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Subtype C gp140 vaccine boosts immune responses primed by SAAVI DNAC2 and MVAC 1 HIV vaccine after more than a two year gap (HVTN 073E/SAAVI 102) 2 3 Glenda E Gray1,2, Kenneth H Mayer3, Marnie L Elizaga4, LindaGail Bekker5, Mary Allen6, 4 Lynn Morris7, David Montefiori8, Stephen C De Rosa4, Alicia Sato4, Niya Gu 4, Georgia D 5 Tomaras8, Timothy Tucker2, Susan W. Barnett9, Nonhlanhla N Mkhize7, Xiaoying Shen8, 6 Katrina Downing5, Carolyn Williamson5,10, Michael Pensiero6, Lawrence Corey4, AnnaLise 7 Williamson5,10, and the NIAIDfunded HIV Vaccine Trials Network 8 9 1: Perinatal HIV Research Unit, Faculty of Health Sciences, University of the 10 Witwatersrand, Johannesburg, South Africa 11 2: South African Medical Research Council, Cape Town, South Africa 12 3: Brown University, Providence, RI, USA 13 4: Fred Hutchinson Cancer Research Center, Seattle, WA, USA 14 5: Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape 15 Town, South Africa 16 6: DAIDS, NIAID, NIH, Washington DC, USA 17 7: National Institute for Communicable Diseases of the National Health Laboratory 18 Services, Johannesburg, South Africa 19 8: Duke University, Durham, NC, USA 20 9: Novartis Vaccines, Cambridge, MA, USA 21 10: National Health Laboratory Service, Cape Town, South Africa 22 23 24
CVI Accepted Manuscript Posted Online 20 April 2016Clin. Vaccine Immunol. doi:10.1128/CVI.00717-15Copyright © 2016 Gray et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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Corresponding Author: 25 26 Professor Glenda Gray 27 Email: [email protected] 28 Contact numbers: +27 21 938 0905/ +27 83 459 2680 29 Address : Francie van Zjil Drive, Parrow Valley , Cape Town, Western Cape South Africa 30 7505 31 32 33 Running Title: Subtype C gp140 vaccine boosts a clade C HIV DNA/MVA vaccine regimen 34 Abstract work Count: 200 35 Article Word Count: 357736
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ABSTRACT 37 Background 38 A Phase I safety and immunogenicity study investigated SAAVI HIV-1 subtype C (HIV-1C) 39 DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with Modified Vaccinia Ankara 40 (MVA) expressing matched antigens. Following the finding of partial protective efficacy in 41 RV144, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the 42 regimen. 43 Methods: 44 48 participants (12 USA; 36 RSA) were randomized to receive 3 intramuscular (IM) doses of 45 SAAVI DNA-C2 4 mg (months 0, 1, 2) and 2 IM doses of SAAVI MVA-C 1.45x109 pfu (months 46 4, 5) (n=40) or placebo (n=8). Approximately two years after vaccination, 27 participants 47 were re-randomized to receive gp140/MF59 100 mcg or placebo, as 2 IM injections, 3 48 months apart. 49 Results: 50 The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen CD4+T-cell 51 and CD8+T cell responses occurred in 74% and 32% of participants respectively. The protein 52 boost increased CD4+T-cell responses to 87% of subjects. All participants developed Tier 1 53 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that 54 recognized linear V3 and C5 peptides. 55 Conclusion: The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular 56 immunogenicity. Boosting with gp140/MF59 enhanced binding and neutralizing antibodies 57 as well as CD4+ T-cell responses to HIV-1 envelope. 58 Trial registration numbers: NCT00574600; NCT01423825; 59 https://clinicaltrials.gov/ct2/show/NCT00574600?term=hvtn+073&rank=2) 60
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https://clinicaltrials.gov/ct2/show/NCT01423825?term=hvtn+073&rank=1) 61 Key words: HIV vaccines, South Africa, clade C, HIV 62 Introduction 63 In response to a devastating HIV-1 subtype C epidemic in southern Africa, the South African 64 AIDS Vaccine Initiative (SAAVI), a lead program of the South African Medical Research 65 Council (SAMRC), in collaboration with the University of Cape Town (UCT) and the USA 66 National Institutes of Health, developed a subtype C HIV (HIV-1C) vaccine regimen consisting 67 of two multigene recombinant vaccines—a DNA vaccine and an MVA vaccine, expressing 68 matched HIV-1C proteins [1]. The HIV-1C gene inserts were selected from representative 69 circulating viral isolates in South Africa [2, 3]. Preclinical immunogenicity studies without 70 the HIV-1 C protein boost in both mice [4] and baboons demonstrated that the DNA/MVA 71 regimen elicited potent T-cell lymphocyte responses, as well as binding antibody responses 72 to HIV-1C gp120 [5]. 73 74 This first-in-human study using the SAAVI DNA-C2 and SAAVI MVA-C vaccines evaluated the 75 safety and immunogenicity of the DNA/MVA prime-boost regimen in both South Africa 76 (RSA) and the USA (HVTN 073/ SAAVI 102). In an attempt to improve HIV-specific antibody 77 responses, a V2 deleted envelope subunit HIV-1C protein vaccine adjuvanted with MF59 78 was used as an additional boost (HVTN 073E/SAAVI 102E), based on recent promising pre-79 clinical and clinical immunogenicity studies [6]. We investigated the effect of the protein 80 boost on both cellular and humoral immunity. 81 82 Methods 83 Study Design 84
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HVTN 073/ SAAVI 102, a phase I randomized, double-blind placebo controlled trial designed 85 to evaluate the safety and immunogenicity of the SAAVI DNA-C2 and SAAVI MVA-C vaccines 86 (Table 1) was conducted in HIV uninfected healthy vaccinia virus-naive adult participants at 87 two RSA sites (Perinatal HIV Research Unit, Soweto, and the Desmond Tutu HIV Centre, 88 Cape Town), and two US sites (Brigham & Women’s Hospital, and Fenway Health, Boston, 89 MA). The trial design is shown in Table 1A and was extended to evaluate a subtype C V2 90 deleted gp140 vaccine with MF59 adjuvant (Table 1B) after the RV144 study suggested that 91 the addition of a protein boost could enhance viral-vector-mediated immunogenicity. 92 93 Vaccines 94 SAAVI DNA-C2 consisted of two DNA plasmids: pVRCgrttnC, expressing HIV-1C Gag-Reverse 95 Transcriptase-Tat-Nef (grttnC) polyprotein from the Du422 isolate, and pVRCgp150CT, 96 expressing an HIV-1C truncated Env from the Du151 isolate [3], manufactured by Althea 97 Technologies, Inc. (San Diego, California, USA) and mixed in equal weight (1:1 w/w) for the 98 vaccine. SAAVI MVA-C contained grttnC under the control of the vaccinia virus 40K 99 promoter inserted into the Del III region, and gp150CT, under the control of the vaccinia 100 virus 13 promoter inserted into the 49/50 region [5]. The SAAVI MVA-C vaccine was 101 manufactured by Therion Biologics Corporation (defunct, Cambridge, Massachusetts, USA) 102 [5]. The Novartis HIV-1C gp140 vaccine (manufactured in Emeryville, California, USA) was a 103 recombinant oligomeric V2 deleted gp140 (gp140∆V2.TV1) produced in Chinese Hamster 104 Ovary cells. The gp140 was derived from a South African subtype C primary isolate, TV1, 105 and given with MF59 [6]. Placebo was 0.9% Sodium Chloride for Injection. 106 Study Population 107
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Participants were healthy volunteers aged 18-45 years, based on medical history, physical 108 examination, laboratory tests, troponin levels and electrocardiograms. Participants were at 109 low risk for HIV infection, according to risk assessment and risk criteria developed by each 110 site. Participants were randomized 5:1 to the treatment group or placebo control (Table 111 1A). For the study extension, consenting eligible participants were re-randomized in a 2:1 112 ratio to receive the subtype C gp140/MF59 or placebo, given twice, 3 months apart, around 113 2 years (median 2.3 yrs; range 2.0-2.4 yrs) after completion of the initial regimen (Table 1B). 114 Safety Assessment 115 Safety evaluations included physical examinations, standard serum chemistry and 116 hematological tests, cardiac troponin T, and 12 lead EKG to identify potential cardiac 117 adverse events after receipt of MVA. 118 Reactogenicity symptoms were assessed for 3 days following each vaccination until 119 resolution. Adverse events (AEs) were recorded for each participant for the 12 months of 120 the original study, and for 15 months in the study extension. Reactions and AEs were graded 121 as mild, moderate or severe according to standard criteria (http://rcc.tech-122 res.com/safetyandpharmacovigilance/). Risk reduction counseling was provided at each 123 visit. 124 125 Immunogenicity assessment 126 Immunogenicity endpoints for both humoral and cellular responses were measured at the 127 primary immunogenicity timepoints, 2 weeks after each MVA-C/placebo and each 128 protein/placebo vaccination, i.e. after the fourth, fifth, sixth, and seventh vaccinations, and 129 at the durability timepoint which was 6 months after the final MVA/placebo or 130 protein/placebo (fifth and seventh) vaccinations. Endpoint assays for assessing the humoral 131
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responses included binding antibodies to Env and Gag by ELISA and binding antibody 132 multiplex assay (BAMA), and HIV-1 specific neutralizing antibodies. The endpoint assay for 133 assessing cellular responses was flow cytometry with intracellular cytokine staining (ICS) for 134 IFN-γ, IL-2 and TNF-α. 135 Intracellular cytokine staining assay (ICS) 136 Flow cytometry was used to examine HIV-1 specific CD4+ and CD8+ T-cell responses [7, 8]. 137 Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved within 8 hours 138 of venipuncture from sodium heparin-anticoagulated blood obtained at the primary 139 immunogenicity timepoints [9]. PBMC were stimulated with peptide pools of HIV-1 global 140 potential T-cell epitopes (PTEG) [10] that spanned the proteins encoded by the vaccine 141 construct. 142 Binding antibodies 143 Binding antibodies to protein antigens were assessed by both validated ELISA and binding 144 antibody multiplex assays (BAMA). ELISAs were performed using serum dilutions of 1:20, 145 for participants in the main study, to ConS gp140 and p55 Gag; and for participants in the 146 study extension, to ConS, Du151.2 gp140, and gp140∆V2.TV1. Samples that were saturated 147 at this dilution were further diluted to 1:2000 [11]. 148 Serum HIV-1 specific IgG responses (1:20 or 1:50) were also evaluated by BAMA against 149 ConS gp140 CFI, gp41 Env, p24 Gag and gp140∆V2.TV1 at baseline, primary immunogenicity 150 timepoints, just prior to the first protein/placebo immunization and 6 months post final 151 boost [11, 12]. Antibody titers (AUC) were calculated from serum serial dilutions (1.50-152 1:388,800). 153
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Linear epitope mapping was evaluated on a subset of vaccines by peptide microarray with 154 15-mer peptides overlapping by 12, covering consensus Env (gp160) and vaccine strains 155 (gp120) as previously described[13, 14, 15] . 156 Neutralizing antibody assay 157 HIV-1 specific neutralizing antibody assays were performed at baseline and at the primary 158 immunogenicity timepoints. Neutralizing antibodies were measured as a function of 159 reductions in Tat-regulated luciferase reporter gene expression after a single round of 160 infection in TZM-bl cells, against Tier 1 and Tier 2 HIV-1 isolates [16]. 161 Statistical Methods 162 All data from enrolled participants who received at least one vaccination were analyzed 163 using SAS and R statistical software. 164 For ICS, two by two contingency tables were constructed comparing the HIV-1 peptide 165 stimulated and negative control for each peptide pool for the two T-cell subsets (CD4+ or 166 CD8+) expressing IFN-γ and/or IL-2. A one-sided Fisher’s Exact Test applied to each table 167 tested whether the number of cytokine-producing cells for the stimulated data was equal to 168 that for the negative control data. Since multiple individual tests (for each peptide pool) 169 were conducted simultaneously, a multiplicity adjustment was made to the individual 170 peptide pool p-values using the Bonferroni-Holm adjustment method [17]. The adjusted p-171 values were used to determine positivity, with values ≤0.00001 indicating a positive 172 response. If one peptide pool for a specific gene was positive, then the overall response to 173 the gene was considered positive. If any peptide pool was positive for a T-cell subset, then 174 the overall response rate for that T-cell subset was considered positive. For the ICS, two 175 sided 95% confidence intervals were calculated using the score test method of Agresti and 176 Coull [18]. 177
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For the ELISA response, a response to a peptide was considered positive if the difference in 178 duplicate antigen-containing and non-antigen containing wells had an optical density 179 (OD)>0.2 and the OD is ≥ 3 times the day 0 (baseline) OD. 180 For the BAMA assay, post-enrollment samples were considered positive if they met three 181 conditions: (1) the MFI minus blank values were >= antigen-specific cutoff (based on the 182 average + 3 standard deviations of 60 seronegative plasma samples), (2) the MFI minus 183 blank values were greater than 3 times the baseline (day 0) MFI minus blank values, and (3) 184 the MFI values were greater than 3 times the baseline MFI values. The MFI minus blank 185 responses were used to summarize the magnitude at a given timepoint. 186 For neutralizing antibodies, a response to an isolate was considered positive if the titer was 187 ≥ 10, where a titer was defined as the sample dilution that reduces infection by half relative 188 to untreated virus or virus treated with pre-vaccination serum. 189 190 Results 191 Participant Accrual, Demographics and Vaccine Safety 192 Forty-eight low-risk participants, 36 in RSA and 12 in the USA, were enrolled over a period of 193 8 months. Overall, 22 (46%) participants were female, and 37 (77%) were Black Africans or 194 African American, with a median age of 24 years (range, 18-39) (Table 2). Forty-five (95%) 195 participants received all 5 vaccinations in the original study (Table 1A). Three participants 196 discontinued the vaccination schedule. Twenty-seven participants (6 USA, 21 RSA; 15 male, 197 12 female) continued in the study extension, with the treatment assignments indicated in 198 Table 1B. The vaccine regimen was found to be safe and well tolerated. Most reactogenicity 199 symptoms were graded mild to moderate (Figure 1). Four people reported severe 200 reactogenicity symptoms: after DNA vaccination, one participant had severe 201
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malaise/fatigue; after MVA vaccination, one person had severe pain at the injection site, 202 one had severe malaise/fatigue, and one reported a severe headache. There were no severe 203 local or systemic reactogenicity symptoms reported after protein vaccination. The 3 204 pregnancies reported all occurred more than 4 months following the vaccination series, and 205 resulted in one full term live birth, one spontaneous abortion, and one elective abortion. 206 207 There were no severe or life-threatening adverse events, or cardiac adverse events such as 208 myocarditis attributable to the study products. Two participants discontinued vaccinations 209 due to adverse events: one participant for a schizophrenia relapse after the first DNA 210 vaccination, deemed probably not product-related; another due to mild right-sided tongue 211 swelling occurring within 90 minutes of the second MVA vaccination, which resolved 212 spontaneously, deemed possibly related to study product. Two other participants 213 discontinued vaccinations early: one refused after the first vaccination, and another was not 214 able to receive the final protein vaccination within the prescribed vaccination window. No 215 participant acquired HIV during the study. 216 217 Immunogenicity 218 HIV1 Specific Tcell Responses 219 Vaccine-induced HIV-specific CD4+ T-cell responses expressing IFN-γ and/or IL-2 to any HIV 220 protein tested were detected in 22/32 (69%) of participants after the first MVA vaccination 221 (Figure 2, upper panel). While the second MVA boost provided a modest increase in 222 proportion of individuals with CD4+ T cell responses to HIV-1 (74%), there was a decrease in 223 magnitude of the response. For the antigen-specific responses, most participants had CD4+ 224 T-cell responses to Env (66%) after the first MVA, which persisted (71%), with reduced 225
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magnitude following the second MVA boost (Figure 2, upper panel; Supplementary Table 1). 226 Fewer responses were detected to Gag (41% after the first MVA; 21% after the second MVA 227 boost) with even fewer participants having CD4+ responses to Pol. 228 CD8+ T-cell responses were much less frequent than CD4+ T-cell responses; 17% of 229 participants had responses to any HIV protein tested after the first MVA, increasing to 32% 230 after the second MVA boost (Figure 2, lower panel). CD8+ T-cell responses were most often 231 seen to Pol (24% of participants), with fewer responses to Env (11%), and Gag (5%) after the 232 second MVA (Figure 2, lower panel). 233 CD4+ T-cell responses to any HIV protein after the second MVA boost were detected in 89% 234 of USA participants compared to 68% of RSA participants, and CD8+ T-cell responses in 44% 235 in the USA compared to 29% in RSA. These differences were not due to differences in cell 236 processing or viability and were not statistically significant (Supplementary Table 2). 237 Based on examination of the co-expression for the 3 cytokines measured, IFN-γ, IL-2 and 238 TNF-α, CD4+ vaccine-induced T-cell responses following both MVA vaccination timepoints 239 were approximately evenly divided between cells producing 1, 2 or 3 cytokines, with slight 240 enrichment for 2 cytokines (data not shown). IL-2 or TNF-α were the dominant cytokines for 241 cells producing one cytokine; the co-expression of these was dominant for cells producing 2 242 cytokines. For CD8+ T-cells, cells producing 1, 2, or 3 cytokines were detected after the first 243 and second MVA boosts. IFN-γ and TNF-α were the major cytokines expressed, either as a 244 single cytokine or as both combined. 245 At the time of the gp140/MF59 protein boost, approximately two years following the initial 246 vaccine regimen, HIV-specific CD4+ T-cell responses remained detectable in 4/15 (27%) of 247 those participants randomized to receive the gp140/MF59 (DDDMMPP) and this increased 248 to 13/15 (87%) of these participants following the first protein boost and was maintained 6 249
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months after the last protein vaccination (73%) (Figure 3, upper panel; Supplementary Table 250 1). The median magnitude of response following the second protein was similar to the 251 magnitude after the first MVA. These response rates were significantly different (p=0.0005) 252 from the subset of placebo recipients following DNA/MVA vaccination (DDDMMCC), among 253 whom only 20% had responses after the first placebo injection, and 17% after the second 254 (Figure 3, upper panel; Supplementary Table 1). Protein boosting offered no enhancement 255 of CD8+ T-cell responses (Figure 3, lower panel; Supplementary Table 1). 256 257 HIV1 Specific Antibody Responses 258 Binding Antibody Responses 259 DNA/MVA vaccination elicited low levels of HIV-1 specific binding antibodies, which were 260 enhanced after boosting with gp140/MF59. At two weeks after the second MVA boost, 261 40.5% (15/37) of subjects had binding antibody responses to ConS gp140, 67.6% to gp41, 262 and 51.4% to p55 antigen (Supplementary Table 3). After boosting with gp140/MF59 all 263 (100%) recipients had vaccine-induced binding antibody responses to ConS gp140 (Figure 4, 264 upper panel; Supplementary Table 3, and by ELISA (not shown)), gp41 (Supplementary Table 265 3) and gp140∆V2.TV1 (Figure 4, lower panel; Supplementary Table 3) after each protein 266 boost, which were still present 6 months after the final vaccination. The Env IgG titer (AUC) 267 increased after the second protein boost (median AUC (maximum, minimum) for each: ConS 268 gp140 37367 (16197, 66311); gp41 37799 (18263, 63540); gp140∆V2.TV1 36658 (11365, 269 62649) but waned in the 6 months following: a mean fold decline of 1.8 for ConS gp140, 1.4 270 for gp41 and 2.1 for gp140∆V2.TV1 (data not shown). By ELISA, 93% of vaccinees also had 271 responses to the subtype C HIV-1 envelope, Du151.2 gp140 the magnitude of which waned 272 after six months (p-value =0.022, Wilcoxon rank sum test). At the initiation of the study 273
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extension, 26.7% of the participants had antibody to p24, and this did not change whether 274 boosted with either protein or placebo. The sole C1/T2 participant developed binding 275 antibodies following the second protein injection, to ConS, gp41, and gp140∆V2.TV1 (data 276 not shown). 277 To evaluate binding specificity, a subset of 12 vaccinees, chosen based on neutralization 278 titers, were examined by IgG linear epitope mapping (Figure 5). The binding antibodies 279 targeted linear epitopes within C1, C2, V3 and C5 regions of gp120 and the 280 immunodominant (ID) region of gp41 (Figure 5A). The dominant responses were against V3 281 and C5, present in 12 out of 12 subjects and contributing to 42% and 43% of the overall 282 binding to linear Env epitopes, respectively (Figure 5A). Notably, IgG responses to the V3 283 region of the HIV-1 envelope glycoprotein were cross-reactive across multiple HIV subtypes 284 in contrast to the C5 epitope specific responses that were more focused on Clade C 285 sequences (Figure 5B). 286 287 Neutralizing Antibody Responses 288 No neutralizing antibodies were detected with the DNA/MVA regimen before the protein 289 boosts. After the first protein boost, neutralizing antibodies were detected to Tier 1 viruses 290 MN.3, SF162.LS, and MW965.26 (Supplementary Table 4). Responses were most frequent 291 against MW965.26, a subtype C virus, and were detected in 56% of participants. After the 292 second protein boost, neutralizing antibody responses to MW965.26 were seen in 100%, 293 and persisted in 75% of T1/T2 participants for at least 6 months (Figure 6). No significant 294 responses were seen to the Tier 2 viruses, Du151.2, and TV1.21 represented in the vaccines 295 (Supplementary Table 4). 296 297
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Discussion 298 Our study indicates that the DNA/MVA vaccine regimen produced a high frequency of 299 CD4+T cell responses to HIV-1 in healthy HIV-uninfected adults. The protein boost increased 300 the CD4+ T-cell response to HIV-1 envelope and induced binding and neutralizing antibody 301 responses in all participants. The dose of MVA at 1.45x109 pfu is the highest ever used in a 302 clinical trial. The combination of the three vaccines given in series was generally safe and 303 well tolerated and contributes to the accumulating safety data on DNA/MVA based vaccines 304 [19, 2021] and the TV1 gp140 protein boost with MF59 [22]. 305 306 The DNA/MVA regimen induced CD4+ T cell responses mostly to Env, which persisted in 307 about a quarter of vaccinated individuals for more than two years and were enhanced with 308 the addition of the protein immunogen. Response rates are overall higher than reported by 309 Goepfert et al [20] after a similar regimen with two DNA primes followed by two MVA 310 boosts, and similar to those seen in RV144, in which 72% of participants had CD4+ T cell 311 responses after the last immunization [23]. Six months following the protein boosts, the 312 CD4+T cell responses persisted in the majority of participants, although the magnitude 313 slightly decreased. The CD4+ T cell responses were measured using PTEG peptides for 314 detection rather than peptides specific for the clade C sequences encoded in the DNA/MVA 315 vaccines; the use of clade–matched peptides may have further increased the response rates 316 reported here. 317 In a recent study, published by the HVTN, that examined the data of 1218 subjects from 10 318 phase 1 clinical trials who only received DNA plasmid HIV vaccination, no evidence of 319 tolerance was found [24]. The immunogenicity of the DNA plasmid vaccination was 320 influenced by the doses of the DNA, the number of doses received, gender, body mass index 321
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and age. Doses of DNA plasmid HIV vaccines appear to be optimal at the 3-6 mg range, and 322 thus we believe our 4mg choice delivered an appropriate response. As our DNA prime was 323 followed up a MVA boost, we do not have data on the CD4+ T cell and CD8+ T cell responses 324 after the DNA prime. Although we did see a reduction in the magnitude of cellular immune 325 responses after the DNA/MVA dosing series, it is difficult to extrapolate the role of DNA in 326 this reduction in magnitude of response, but not in overall response. 327 328 329 Tier 1 neutralizing antibody responses were present only after the protein boost, and were 330 strongest against the HIV-1C isolate. Similar to findings in other vaccine trials, including 331 RV144, Tier 2 isolates were not neutralized by the antibody responses [20]. The durability of 332 neutralizing antibodies were not sustained, declining significantly in the 6 months following 333 the last vaccination, signifying the need for subsequent boosts or other strategies to 334 maintain antibody responses in future trials, if they are found to be correlated with vaccine 335 efficacy. 336 337 Binding antibody responses were initially weak after DNA-MVA vaccination. Env binding 338 antibody response rates after the second MVA immunization were not as high those 339 reported by Goepfert et al [20]. However, following the second protein boost, all our 340 participants responded to these antigens and this response was sustained 6 months after 341 the last vaccination. The protein boost also increased binding antibody titers (magnitude), 342 which were well-maintained over 6 months. Goepfert et al [20] reported a <3 fold decline in 343 magnitude in the first 6 months after vaccination whereas in our study there was <2 fold 344
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decline. The durability of these responses was also considerably better than the 10-fold 345 drop over 26 weeks seen in the RV144 trial [25]. 346 347 In this study, there were two dominant linear Env epitope specificities, namely V3 and C5. 348 This is in contrast to RV144, where epitope specificities included C1 and V2, in addition to 349 V3 and C5 regions of gp120. IgG to V2 was significantly inversely correlated with infection 350 risk in RV144 [15]. However, as the protein in this study had a V2 deletion, antibodies to V2 351 were not observed after the boost. In RV144 the response to V3 was also inversely 352 correlated with infection risk but only in vaccinees who had lower levels of other antibodies. 353 Responses to C5 showed no significant correlation with infection risk. Interestingly in HIV-1 354 infected subjects, dominant responses targeted the V3 and C5 regions of gp120 [15]. 355 356 Grttn as a polyprotein had relatively low immunogenicity compared to Env, with minimal 357 responses to Nef and no responses to Tat. Env T-cell responses predominated in our study 358 which has also been reported in a number of studies testing poxviruses with multiple HIV 359 gene inserts. CD8+ responses to Pol were more frequent than to Gag (25 vs 5.6%). 360 Conversely, CD4+ Gag responses were more often detected than those to Pol (34 vs 9.4% at 361 D126). Gag CD4+ responses were somewhat lower than Env and lower than that reported 362 by Goepfert et al [20]. This may have been due to the Geovax vaccines producing Gag virus-363 like particles, whereas in this study, Gag was part of a polyprotein which did not bud due to 364 the removal of the myristylation signal [3]. T-cell responses to Gag have been correlated 365 with HIV control and may be desirable in a HIV vaccine regimen [26]. 366 367
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In summary, the delayed protein boost enhanced neutralization, CD4+ T-cell and binding 368 antibody responses. As with most recombinant pox vector vaccines, CD8+ T-cell responses 369 remain limited. Neutralizing antibody responses waned significantly after 6 months, 370 indicating the need for additional boosting or more potent protein boosts. Although binding 371 antibody responses persisted for 6 months, whether this would translate into sustained 372 protection remains to be determined. Evaluating various combinations of different vaccines 373 in prime-boost regimens is necessary in order to design optimal HIV vaccine regimens. 374 375 Acknowledgments 376 We are indebted to the participants and the staff at the four clinical sites for their 377 dedication to the study. We thank Maphuti Madiga and Tandile Hermanus from the NICD, 378 and Ryan Duffy, Jennifer Martin, Corinne Dary, and Vicki Ashley from Duke University for 379 technical assistance, and Marcella Sarzotti Kelsoe from Duke University for quality 380 assurance oversight. We thank protocol team members Zhixue (Maggie) Wang, Li Qin, Niles 381 Eaton, Jin Bae, Eva Chung, Scharla Estep, Carter Bentley, Laura Saganic, Renee Holt, 382 Christine Cooper-Trenbeath, Yevgeny Grigoriev, Jill Royer, Joy St. Louis, and Lipson Oupa 383 Mafumane. We also appreciate support from Elise Levendal from SAAVI, Bill Blattner from 384 the University of Maryland, and Chris Butler from NIAID. 385 386 Funding Statement 387 This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) 388 U.S. Public Health Service Grants UM1 AI068614 [LOC: HIV Vaccine Trials Network], UM1 389 AI068635 [HVTN SDMC FHCRC], UM1 AI068618 [HVTN Laboratory program FHCRC]; 390 AI069412 [to Brigham and Women's Hospital and Fenway Health], AI069453 [to the 391
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Perinatal HIV Research Unit, Soweto], and AI069519 [to the Desmond Tutu HIV Centre, Cape 392 Town]. The SAMRC funded the HIV vaccine development through SAAVI. Funding for the 393 production of gp140∆V2.TV1 was provided to Novartis Vaccines from NIAID-NIH contract 394 N01-AI-25473, TO#1. Some of this work is based upon research supported by the South 395 African Research Chairs Initiative of the Department of Science and Technology and 396 National Research Foundation. 397 398
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12. Yates NL, Liao HX, Fong Y, deCamp A, Vandergrift NA, Williams WT, Alam SM, Ferrari 448 G, Yang ZY, Seaton KE, Berman PW, Alpert MD, Evans DT, O'Connell RJ, Francis D, 449 Sinangil F, Lee C, Nitayaphan S, Rerks-Ngarm S, Kaewkungwal J, Pitisuttithum P, 450 Tartaglia J, Pinter A, Zolla-Pazner S, Gilbert PB, Nabel GJ, Michael NL, Kim JH, Montefiori 451 DC, Haynes BF, Tomaras GD. Vaccine-induced Env V1-V2 IgG3 correlates with lower 452 HIV-1 infection risk and declines soon after vaccination. Science Translational Medicine 453 2014; 19:6(228):228ra39. 454 13. Tomaras GD, Binley JM, Gray ES, Crooks ET, Osawa K, Moore PL, Tumba N, Tong T, 455 Shen X, Yates NL, Decker J, Wibmer CK, Gao F, Alam SM, Easterbrook P, Abdool Karim S, 456 Kamanga G, Crump JA, Cohen M, Shaw GM, Mascola JR, Haynes BF, Montefiori DC, Morris 457 L. Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-458 infected individuals. Journal of virology 2011; 85:11502-19. 459 14. Shen X, Duffy R, Howington R, Cope A3, Sadagopal S4, Park H5, Pal R6, Kwa S4, Ding 460 S7, Yang OO8, Fouda GG9, Le Grand R10, Bolton D11, Esteban M12, Phogat S13, 461 Roederer M11, Amara RR4, Picker LJ5, Seder RA11, McElrath MJ14, Barnett S15, Permar 462 SR16, Shattock R3, DeVico AL17, Felber BK18, Pavlakis GN19, Pantaleo G7, Korber 463 BT20, Montefiori DC21, Tomaras GD22. Vaccine Induced Epitope Specific Antibodies to 464 SIVmac239 Envelope Are Distinct from Those Induced to the HIV-1 Envelope in Non-465 Human Primates. Journal of virology 2015. 466 15. Gottardo R, Bailer RT, Korber BT, Gnanakaran S, Phillips J, Shen X, Tomaras GD, Turk 467 E, Imholte G, Eckler L, Wenschuh H, Zerweck J, Greene K, Gao H, Berman PW, Francis D, 468 Sinangil F, Lee C, Nitayaphan S, Rerks-Ngarm S, Kaewkungwal J, Pitisuttithum P, 469 Tartaglia J, Robb ML, Michael NL, Kim JH, Zolla-Pazner S, Haynes BF, Mascola JR, Self S, 470 Gilbert P, Montefiori DC. Plasma IgG to linear epitopes in the V2 and V3 regions of HIV-471 1 gp120 correlate with a reduced risk of infection in the RV144 vaccine efficacy trial. 472 PloS one 2013; 8:e75665. 473 16. Sarzotti-Kelsoe M, Bailer RT, Turk E, Lin CL2, Bilska M3, Greene KM3, Gao H3, Todd 474 CA3, Ozaki DA3, Seaman MS4, Mascola JR2, Montefiori DC3. Optimization and validation 475 of the TZM-bl assay for standardized assessments of neutralizing antibodies against 476 HIV-1. Journal of immunological methods 2014; 409:131-46. 477 17. Holm S. A simple sequentially rejective multiple test procedure. Scandinavian 478 Journal of Statistics 1979; 6:2:65-70. 479 18. Agresti A, Coull BA.Approximate Is Better than "Exact" for Interval Estimation of 480 Binomial Proportions. 481 The American Statistician 1998; 52:2:119-12619. Elizaga ML, Vasan S, Marovich MA, 482 Sato AH, Lawrence DN, Chaitman BR, Frey SE, Keefer MC; MVA Cardiac Safety Working 483 Group . Prospective surveillance for cardiac adverse events in healthy adults receiving 484 modified vaccinia Ankara vaccines: a systematic review. PloS One 2013;26:8(9): 485 e75665. 486 20. Goepfert PA, Elizaga ML, Seaton K, omaras GD3, Montefiori DC3, Sato A2, Hural J2, 487 DeRosa SC4, Kalams SA5, McElrath MJ4, Keefer MC6, Baden LR7, Lama JR8, Sanchez J8, 488 Mulligan MJ9, Buchbinder SP10, Hammer SM11, Koblin BA12, Pensiero M13, Butler C13, 489 Moss B14, Robinson HL15; HVTN 205 Study Group; National Institutes of Allergy and 490 Infectious Diseases HIV Vaccines Trials Network. Specificity and 6-month durability of 491 immune responses induced by DNA and recombinant modified vaccinia Ankara 492 vaccines expressing HIV-1 virus-like particles. The Journal of Infectious Diseases 2014; 493 210:99-110. 494 21. Spearman P, Lally MA, Elizaga M, Montefiori D, Tomaras GD, McElrath MJ, Hural J, De 495 Rosa SC, Sato A, Huang Y, Frey SE, Sato P, Donnelly J, Barnett S, Corey LJ; HIV Vaccine 496
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Trials Network of NIAID. A trimeric, V2-deleted HIV-1 envelope glycoprotein vaccine 497 elicits potent neutralizing antibodies but limited breadth of neutralization in human 498 volunteers. The Journal of Infectious Diseases 2011; 203:1165-73. 499 22. Palma P, Romiti ML, Bernardi S, Pontrelli G, Mora N, Santilli V, Tchidjou HK, Aquilani 500 A, Cotugno N, Alghisi F, Lucidi V, Rossi P, Douagi I. Safety and immunogenicity of a 501 monovalent MF59(R)-adjuvanted A/H1N1 vaccine in HIV-infected children and young 502 adults. Biologicals : Journal of the International Association of Biological 503 Standardization 2012; 40:134-9. 504 23. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, Evans DT, 505 Montefiori DC, Karnasuta C, Sutthent R, Liao HX, DeVico AL, Lewis GK, Williams C, Pinter 506 A, Fong Y, Janes H, DeCamp A, Huang Y, Rao M, Billings E, Karasavvas N, Robb ML, Ngauy 507 V, de Souza MS, Paris R, Ferrari G, Bailer RT, Soderberg KA, Andrews C, Berman PW, 508 Frahm N, De Rosa SC, Alpert MD, Yates NL, Shen X, Koup RA, Pitisuttithum P, 509 Kaewkungwal J, Nitayaphan S, Rerks-Ngarm S, Michael NL, Kim JH. Immune-correlates 510 analysis of an HIV-1 vaccine efficacy trial. The New England Journal of Medicine 2012; 511 366:1275-86. 512 24. Jin X, Morgan C, Yu X, DeRosa S, Tomaras GD, Montefiori DC, Kublin J, Corey L, 513 Keefer MC; NIAID HIV Vaccine Trials Network. Multiple factors affect 514 immunogenicity of DNA plasmid HIV vaccines in human clinical trials. Vaccine. 515 2015 May 11;33(20):2347-53. 516 25. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, 517 Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, 518 McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, 519 Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH; MOPH-TAVEG Investigators. 520 Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. The New 521 England Journal of Medicine 2009; 361:2209-20. 522 26. Pereyra F, Addo MM, Kaufmann DE, Liu Y, Miura T, Rathod A, Baker B, Trocha A, 523 Rosenberg R, Mackey E, Ueda P, Lu Z, Cohen D, Wrin T, Petropoulos CJ, Rosenberg ES, 524 Walker BD. Genetic and immunologic heterogeneity among persons who control HIV 525 infection in the absence of therapy. The Journal of Infectious Diseases 2008; 197:563-526 71. 527 528 529 530
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Figure Legends : 531 Figure 1: Injection site and systemic reactogenicity. The percentage of participants with 532 local injection site pain and/or tenderness (left panel) and systemic symptoms (right panel) 533 following each administration of placebo (C), DNA (D), MVA (M), or protein (P) is shown by 534 treatment group, for the DNA/MVA prime-boost study (Panels A, B) and the study extension 535 (Panels C, D). The reactogenicity events were graded as mild, moderate, and severe. 536 Figure 2: Intracellular cytokine staining assay for vaccineinduced Tcell responses after 537 MVA or placebo vaccinations. The percentage of CD4+ T cells (upper panel) and CD8+ T 538 cells (lower panel) expressing IL-2 or IFN-γ, in response to global PTE peptide pools 539 representing the HIV antigens specifically indicated on the X-axis, or showing a response to 540 any one of these antigens (ANY). HIV-1-specific CD4+and CD8+ T-cell responses were 541 measured using a validated ICS assay. Results are from 2 weeks after the first MVA 542 vaccination (M1) and 2 weeks after the second MVA vaccination (M2). Results from the 543 placebo control group are combined from timepoints 2 weeks after the fourth and fifth 544 placebo injections (far left). Responders are shown as red dots, and nonresponders are 545 shown as blue triangles. Box plots show the distribution of the magnitude of response in 546 positive responders only. The box indicates the median and interquartile range (IQR); 547 whiskers extend to the furthest point within 1.5 times the IQR from the upper or lower 548 quartile. Numbers at the top of each panel show the percentage and number of responders 549 from each group, out of the evaluable participants. 550 Figure 3: Intracellular cytokine staining assay in study extension participants. The 551 percentage of CD4+ T cells (upper panel) and CD8+ T cells (lower panel) expressing IL-2 or 552 IFN-γ, in response to any antigen represented in the global PTE peptide pools (PTEG), 553 measured using a validated ICS assay. Results from study extension participants who had 554
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received the DNA/MVA regimen are shown over time, from 2 weeks after the first MVA 555 vaccination (M1), 2 weeks after the second MVA vaccination (M2), at extension baseline (B) 556 prior to injection with gp140 or placebo, 2 weeks after each study extension injection, and 6 557 months after the final injection. Participants received either Placebo (DDDMMCC group) or 558 Protein (DDDMMPP). Responders are shown as red dots, and nonresponders are shown as 559 blue triangles. Box plots show the distribution of the magnitude of response in positive 560 responders only. The box indicates the median and interquartile range (IQR); whiskers 561 extend to the furthest point within 1.5 times the IQR from the upper or lower quartile. 562 Numbers at the top of each panel show the percentage and number of responders from 563 each group, out of the evaluable participants. 564 Figure 4: The frequency and magnitude of IgG antibody binding by HIV1 binding antibody 565 multiplex assay (BAMA). Results are shown to ConS gp140 CFI (upper panel) and 566 gp140∆V2.TV1 (lower panel). The MFI minus blank responses summarize the magnitudes at 567 a given timepoint. Results from study extension participants who had received the 568 DNA/MVA regimen are shown over time, from samples obtained 2 weeks after the first 569 MVA vaccination (M1), 2 weeks after the second MVA vaccination (M2), at extension 570 baseline (B) prior to injection with gp140 or placebo, 2 weeks after each study extension 571 injection, and 6 months after the final injection. Participants received either Placebo 572 (DDDMMCC group) or Protein (DDDMMPP). Numbers at the top of each panel show the 573 percentage and number of responders from each group, out of the evaluable participants. 574 Plots include data from responders in red and nonresponders in blue. Box plots show the 575 distribution of the magnitude of response in positive responders only; the mid-line denotes 576 the median and the ends of the box denote the 25th and 75th percentiles. Whiskers extend 577 to the most extreme data points within 1.5 times the interquartile range. 578
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Figure 5: Linear epitope specific response measured by peptide microarray. 579 A. Proportions of linear binding responses to each epitope region. The peptide region for 580 each epitope is listed as the peptide numbers in the array library in parentheses next to the 581 name of the epitope. Sequences of all peptides in the microarray library have been 582 published previously [15]. For each subject, the percentage values for each epitope are 583 calculated as: maximum binding to the epitope / sum of maximum binding to all epitopes. 584 Each pie slice represents the average value of all subjects (N=12) mapped in the study. B. 585 Clade/strain preferences of the two dominant specificities V3 and C5. Binding magnitudes 586 (maximum binding intensity) to V3 and C5 peptides of each clade/strain are plotted. Each 587 bar represents the average value of all 12 subjects mapped. 588 589 Figure 6: Neutralization ID50 titers in the TZMbl assay against the HIV1C virus 590 MW965.26. The titer distributions for each treatment group are shown by timepoint, from 591 samples obtained 2 weeks after the first MVA vaccination (M1), 2 weeks after the second 592 MVA vaccination (M2), at extension baseline (B) prior to the first injection with gp140 or 593 placebo, 2 weeks after each study extension injection, and 6 months after the final injection. 594 Participants received either Placebo (DDDMMCC group) or Protein (DDDMMPP). Numbers 595 at the top of each panel show the percentage and number of responders from each group, 596 out of the evaluable participants. Plots include data from responders in red and 597 nonresponders in blue. Box plots show the distribution of the magnitude of response in 598 positive responders only; the mid-line denotes the median and the ends of the box denote 599 the 25th and 75th percentiles. Whiskers extend to the most extreme data points within 1.5 600 times the interquartile range.601
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603 604 605 606 607 608 609 610 611 612 613
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Table 1: Study design of the HVTN 073/073E trial
Table 1A. Trial schema for initial DNA/MVA regimen (HVTN 073)
Number
(48)
Treatment
Arm
Dose Months (days) after first injection
DNA-C2 (mg)
MVA-C (pfu)
0(0) 1(28) 2(56) 4(112) 5 (140)
40 T1 4 1.45x109 DNA-C2 DNA-C2 DNA-C2 MVA-C MVA-C
8 C1 0 0 Placebo Placebo Placebo Placebo Placebo
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Table 1B. Trial schema for study extension (HVTN 073E)
Previous regimen Treatment Group
Dose Months (days) after first extension injection
Number
(27)
DNA-C2 (D) and MVA-C (M), or Placebo (C)
Subtype C gp140/MF59
0(0) 3(84)
16 DDDMM T1/T2 100mcg gp140/MF59 gp140/MF59
6 DDDMM T1/C2 0 Placebo Placebo
1 CCCC C1/T2 100mcg gp140/MF59 gp140/MF59
4 CCCC C1/C2 0 Placebo Placebo
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Table 2: Demographics and Vaccination Frequencies
C1
(n=8)
T1
(n=40)
Total
(n=48)
C2
(n=10)
T2
(n=17)
Total
(n=27)
Sex
Male
Female
5 (63%)
3 (38%)
21 (53%)
19 (48%)
26 (54%)
22 (46%)
6(60%)
4(40%)
9(53%)
8(47%)
15(56%)
12(44%)
Race
White
Black/African American
2 (25%)
6 (75%)
7 (18%)
31 (78%)
9 (19%)
37 (77%)
0(0%)
9(100%)
4(24%)
13(76%)
4(15%)
22(81%)
Age (years)
18-20
21-30
31-40
Median
Range
4 (50%)
2 (25%)
2 (25%)
21.5
18-35
8 (20%)
25 (63%)
7 (63%)
24.0
18-39
12 (25%)
27 (25%)
9 (19%)
24.0
18-39
4(40%)
3(30%)
3(30%)
21.0
18-37
5(29%)
9(53%)
3(18%)
22.0
18-39
9(33%)
12(44%)
6(22%)
22.0
18-39
Vaccination Frequencies
Day 0
Day 28
Day 56
Day 112
Day 140
Extension Day 0
Extension Day 84
8 (100%)
8 (100%)
8 (100%)
8 (100%)
8 (100%)
40 (100%)
38 (95%)
37 (93%)
38 (95%)
37 (93%)
48 (100%)
46 (96%)
45 (94%)
46 (96%)
45 (94%)
10 (100%)
10 (100%)
17 (100%)
16 (94%)
27 (100%)
26 (96%)
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Bin
din
g I
nte
ns
ity
V 3 C 5
0
1 .01 0 4
2 .01 0 4
3 .01 0 4
4 .01 0 4 A C o n
B C o n
B .M N
C C o n
C .Z M 6 5 1
C .T V 1
C .1 0 8 6
D C o n
M C o n
C R F 0 1 C o n
1 .T H 0 23
1 .A 2 4 4
C R F 0 2 C o n
A
B
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