of binding IgG antibodies against consensus HIV-1 gp140 protein of group M (Fig. 5A), gp120 from isolate TV1 (Fig. 5B), gp120 from isolate 1086 (Fig. 5C), and the MuLV gp70-scaffolded V1/V2 protein (Fig. 5D), although there were no statistical differences between groups. 14

455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 Similarly to the results in Figure 5, cross-clade binding IgG antibody levels induced by both immunization groups were low by week 6 but increased markedly by week 26, and waned with time at week 36 but remained to levels similar or higher to week 6 (data not shown). Overall, both immunization groups induced similar high levels of cross-clade binding IgG antibodies against consensus HIV-1 gp140 of clade A, clade B, clade A gp140 (00MSA) and clade B gp140 (JRFL), with no statistically significant differences between the groups. A trend to higher cross-clade binding IgG antibodies for NYVAC-C-KC was noted at week 14. In addition, rectal IgG binding responses against group M HIV-1 gp140 consensus, and clade C gp120 from isolates 1086 and TV1 were detected in both immunization groups, with a peak of response at week 26, and a decline at week 36 (Fig. 5E), although differences were not significant. The rates of responders at week 26 were higher in the NYVAC-C-KC-ΔB19R immunization group, while at week 36 was higher in the NYVAC-C-KC immunization group. Because the RV144 phase III clinical trial showed that high levels of binding plasma IgA antibodies to HIV-1 Env correlated directly with an increased risk of infection (4, 5), we next analyzed the binding IgA antibodies induced after immunization with the two NYVAC-C-KC vectors in serum and in rectal mucosal (data not shown). The results in serum showed that both immunization groups induced very low levels (barely above the background for most time-points) of binding IgA antibodies against the consensus HIV-1 gp140 protein of clade C, gp120 from isolates TV1, and gp120, and the MuLV gp70-scaffolded protein. Furthermore, similar negligible levels of binding IgA antibodies to consensus HIV-1 gp140 proteins of clade A, B, group M, clade A gp140 (00MSA) and clade B gp140 (JRFL) were observed from the two vaccinated groups. There were no rectal IgA binding antibodies (data not shown). ADCC responses. Since ADCC can mediate non-neutralizing antibody effector functions dependent on Fc receptor engagement, and has been suggested as a correlate of protection in the RV144 phase III clinical trial (4, 49), we next analyzed the ability of NYVAC-C-KC and NYVAC-C-KC-ΔB19R to induce ADCC responses against HIV-1 gp120 proteins from isolates TV1 and 1086 at weeks 0, 26, and 36 in individual serum samples obtained from each immunized rhesus macaque (Fig. 6A). The results showed that both immunization groups elicited similar titers of ADCC at week 26 against HIV-1 gp120 from isolates TV1 and 1086, with a decline at week 36. The titers against target cells coated with the HIV-1 gp120 protein of TV1 isolate were greater than those obtained with HIV-1 gp120 of 1086 isolate. Although there were no statistical differences between plasma samples from the two NYVAC-C-KC vectors, 15

492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 immunization with NYVAC-C-KC-ΔB19R induced a trend to higher ADCC titers against HIV-1 gp120 protein of TV1 isolate at week 36 than NYVAC-C-KC. On the other hand, NYVAC-C-KC elicited higher ADCC responses against HIV-1 gp120 1086. Neutralizing antibody responses. The induction of HIV-1 NAb is of special importance to control HIV-1 (50). Thus, we next analyzed the capacity of NYVAC-C-KC and NYVAC-C-KC-ΔB19R to elicit NAb against a panel of different Tier 1 and 2 HIV-1 viruses at weeks 26 and 36 in individual serum samples obtained from each immunized rhesus macaque (Fig. 6B). The results showed that neutralization titers induced by both immunization groups were higher at week 26 than at week 36, with overall no significant differences between both groups in the neutralization against the different Tier 1 and 2 HIV-1 isolates. However, some minor differences were observed. Using the more sensitive A3R5 assay, results showed that NYVAC-C-KC-ΔB19R induced a trend to higher titers of NAb against HIV-1 gp120 TV1 (Tier 2, clade C), and Ce1086 (Tier 2, clade C), at weeks 26 and 36, although there were no statistical differences compared to NYVAC-C-KC. The TZM.bl assay showed that both immunization groups induced similar levels of NAb against a wide range of Tier 1 HIV- 1 isolates. Correlation between T cell and antibody responses. Next, to define if there is a correlation between the two vectors in the elicitation of CD4/CD8 T cell responses versus the induced antibody responses, we performed a statistical correlation analysis at week 26. Figure 7 shows correlation coefficients between T cell responses (ICS assay from both CD4/CD8) and antibody responses (BAMA, ADCC GTL and NAb assays). In general, CD4 + T cell responses showed a higher number of positive correlations with antibody responses as compared to CD8 + T cell responses. Within CD4 + T cell responses, NYVAC-C-KC-ΔB19R showed a more favorable trend for a correlation with antibody responses as compared to NYVAC-C-KC. Most of the correlations fail to be significant (< 0.05) due to low sample size, except between CD4 + and ADCC and NAb within the NYVAC-C-KC-ΔB19R group. DISCUSSION After the modest efficacy obtained in the RV144 phase III clinical trial the scientific community has focused on the generation and optimization of HIV/AIDS vaccine candidates to afford protection to a large percentage of vaccinees. Poxviruses, and in particular the highly attenuated VACV strains MVA and NYVAC, have being widely used as HIV/AIDS vaccine candidates and are included as 16

529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 components of some of the clinical trials planned within the next 5 years on the basis of lessons learned from recent trials (8, 9, 51). However, despite the safety and immunogenicity profiles exhibited by these attenuated VACV strains, more efficient vectors that enhance the magnitude, breadth, polyfunctionality, and durability of the immune responses to exogenously expressed antigens are desirable. Different strategies have been followed for poxviruses to achieve that purpose (35). One of them is the deletion of VACV immunomodulatory genes, that are still present in the vector genome, and whose gene products may be predicted to interfere with the optimal induction of cellular and humoral responses. In this sense, we have previously reported enhanced immunogenicity in mice with MVA and NYVAC-based recombinants by single or multiple deletions of VACV immunomodulatory genes such as B8R and/or B19R (17), A46R (25), C12L (52), C6L and/or K7R (37, 38), A41L and/or B16R (53), N2L (54) and F1L (55). Another strategy is the generation of new vectors with replication competence in human cells in order to increase the timing and levels of expression of the heterologous antigen in the host. To date, some replicationcompetent recombinant VACV-based vaccines have been used for various infectious diseases, demonstrating that they are able to elicit potent humoral and cellular mediated immune responses, and to confer long-lasting protection while maintaining a safety phenotype (23, 56-58). In this study, we aimed to define best-in-class protocols of immunization following lessons learned from the RV144 efficacy clinical trial with poxvirus vectors. We have evaluated and compared the immunogenicity in NHP of two replicationcompetent NYVAC-C recombinants generated by restoration of replication competence in human cells after the re-incorporation of the K1L and C7L host range genes (NYVAC-C-KC) or by combination of restoration of replication competence with removal of the immunomodulatory viral molecule B19 (NYVAC-C-KC-ΔB19R) that blocks type I IFN response. Through extensive analysis of HIV-1 specific immune responses, including the measurement of several immune markers that have been associated in the RV144 phase III clinical trial with immune efficacy (2-7), we observed that both NYVAC-C-KC and NYVAC-C-KC-ΔB19R viruses were able to induce a broad spectrum of HIV-1-specific B and T cell responses against the HIV-1 antigens. Thus, the vaccine-induced T cell immune responses were high in magnitude in both immunization groups, and the deletion of the immunomodulatory gene B19R on NYVAC-C-KC recombinant improved to some extent the magnitude of the HIV-1- specific CD4 + and CD8 + T cell immune responses. Moreover, a low level of IgA binding antibodies, high level of binding IgG antibodies in serum and rectal mucosal samples, ADCC responses and NAb against different tier 1 and 2 HIV-1 isolates were elicited by 17

566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 NYVAC-C-KC and NYVAC-C-KC-ΔB19R, with some minor differences between the two groups. Interestingly, at the peak of response (week 26), the higher levels of HIV- 1-specific CD4 + and CD8 + T cells elicited by NYVAC-C-KC and NYVAC-C-KC-ΔB19R coincides with similar high levels of antibodies to Env as well as to high HIV neutralization titers and of ADCC at the same time, suggesting a correlation between enhanced CD4 + /CD8 + T cell responses and B cell responses, pointing that CD4 + and/or CD8 + T cells might influence the B cell response. In fact, a correlation analysis (Fig. 7) showed that percentages of HIV-1-specific CD4 + T cell responses induced by the NYVAC-C-KC-ΔB19R vector have a more favourable trend for a correlation with antibody responses than for NYVAC-C-KC. However, the correlation between CD8 + T cell and B cell responses was less pronounced than for CD4 + T cells. How does the B19R gene operate? It has been shown that B19 is a type I IFNbinding protein that acts both in solution and when bound to glycosaminoglycans to inhibit the action of a wide range of IFNs from different species, sequestering and suppressing efficiently the function of type I IFNs (59, 60). In cells infected with a NYVAC vector lacking B19, we have shown in human DCs enhanced IFN-alpha production, maturation and expression of IFN-induced pathways and IFN-regulated transcription factors as well as multiple inflammatory cytokines (12). Immunization of mice with NYVAC lacking B19R and expressing Env, Gag, Pol, and Nef clade C (CN54) HIV-1 antigens, improved the magnitude and quality of HIV-1-specific CD8 + T cell adaptive immune responses and impacted their memory phase, changing the contraction, the memory differentiation and the magnitude and functionality profile, while for B cell responses B19 had no apparent effect on antibody levels (17). The immunomodulatory effects of B19 were more pronounced in mice than in NHP. Here in NHP we noted at some time points after boost immunization the influence of B19 by a trend to slightly higher levels of T cells and of some cytokines triggered by NYVAC-C- KC-ΔB19R over NYVAC-C-KC. The overall effect of B19 on Env-specific binding IgG antibodies, IgA, NAb and of ADCC was not different between the NYVAC-C-KC vectors. Thus, it seems that B19 main effect is on T cell responses, and the role of B19R blocking type I IFN could explain the small differences in immunogenicity, in particular on T cell responses, observed in macaques inoculated with NYVAC-C-KC versus NYVAC-C-KC-ΔB19R. In our previous study in NHP we demonstrated that non-replicating NYVAC vector expressing the same HIV-1 antigens and the same protocol as in this current study was superior to the ALVAC vector used in the RV144 phase III clinical trial (13). Comparison of the immunogenicity profiles triggered by the NYVAC-C-KC vectors (this 18

602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 study) with non-replicating NYVAC vector (13) using the same standardized immune assays revealed an overall higher magnitude of HIV-1-specific T cell and humoral immune responses induced by the NYVAC-C-KC vectors. This is likely due to more antigen expression from the NYVAC-C-KC vectors (Fig. 1), as the virus replicates more efficiently in NHP than NYVAC lacking the VACV host range genes K1L and C7L. These results correlated with our previous in vitro findings, which demonstrated that NYVAC-KC vectors acquired specific biological characteristics over non-replicating NYVAC, giving replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and higher virus spread in tissues while maintaining a highly attenuated phenotype (19, 26). While antigen exposure has been defined recently as a determinant of HIV-1 broadly neutralizing antibody (bnab) induction (61), the question remains as to how best to induce bnabs by vaccination approaches. Using the protocols described here we have been able to trigger a broad spectrum of T and B cell immune responses. A balanced of T and B cell immune responses has been correlated with levels of protection by HIV-vaccines in NHP models of HIV infection (62). A number of HIV/SIV vaccine protection studies in NHPs have revealed a role for humoral responses as potential correlates of immunity (63-65), broadly including the types of immune responses elicited by this vaccine regimen. However, it remains to be defined whether the responses elicited by NYVAC-C-KC vectors confer in NHP protection against a SHIV virus challenge and/or are able to induce bnabs or other potentially protective immune responses in a prophylactic phase I clinical trial. The high immunological profiles induced by the NYVAC-C-KC vectors, including induction of CD4 + and CD8 + T cell immune responses, high levels of anti-env binding antibodies to both gp140 and V1/V2 sites, enhanced ADCC responses and neutralizing capacity, together with low levels of IgA antibodies, are all potential correlates of protection, suggesting that these viral vectors could be considered as improved HIV/AIDS vaccine candidates. ACKNOWLEDGMENTS This investigation was supported by the PTVDC/CAVD Program with support from the Bill and Melinda Gates Foundation (BMGF). Humoral immune monitoring data were supported by the BMGF CAVIMC 1032144 grant and the NIH/NIAID Duke Center for AIDS Research (CFAR; 5P30 AI064518). Novartis Vaccines received support for this work under contract number HHSN266200500007C from DAIDS, NIAID, NIH. We thank Dr. Marcella Sarzotti-Kelsoe for quality assurance oversight, William T. Williams, Robert Howington, R. Glenn Overman, Cristina Sánchez and Victoria 19

639 640 641 Jiménez for technical assistance, Sheetal Sawant for BAMA data management and Hua-Xin Liao and Bart Haynes for envelope and V1/V2 protein reagents. 20

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968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys. Cell 155:531-539. 65. Roederer M, Keele BF, Schmidt SD, Mason RD, Welles HC, Fischer W, Labranche C, Foulds KE, Louder MK, Yang ZY, Todd JP, Buzby AP, Mach LV, Shen L, Seaton KE, Ward BM, Bailer RT, Gottardo R, Gu W, Ferrari G, Alam SM, Denny TN, Montefiori DC, Tomaras GD, Korber BT, Nason MC, Seder RA, Koup RA, Letvin NL, Rao SS, Nabel GJ, Mascola JR. 2014. Immunological and virological mechanisms of vaccine-mediated protection against SIV and HIV. Nature 505:502-508. FIGURE LEGENDS Figure 1. Enhanced expression, plaque size phenotype and innate immune profile of NYVAC-C-KC vectors. (A) Expression of HIV-1 gp140 by Western blotting. Monolayers of HeLa cells were mock-infected or infected at 5 PFU/cell with NYVAC- WT, NYVAC-KC, NYVAC-gp140, NYVAC-KC-gp140 or NYVAC-KC-gp140-ΔB19R. At 6 or 24h post infection, cells were lysed in Laemmli buffer, and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blotting using the rabbit polyclonal anti-gp120 antibody. (B) Analysis of the plaque size. Monkey BSC-40 cells were infected with serial dilutions of NYVAC-WT, NYVAC-KC, NYVAC-gp140, NYVAC- KC-gp140 or NYVAC-KC-gp140-ΔB19R. At 48h post infection, cells were fixed and stained with crystal violet to visualize virus plaques. (C) Analysis of the innate immune profile in infected human THP-1 macrophages. Human THP-1 macrophages were mock-infected or infected at 5 PFU/cell with NYVAC-WT, NYVAC-KC, NYVAC-gp140, NYVAC-KC-gp140 or NYVAC-KC-gp140-ΔB19R. At 3 and 6 hours post-infection, RNA was extracted and the mrna levels of IFN-β, MIP-1α, IL-8, IL-1β, and HPRT were analyzed by RT-PCR. Results were expressed as the ratio of the gene of interest to HPRT mrna levels. A.U: arbitrary units. p values indicate significantly higher responses comparing NYVAC-KC-gp140 to NYVAC-KC-gp140-ΔB19R at the same hour (*, p<0.05; **, p<0.005; ***, p<0.001). Figure 2. Immunization schedule for non-human primates. (A) Immunization groups included in the AUP513 study. Eight NHPs (rhesus macaques) were immunized in each group at weeks 0 and 4 with the corresponding replication-competent NYVAC- C-KC poxvirus vector (NYVAC-C-KC or NYVAC-C-KC-ΔB19R) and at weeks 12 and 24 with a combination of replication-competent NYVAC-C-KC poxvirus vector plus a clade C HIV-1 gp120 protein, as detailed in Materials and Methods. The composition of NYVAC-C-KC, NYVAC-C-KC-ΔB19R, and the bivalent clade C HIV-1 gp120 protein is detailed in Materials and Methods. (B) Chronological diagram showing the immunization schedule and the immunogenicity endpoints used in this study. At weeks 27

1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 0, 4, 12 and 24, animals were immunized (see panel A). A dose of 1 x 10 8 PFU of each recombinant replication-competent poxvirus vector (NYVAC-KC-gp140 plus NYVAC- KC-Gag-Pol-Nef for the NYVAC-C-KC vector; and NYVAC-KC-gp140-ΔB19R plus NYVAC-KC-Gag-Pol-Nef-ΔB19R for the NYVAC-C-KC-ΔB19R vector; 2 x 10 8 PFU of total virus) and 50 µg of each clade C HIV-1 gp120 protein (TV1 gp120 plus 1086 gp120; 100 µg of total protein) were used in each immunization. Bivalent clade C gp120 protein was administered together with MF59 adjuvant. At weeks 0, 6, 14, 26, and 36 (at the beginning of the study, 2 weeks after the second, third, and fourth immunizations, and at the end of the study, respectively), PBMCs, serum and rectal mucosal samples were obtained from each immunized animal, and HIV-1-specific T cell and humoral immune responses were analyzed. Figure 3. Magnitude of HIV-1-specific T cell immune responses. PBMCs were collected at weeks 0, 6, 14, 26, and 36 from each rhesus macaque (n=8 per group) immunized with NYVAC-C-KC or NYVAC-C-KC-ΔB19R, and the total magnitude of HIV-1-specific T cell immune responses triggered by the different immunization groups were measured by ELISpot (A) or ICS (B and C) assays following stimulation of PBMCs with HIV-1 Env, Gag, Pol and Nef peptide pools. The total magnitude of SFUs per million of cells is shown (A), with values representing the number of IFN-γ positive T cells against Env, Gag, Pol, and Nef peptide pools. The total magnitudes of HIV-1- specific CD4 + (B) and CD8 + (C) T cell responses elicited by the different immunization groups are shown, with the values representing the sums of the percentages of T cells producing IFN-γ and/or TNF-α and/or IL-2 against Env, Gag, Pol, and Nef peptide pools. Values from unstimulated controls were subtracted in all cases. Each dot represents the value for one immunized macaque. Box plots represent the distribution of data values, with the line inside the box indicating the median value. p values and the number of responding rhesus macaques are indicated. Figure 4. Magnitude of HIV-1-specific CD4 + and CD8 + T cells producing cytokines. The overall magnitudes of HIV-1-specific CD4 + (panels A to C) and CD8 + (panels D to F) T cells elicited by the different immunization groups and producing IFNγ (A and D), TNF-α (B and E), or IL-2 (C and F) are shown. PBMCs were collected at weeks 6, 14, 26, and 36 from each rhesus macaque (n=8 per group) immunized with NYVAC-C-KC or NYVAC-C-KC-ΔB19R. HIV-1-specific CD4 + and CD8 + T cell immune responses triggered by both immunization groups were measured by ICS assay following stimulation of PBMCs with HIV-1 Env, Gag, Pol, and Nef peptide pools. The values represent the sums of the percentages of T cells producing IFN-γ (A and D) or 28

1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 TNF-α (B and E) or IL-2 (C and F) against Env, Gag, Pol, and Nef peptide pools. Values from unstimulated controls were subtracted in all cases. Each dot represents the value for one immunized macaque. Box plots represent the distribution of data values, with the line inside the box indicating the median value. p values and the number of responding rhesus macaques are indicated. Figure 5. Binding IgG antibody responses. Total plasma binding IgG antibody levels against HIV-1 gp140 group M consensus (scon) (A), gp120 from isolate TV1 (B), gp120 from isolate 1086 (C), and MuLV gp70-scaffolded V1/V2 proteins (D) induced by the different immunization groups. (E) Rectal binding IgG antibody levels against HIV-1 gp140 group M consensus (scon), and gp120 from isolates 1086 and TV1 induced by the different immunization groups. Individual serum or rectal mucosal samples were obtained at weeks 6, 14, 26, and 36 from each rhesus macaque (n=8 per group) immunized with NYVAC-C-KC or NYVAC-C-KC-ΔB19R. Binding IgG antibodies were measured by BAMA (A to D), or by analyzing the binding magnitude normalized to total rhesus IgG (specific activity) (E), as indicated in Materials and Methods. The magnitudes of the total binding IgG antibody responses are expressed as the AUC from serial dilutions of plasma. Each dot represents the value for one immunized macaque. Box plots represent the distribution of data values, with the line inside the box indicating the median value. p values and the number of responding rhesus macaques are indicated. Figure 6. ADCC and NAb responses against HIV-1. (A) ADCC activity induced by the different immunization groups. Individual plasma samples were obtained at weeks 0, 26, and 36 from each rhesus macaque (n=8 per group) immunized with NYVAC-C- KC or NYVAC-C-KC-ΔB19R. ADCC activity against gp120 TV1 and 1086 proteins was measured as indicated in Materials and Methods. (B) Neutralization titers induced by the different immunization groups. Individual serum samples were obtained at weeks 26, and 36 from each rhesus macaque (n=8 per group) immunized with NYVAC-C-KC or NYVAC-C-KC-ΔB19R. NAb against different tier 1 and 2 HIV-1 strains were measured using the TZM-bl or A3R5 assays, as described in Materials and Methods. Each dot represents the value for one immunized macaque. Box plots represent the distribution of data values, with the line inside the box indicating the median value. p values and the number of responding rhesus macaques are indicated. Figure 7. Correlation between T cell and antibody responses. (A) Correlation plot summarizing Spearman s rank-based correlations of CD4 + T cell responses with B-cell 29

1081 1082 1083 1084 1085 1086 1087 1088 1089 assays for NYVAC-C-KC and NYVAC-C-KC-ΔB19R at week 26, and ordered by overall median correlation for each cytokine and pooled antigen combination. (B) Correlation plot summarizing Spearman s rank-based correlations of CD8 + T cell responses with B-cell assays for NYVAC-C-KC and NYVAC-C-KC-ΔB19R at week 26, and ordered by overall median correlation for each cytokine and pooled antigen combination. CD4 + and CD8 + T cell responses were assessed for correlation with the gp120.tv1 antigen in the BAMA and ADCC GTL assays, and the MW965.26 isolate in the TZM.bl NAb assay. In each case, reported p-values are calculated using a onesided test with alternative that the true correlation coefficient is greater than 0. 30