Safety and immunogenicity of a subtype C ALVAC-HIV (vCP2438) vaccine prime plus bivalent subtype C gp120 vaccine boost adjuvanted with MF59 or alum in healthy adults without HIV (HVTN 107): A phase 1/2a randomized trial.

BACKGROUND: Adjuvants are widely used to enhance and/or direct vaccine-induced immune responses yet rarely evaluated head-to-head. Our trial directly compared immune responses elicited by MF59 versus alum adjuvants in the RV144-like HIV vaccine regimen modified for the Southern African region. The RV144 trial of a recombinant canarypox vaccine vector expressing HIV env subtype B (ALVAC-HIV) prime followed by ALVAC-HIV plus a bivalent gp120 protein vaccine boost adjuvanted with alum is the only trial to have shown modest HIV vaccine efficacy. Data generated after RV144 suggested that use of MF59 adjuvant might allow lower protein doses to be used while maintaining robust immune responses. We evaluated safety and immunogenicity of an HIV recombinant canarypox vaccine vector expressing HIV env subtype C (ALVAC-HIV) prime followed by ALVAC-HIV plus a bivalent gp120 protein vaccine boost (gp120) adjuvanted with alum (ALVAC-HIV+gp120/alum) or MF59 (ALVAC-HIV+gp120/MF59) or unadjuvanted (ALVAC-HIV+gp120/no-adjuvant) and a regimen where ALVAC-HIV+gp120 adjuvanted with MF59 was used for the prime and boost (ALVAC-HIV+gp120/MF59 coadministration). METHODS AND FINDINGS: Between June 19, 2017 and June 14, 2018, 132 healthy adults without HIV in South Africa, Zimbabwe, and Mozambique were randomized to receive intramuscularly: (1) 2 priming doses of ALVAC-HIV (months 0 and 1) followed by 3 booster doses of ALVAC-HIV+gp120/MF59 (months 3, 6, and 12), n = 36; (2) 2 priming doses of ALVAC-HIV (months 0 and 1) followed by 3 booster doses of ALVAC-HIV+gp120/alum (months 3, 6, and 12), n = 36; (3) 4 doses of ALVAC-HIV+gp120/MF59 coadministered (months 0, 1, 6, and 12), n = 36; or (4) 2 priming doses of ALVAC-HIV (months 0 and 1) followed by 3 booster doses of ALVAC-HIV+gp120/no adjuvant (months 3, 6, and 12), n = 24. Primary outcomes were safety and occurrence and mean fluorescence intensity (MFI) of vaccine-induced gp120-specific IgG and IgA binding antibodies at month 6.5. All vaccinations were safe and well-tolerated; increased alanine aminotransferase was the most frequent related adverse event, occurring in 2 (1.5%) participants (1 severe, 1 mild). At month 6.5, vaccine-specific gp120 IgG binding antibodies were detected in 100% of vaccinees for all 4 vaccine groups. No significant differences were seen in the occurrence and net MFI of vaccine-specific IgA responses between the ALVAC-HIV+gp120/MF59-prime-boost and ALVAC-HIV+gp120/alum-prime-boost groups or between the ALVAC-HIV+gp120/MF59-prime-boost and ALVAC-HIV+gp120/MF59 coadministration groups. Limitations were the relatively small sample size per group and lack of evaluation of higher gp120 doses. CONCLUSIONS: Although MF59 was expected to enhance immune responses, alum induced similar responses to MF59, suggesting that the choice between these adjuvants may not be critical for the ALVAC+gp120 regimen. TRIAL REGISTRATION: HVTN 107 was registered with the South African National Clinical Trials Registry (DOH-27-0715-4894) and ClinicalTrials.gov (NCT03284710).

Protein dose-sparing effect of AS01B adjuvant in a randomized preventive HIV vaccine trial of ALVAC-HIV (vCP2438) and adjuvanted bivalent subtype C gp120.

BACKGROUND: HVTN 120 is a phase 1/2a randomized double-blind placebo-controlled HIV vaccine trial that evaluated the safety and immunogenicity of ALVAC-HIV (vCP2438) and MF59- or AS01B-adjuvanted bivalent subtype C gp120 Env protein at two dose levels in healthy HIV-uninfected adults. Trial registration URL https://clinicaltrials.gov/ct2/show/NCT03122223 and registration number NCT03122223. METHODS: Participants received ALVAC-HIV (vCP2438) alone or placebo at months 0 and 1. At months 3 and 6, participants received either placebo, ALVAC-HIV (vCP2438) with 200µg of bivalent subtype C gp120 adjuvanted with MF59 or AS01B, or ALVAC-HIV (vCP2438) with 40µg of bivalent subtype C gp120 adjuvanted with AS01B. Primary outcomes were safety and immune responses. RESULTS: We enrolled 160 participants, 55% females, 18-40 years old (median age 24 years) of whom 150 received vaccine and 10 placebo. Vaccines were generally safe and well tolerated. At months 6.5 and 12, CD4+ T-cell response rates and magnitudes were higher in the AS01B-adjuvanted groups than in the MF59-adjuvanted group. At month 12, HIV-specific Env-gp120 binding antibody response magnitudes in the 40µg gp120/AS01B group were higher than in either of the 200µg gp120 groups. CONCLUSIONS: The 40µg dose gp120/AS01B regimen elicited the highest CD4+ T-cell and binding antibody responses.

Analysis of Neutralizing Antibodies as a Correlate of Instantaneous Risk of Hospitalized Dengue in Placebo Recipients of Dengue Vaccine Efficacy Trials.

BACKGROUND: In the CYD14 (NCT01373281) and CYD15 (NCT01374516) dengue vaccine efficacy trials, month 13 neutralizing antibody (nAb) titers correlated inversely with risk of symptomatic, virologically confirmed dengue (VCD) between month 13 (1 month after final dose) and month 25. We assessed nAb titer as a correlate of instantaneous risk of hospitalized VCD (HVCD), for which participants were continually surveilled for 72 months. METHODS: Using longitudinal nAb titers from the per-protocol immunogenicity subsets, we estimated hazard ratios (HRs) of HVCD by current nAb titer value for 3 correlate/endpoint pairs: average titer across all 4 serotypes/HVCD of any serotype (HVCD-Any), serotype-specific titer/homologous HVCD, and serotype-specific titer/heterologous HVCD. RESULTS: Baseline-seropositive placebo recipients with higher average titer had lower instantaneous risk of HVCD-Any in 2- to 16-year-olds and in 9- to 16-year-olds (HR, 0.26 or 0.15 per 10-fold increase in average titer by 2 methods [95% confidence interval {CI}, .14-.45 and .07-.34, respectively]) pooled across both trials. Results were similar for homologous HVCD. There was evidence suggesting increased HVCD-Any risk in participants with low average titer (1:10 to 1:100) compared to seronegative participants (HR, 1.85 [95% CI, .93-3.68]). CONCLUSIONS: Natural infection-induced nAbs were inversely associated with hospitalized dengue, upon exceeding a relatively low threshold.

Analysis of the HIV Vaccine Trials Network 702 Phase 2b-3 HIV-1 Vaccine Trial in South Africa Assessing RV144 Antibody and T-Cell Correlates of HIV-1 Acquisition Risk.

BACKGROUND: The ALVAC/gp120 + MF59 vaccines in the HIV Vaccine Trials Network (HVTN) 702 efficacy trial did not prevent human immunodeficiency virus-1 (HIV-1) acquisition. Vaccine-matched immunological endpoints that were correlates of HIV-1 acquisition risk in RV144 were measured in HVTN 702 and evaluated as correlates of HIV-1 acquisition. METHODS: Among 1893 HVTN 702 female vaccinees, 60 HIV-1-seropositive cases and 60 matched seronegative noncases were sampled. HIV-specific CD4+ T-cell and binding antibody responses were measured 2 weeks after fourth and fifth immunizations. Cox proportional hazards models assessed prespecified responses as predictors of HIV-1 acquisition. RESULTS: The HVTN 702 Env-specific CD4+ T-cell response rate was significantly higher than in RV144 (63% vs 40%, P = .03) with significantly lower IgG binding antibody response rate and magnitude to 1086.C V1V2 (67% vs 100%, P < .001; Pmag < .001). Although no significant univariate associations were observed between any T-cell or binding antibody response and HIV-1 acquisition, significant interactions were observed (multiplicity-adjusted P ≤.03). Among vaccinees with high IgG A244 V1V2 binding antibody responses, vaccine-matched CD4+ T-cell endpoints associated with decreased HIV-1 acquisition (estimated hazard ratios = 0.40-0.49 per 1-SD increase in CD4+ T-cell endpoint). CONCLUSIONS: HVTN 702 and RV144 had distinct immunogenicity profiles. However, both identified significant correlations (univariate or interaction) for IgG V1V2 and polyfunctional CD4+ T cells with HIV-1 acquisition. Clinical Trials Registration . NCT02968849.

CYD Tetravalent Dengue Vaccine Performance by Baseline Immune Profile (Monotypic/Multitypic) in Dengue-Seropositive Individuals.

BACKGROUND: The immune profile of dengue-experienced individuals is a determinant of dengue reinfection severity risk. Individuals with a single prior dengue infection (monotypic) are at highest risk for severe disease, while individuals with ≥ 2 prior dengue infections (multitypic) are at lower risk. The tetravalent dengue vaccine (CYD-TDV) has shown efficacy in the prevention of dengue in individuals with prior dengue infection. We estimated efficacy in individuals with monotypic or multitypic immune profiles. METHODS: Participants enrolled in the immunogenicity subsets of 2 randomized placebo-controlled phase 3 studies (CYD14, NCT01373281; CYD15, NCT01374516) were classified as either monotypic or multitypic, based on measured baseline dengue plaque reduction neutralization test. Vaccine efficacy (VE) against symptomatic virologically confirmed dengue (VCD) was assessed over 25 months and against VCD hospitalization over 6 years. RESULTS: Of 3927 participants in the immunogenicity subsets, 496 and 257 in the CYD-TDV and placebo groups, respectively, were classified as monotypic immune, and 1227 and 612, respectively, as multitypic immune. VE against symptomatic VCD was 77.4% (95% CI, 56.4%-88.2%) for monotypic and 89.2% (95% CI, 71.5%-95.9%) for multitypic profiles, with corresponding absolute risk reductions (ARRs) of 4.48% (95% CI, 2.32%-6.65%) for monotypics and 1.67% (95% CI, .89%-2.46%) for multitypics. VE against hospitalized VCD was 75.3% (95% CI, 42.7%-90.2%) in monotypics and 81.2% (95% CI, 21.7%-96.8%) in multitypics, with ARRs of 0.95% (95% CI, .37%-1.53%) for monotypics and 0.18% (95% CI, .02%-.34%) for multitypics. CONCLUSIONS: CYD-TDV benefits individuals with monotypic and multitypic immune profiles. Larger public health benefit is expected to derive from the protection of individuals with a monotypic immune profile.

Analysis of Hospitalized and Severe Dengue Cases Over the 6 years of Follow-up of the Tetravalent Dengue Vaccine (CYD-TDV) Efficacy Trials in Asia and Latin America.

BACKGROUND: CYD-TDV, a live, attenuated, tetravalent dengue vaccine, has been approved for the prevention of symptomatic dengue in previously dengue exposed individuals. This post hoc analysis assessed hospitalized and severe virologically confirmed dengue (VCD) over the complete 6-year follow-up of 3 CYD-TDV efficacy studies (CYD14, CYD15, and CYD23/CYD57). METHODS: The main outcomes were hazard ratios (HRs) for hospitalized or severe VCD by baseline dengue serostatus, focusing on those who were seropositive, and by age at immunization (<9 years/≥9 years). Baseline dengue serostatus was measured or inferred using several methods. Hospitalized VCD cases were characterized in terms of clinical signs and symptoms and wild-type viremia level. Antibody persistence was assessed up to 5 years after the last injection. RESULTS: In those aged ≥9 years and baseline seropositive, CYD-TDV protected against hospitalized and severe VCD over 6 years compared to placebo (HR [95% confidence interval] multiple imputation from month 0 method, .19 [.12-.30] and .15 [.06-.39]; other methods were consistent). Vaccine protection was observed over the different study periods, being highest during the first 2 years. Evidence for a decreased risk of hospitalized and severe VCD was also observed in seropositive participants aged 6-8 years. Clinical signs and symptoms, and quantified dengue viremia from participants with hospitalized VCD were comparable between groups. CONCLUSIONS: CYD-TDV demonstrated robust protection against hospitalized and severe VCD over the entire 6-year follow-up in participants who were seropositive and ≥9 years old. Protection was also observed in seropositive 6-8 year-olds. Clinical Trials Registration: NCT00842530, NCT01983553, NCT01373281, NCT01374516.

Effect of Dengue Serostatus on Dengue Vaccine Safety and Efficacy.

BACKGROUND: In efficacy trials of a tetravalent dengue vaccine (CYD-TDV), excess hospitalizations for dengue were observed among vaccine recipients 2 to 5 years of age. Precise risk estimates according to observed dengue serostatus could not be ascertained because of the limited numbers of samples collected at baseline. We developed a dengue anti-nonstructural protein 1 (NS1) IgG enzyme-linked immunosorbent assay and used samples from month 13 to infer serostatus for a post hoc analysis of safety and efficacy. METHODS: In a case-cohort study, we reanalyzed data from three efficacy trials. For the principal analyses, we used baseline serostatus determined on the basis of measured (when baseline values were available) or imputed (when baseline values were missing) titers from a 50% plaque-reduction neutralization test (PRNT50), with imputation conducted with the use of covariates that included the month 13 anti-NS1 assay results. The risk of hospitalization for virologically confirmed dengue (VCD), of severe VCD, and of symptomatic VCD according to dengue serostatus was estimated by weighted Cox regression and targeted minimum loss-based estimation. RESULTS: Among dengue-seronegative participants 2 to 16 years of age, the cumulative 5-year incidence of hospitalization for VCD was 3.06% among vaccine recipients and 1.87% among controls, with a hazard ratio (vaccine vs. control) through data cutoff of 1.75 (95% confidence interval [CI], 1.14 to 2.70). Among dengue-seronegative participants 9 to 16 years of age, the cumulative incidence of hospitalization for VCD was 1.57% among vaccine recipients and 1.09% among controls, with a hazard ratio of 1.41 (95% CI, 0.74 to 2.68). Similar trends toward a higher risk among seronegative vaccine recipients than among seronegative controls were also found for severe VCD. Among dengue-seropositive participants 2 to 16 years of age and those 9 to 16 years of age, the cumulative incidence of hospitalization for VCD was 0.75% and 0.38%, respectively, among vaccine recipients and 2.47% and 1.88% among controls, with hazard ratios of 0.32 (95% CI, 0.23 to 0.45) and 0.21 (95% CI, 0.14 to 0.31). The risk of severe VCD was also lower among seropositive vaccine recipients than among seropositive controls. CONCLUSIONS: CYD-TDV protected against severe VCD and hospitalization for VCD for 5 years in persons who had exposure to dengue before vaccination, and there was evidence of a higher risk of these outcomes in vaccinated persons who had not been exposed to dengue. (Funded by Sanofi Pasteur; ClinicalTrials.gov numbers, NCT00842530 , NCT01983553 , NCT01373281 , and NCT01374516 .).

Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination.

BACKGROUND: Recent Mycobacterium tuberculosis infection confers a predisposition to the development of tuberculosis disease, the leading killer among global infectious diseases. H4:IC31, a candidate subunit vaccine, has shown protection against tuberculosis disease in preclinical models, and observational studies have indicated that primary bacille Calmette-Guérin (BCG) vaccination may offer partial protection against infection. METHODS: In this phase 2 trial, we randomly assigned 990 adolescents in a high-risk setting who had undergone neonatal BCG vaccination to receive the H4:IC31 vaccine, BCG revaccination, or placebo. All the participants had negative results on testing for M. tuberculosis infection on the QuantiFERON-TB Gold In-tube assay (QFT) and for the human immunodeficiency virus. The primary outcomes were safety and acquisition of M. tuberculosis infection, as defined by initial conversion on QFT that was performed every 6 months during a 2-year period. Secondary outcomes were immunogenicity and sustained QFT conversion to a positive test without reversion to negative status at 3 months and 6 months after conversion. Estimates of vaccine efficacy are based on hazard ratios from Cox regression models and compare each vaccine with placebo. RESULTS: Both the BCG and H4:IC31 vaccines were immunogenic. QFT conversion occurred in 44 of 308 participants (14.3%) in the H4:IC31 group and in 41 of 312 participants (13.1%) in the BCG group, as compared with 49 of 310 participants (15.8%) in the placebo group; the rate of sustained conversion was 8.1% in the H4:IC31 group and 6.7% in the BCG group, as compared with 11.6% in the placebo group. Neither the H4:IC31 vaccine nor the BCG vaccine prevented initial QFT conversion, with efficacy point estimates of 9.4% (P=0.63) and 20.1% (P=0.29), respectively. However, the BCG vaccine reduced the rate of sustained QFT conversion, with an efficacy of 45.4% (P=0.03); the efficacy of the H4:IC31 vaccine was 30.5% (P=0.16). There were no clinically significant between-group differences in the rates of serious adverse events, although mild-to-moderate injection-site reactions were more common with BCG revaccination. CONCLUSIONS: In this trial, the rate of sustained QFT conversion, which may reflect sustained M. tuberculosis infection, was reduced by vaccination in a high-transmission setting. This finding may inform clinical development of new vaccine candidates. (Funded by Aeras and others; C-040-404 ClinicalTrials.gov number, NCT02075203 .).

Safety and immune responses after a 12-month booster in healthy HIV-uninfected adults in HVTN 100 in South Africa: A randomized double-blind placebo-controlled trial of ALVAC-HIV (vCP2438) and bivalent subtype C gp120/MF59 vaccines.

BACKGROUND: HVTN 100 evaluated the safety and immunogenicity of an HIV subtype C pox-protein vaccine regimen, investigating a 12-month booster to extend vaccine-induced immune responses. METHODS AND FINDINGS: A phase 1-2 randomized double-blind placebo-controlled trial enrolled 252 participants (210 vaccine/42 placebo; median age 23 years; 43% female) between 9 February 2015 and 26 May 2015. Vaccine recipients received ALVAC-HIV (vCP2438) alone at months 0 and 1 and with bivalent subtype C gp120/MF59 at months 3, 6, and 12. Antibody (IgG, IgG3 binding, and neutralizing) and CD4+ T-cell (expressing interferon-gamma, interleukin-2, and CD40 ligand) responses were evaluated at month 6.5 for all participants and at months 12, 12.5, and 18 for a randomly selected subset. The primary analysis compared IgG binding antibody (bAb) responses and CD4+ T-cell responses to 3 vaccine-matched antigens at peak (month 6.5 versus 12.5) and durability (month 12 versus 18) timepoints; IgG responses to CaseA2_gp70_V1V2.B, a primary correlate of risk in RV144, were also compared at these same timepoints. Secondary and exploratory analyses compared IgG3 bAb responses, IgG bAb breadth scores, neutralizing antibody (nAb) responses, antibody-dependent cellular phagocytosis, CD4+ polyfunctionality responses, and CD4+ memory sub-population responses at the same timepoints. Vaccines were generally safe and well tolerated. During the study, there were 2 deaths (both in the vaccine group and both unrelated to study products). Ten participants became HIV-infected during the trial, 7% (3/42) of placebo recipients and 3% (7/210) of vaccine recipients. All 8 serious adverse events were unrelated to study products. Less waning of immune responses was seen after the fifth vaccination than after the fourth, with higher antibody and cellular response rates at month 18 than at month 12: IgG bAb response rates to 1086.C V1V2, 21.0% versus 9.7% (difference = 11.3%, 95% CI = 0.6%-22.0%, P = 0.039), and ZM96.C V1V2, 21.0% versus 6.5% (difference = 14.5%, 95% CI = 4.1%-24.9%, P = 0.004). IgG bAb response rates to all 4 primary V1V2 antigens were higher 2 weeks after the fifth vaccination than 2 weeks after the fourth vaccination: 87.7% versus 75.4% (difference = 12.3%, 95% CI = 1.7%-22.9%, P = 0.022) for 1086.C V1V2, 86.0% versus 63.2% (difference = 22.8%, 95% CI = 9.1%-36.5%, P = 0.001) for TV1c8.2.C V1V2, 67.7% versus 44.6% (difference = 23.1%, 95% CI = 10.4%-35.7%, P < 0.001) for ZM96.C V1V2, and 81.5% versus 60.0% (difference = 21.5%, 95% CI = 7.6%-35.5%, P = 0.002) for CaseA2_gp70_V1V2.B. IgG bAb response rates to the 3 primary vaccine-matched gp120 antigens were all above 90% at both peak timepoints, with no significant differences seen, except a higher response rate to ZM96.C gp120 at month 18 versus month 12: 64.5% versus 1.6% (difference = 62.9%, 95% CI = 49.3%-76.5%, P < 0.001). CD4+ T-cell response rates were higher at month 18 than month 12 for all 3 primary vaccine-matched antigens: 47.3% versus 29.1% (difference = 18.2%, 95% CI = 2.9%-33.4%, P = 0.021) for 1086.C, 61.8% versus 38.2% (difference = 23.6%, 95% CI = 9.5%-37.8%, P = 0.001) for TV1.C, and 63.6% versus 41.8% (difference = 21.8%, 95% CI = 5.1%-38.5%, P = 0.007) for ZM96.C, with no significant differences seen at the peak timepoints. Limitations were that higher doses of gp120 were not evaluated, this study was not designed to investigate HIV prevention efficacy, and the clinical significance of the observed immunological effects is uncertain. CONCLUSIONS: In this study, a 12-month booster of subtype C pox-protein vaccines restored immune responses, and slowed response decay compared to the 6-month vaccination. TRIAL REGISTRATION: ClinicalTrials.gov NCT02404311. South African National Clinical Trials Registry (SANCTR number: DOH--27-0215-4796).

Efficacy of high-dose versus standard-dose influenza vaccine in older adults.

BACKGROUND: As compared with a standard-dose vaccine, a high-dose, trivalent, inactivated influenza vaccine (IIV3-HD) improves antibody responses to influenza among adults 65 years of age or older. This study evaluated whether IIV3-HD also improves protection against laboratory-confirmed influenza illness. METHODS: We conducted a phase IIIb-IV, multicenter, randomized, double-blind, active-controlled trial to compare IIV3-HD (60 µg of hemagglutinin per strain) with standard-dose trivalent, inactivated influenza vaccine (IIV3-SD [15 µg of hemagglutinin per strain]) in adults 65 years of age or older. Assessments of relative efficacy, effectiveness, safety (serious adverse events), and immunogenicity (hemagglutination-inhibition [HAI] titers) were performed during the 2011-2012 (year 1) and the 2012-2013 (year 2) northern-hemisphere influenza seasons. RESULTS: A total of 31,989 participants were enrolled from 126 research centers in the United States and Canada (15,991 were randomly assigned to receive IIV3-HD, and 15,998 to receive IIV3-SD). In the intention-to-treat analysis, 228 participants in the IIV3-HD group (1.4%) and 301 participants in the IIV3-SD group (1.9%) had laboratory-confirmed influenza caused by any viral type or subtype associated with a protocol-defined influenza-like illness (relative efficacy, 24.2%; 95% confidence interval [CI], 9.7 to 36.5). At least one serious adverse event during the safety surveillance period was reported by 1323 (8.3%) of the participants in the IIV3-HD group, as compared with 1442 (9.0%) of the participants in the IIV3-SD group (relative risk, 0.92; 95% CI, 0.85 to 0.99). After vaccination, HAI titers and seroprotection rates (the percentage of participants with HAI titers ≥ 1:40) were significantly higher in the IIV3-HD group. Conclusions: Among persons 65 years of age or older, IIV3-HD induced significantly higher antibody responses and provided better protection against laboratory-confirmed influenza illness than did IIV3-SD. (Funded by Sanofi Pasteur; ClinicalTrials.gov number, NCT01427309.).

Effect of Previous-Year Vaccination on the Efficacy, Immunogenicity, and Safety of High-Dose Inactivated Influenza Vaccine in Older Adults.

BACKGROUND: High-dose inactivated influenza vaccine (IIV-HD) is an alternative to the standard-dose inactivated influenza vaccine (IIV-SD) in the United States for influenza prevention in older adults. IIV-HD improved efficacy relative to IIV-SD in a randomized controlled trial. Recent observational studies suggest that previous influenza vaccination may influence the immunogenicity and effectiveness of current-season vaccination. METHODS: The original study was a double-blind, randomized trial comparing IIV-HD to IIV-SD in adults aged ≥65 years over 2 influenza seasons. A subset of year 1 (Y1) participants reenrolled in year 2 (Y2), receiving vaccine by random assignment in both years. We evaluated the effect of Y1 vaccination on Y2 relative vaccine efficacy (VE), immunogenicity (hemagglutination inhibition [HAI] titers), and safety among reenrolled participants. RESULTS: Of 14 500 Y1 participants, 7643 reenrolled in Y2. Relative to participants who received IIV-SD both seasons, VE was higher for IIV-HD vaccinees in Y2 (28.3% overall; 25.1% for Y1 IIV-HD, Y2 IIV-HD; and 31.6% for Y1 IIV-SD, Y2 IIV-HD). In multivariate logistic regression models, Y1 vaccine was not a significant modifier of Y2 VE (P= .43), whereas Y2 IIV-HD remained significantly associated with lower influenza risk (P= .043). Compared to administration of IIV-SD in both years, postvaccination HAI titers were significantly higher for patterns that included IIV-HD in Y2. No safety concerns were raised with IIV-HD revaccination. CONCLUSIONS: IIV-HD is likely to provide clinical benefit over IIV-SD irrespective of previous-season vaccination with IIV-HD or IIV-SD. IIV-HD consistently improved immune responses, and no safety concerns emerged in the context of IIV-HD revaccination.

Safety and immunogenicity after a 30-month boost of a subtype C ALVAC-HIV (vCP2438) vaccine prime plus bivalent subtype C gp120/MF59 vaccine boost (HVTN 100): A phase 1-2 randomized double-blind placebo-controlled trial.

Induction of broad, durable immune responses is a challenge in HIV vaccine development. HVTN 100 Part A administered subtype C-containing ALVAC-HIV at months 0 and 1, and ALVAC-HIV with bivalent subtype C gp120/MF59 at months 3, 6 and 12. As IgG binding antibody and T-cell responses were similar or greater at month 12.5 vs. month 6.5, but waned by month 18, we investigated vaccine-elicited immune responses after a month 30 boost in this study, HVTN 100 Part B. From 13 September 2017 to 7 August 2018, a subgroup of vaccinees was randomized to receive intramuscular injections of ALVAC+gp120/MF59 (n = 32) or gp120/MF59 alone (n = 31) and a subgroup of placebo recipients was administered placebo (n = 7) at month 30. Primary outcomes were safety, IgG binding antibodies (bAbs) to vaccine-specific and V1V2 Env proteins and vaccine-specific CD4+ T cells at month 30.5. Secondary outcomes included neutralizing and antibody dependent cellular cytotoxicity functions and durability at months 30 and 36. Both vaccine groups had an acceptable safety profile. There were no statistically significant differences in the occurrence or level of IgG bAbs between the vaccine boost groups for any vaccine-specific or V1V2 antigens. IgG responses were higher to vaccine-matched gp120 than to V1V2. The booster vaccination restored the magnitude-breadth IgG bAb response to V1V2 antigens at month 30.5. However, it rapidly waned by month 36. CD4+ T-cell response rates to the 3 vaccine-matched Env antigens for the combined vaccine groups ranged from 37% at month 30, boosted to as high as 91% at month 30.5, and waned by month 36 to as low as 44%, with no significant differences between the vaccine boost groups. Because these responses waned after 6 months, additional strategies may be needed to maintain the durability of prime-boost vaccine regimens and to generate these or other immune responses that confer protection. Trial registration: South African National Clinical Trials Register (SANCTR number: DOH-27-0215-4796) and ClinicalTrials.gov (NCT02404311).

Efficacy of a bivalent (D614 + B.1.351) SARS-CoV-2 Protein Vaccine.

Background: COVID-19 vaccines with alternative strain compositions are needed to provide broad protection against newly emergent SARS-CoV-2 variants of concern. Methods: We conducted a global Phase 3, multi-stage efficacy study (NCT04904549) among adults aged ≥18 years. Participants were randomized 1:1 to receive two intramuscular injections 21 days apart of a bivalent SARS-CoV-2 recombinant protein vaccine with AS03-adjuvant (5 µg of ancestral (D614) and 5 µg of B.1.351 [beta] variant spike protein) or placebo. Symptomatic COVID-19 was defined as laboratory-confirmed COVID-19 with COVID-19-like illness (CLI) symptoms. The primary efficacy endpoint was the prevention of symptomatic COVID-19 ≥14 days after the second injection (post-dose 2 [PD2]). Results: Between 19 Oct 2021 and 15 Feb 2022, 12,924 participants received ≥1 study injection. 75% of participants were SARS-CoV-2 non-naïve. 11,416 participants received both study injections (efficacy-evaluable population [vaccine, n=5,736; placebo, n=5,680]). Up to 15 March 2022, 121 symptomatic COVID-19 cases were reported (32 in the vaccine group and 89 in the placebo group) ≥14 days PD2 with a vaccine efficacy (VE) of 64.7% (95% confidence interval [CI] 46.6; 77.2%). VE was 75.1% (95% CI 56.3; 86.6%) in non-naïve and 30.9% (95% CI -39.3; 66.7%) in naïve participants. Viral genome sequencing identified the infecting strain in 68 cases (Omicron [BA.1 and BA.2 subvariants]: 63; Delta: 4; Omicron and Delta: 1). The vaccine was well-tolerated and had an acceptable safety profile. Conclusions: A bivalent vaccine conferred heterologous protection against symptomatic infection with newly emergent Omicron (BA.1 and BA.2) in non-naïve adults 18-59 years of age.

Efficacy of a bivalent (D614 + B.1.351) SARS-CoV-2 recombinant protein vaccine with AS03 adjuvant in adults: a phase 3, parallel, randomised, modified double-blind, placebo-controlled trial.

BACKGROUND: COVID-19 vaccines with alternative strain compositions are needed to provide broad protection against newly emergent SARS-CoV-2 variants of concern. This study aimed to describe the clinical efficacy and safety of a bivalent SARS-CoV-2 recombinant protein vaccine as a two-injection primary series during a period of circulation of the omicron (B.1.1.529) variant. METHODS: We conducted a phase 3, parallel, randomised, modified double-blind, placebo-controlled trial in adults aged 18 years or older at 54 clinical research centres in eight countries (Colombia, Ghana, India, Kenya, Mexico, Nepal, Uganda, and Ukraine). Participants were recruited from the community and randomly assigned (1:1) by use of an interactive response technology system to receive two intramuscular 0·5 mL injections, 21 days apart, of the bivalent vaccine (5 µg of ancestral [D614] and 5 µg of beta [B.1.351] variant spike protein, with AS03 adjuvant) or placebo (0·9% normal saline). All participants, outcome assessors, and laboratory staff performing assays were masked to group assignments; those involved in the preparation and administration of the vaccines were unmasked. Participants were stratified by age (18-59 years and ≥60 years) and baseline SARS-CoV-2 rapid serodiagnostic test positivity. Symptomatic COVID-19 was defined as laboratory-confirmed (via nucleic acid amplification test or PCR test) COVID-19 with COVID-19-like illness symptoms. The primary efficacy endpoint was the clinical efficacy of the bivalent vaccine for prevention of symptomatic COVID-19 at least 14 days after the second injection (dose 2). Safety was assessed in all participants receiving at least one injection of the study vaccine or placebo. This trial is registered with ClinicalTrials.gov (NCT04904549) and is closed to recruitment. FINDINGS: Between Oct 19, 2021, and Feb 15, 2022, 13 002 participants were enrolled and randomly assigned to receive the first dose of the study vaccine (n=6512) or placebo (n=6490). 12 924 participants (6472 in the vaccine group and 6452 in the placebo group) received at least one study injection, of whom 7542 (58·4%) were male and 9693 (75·0%) were SARS-CoV-2 non-naive. Of these 12 924 participants, 11 543 (89·3%) received both study injections (5788 in the vaccine group and 5755 in the placebo group). The efficacy-evaluable population after dose 2 comprised 11 416 participants (5736 in the vaccine group and 5680 in the placebo group). The median duration of follow-up was 85 days (IQR 50-95) after dose 1 and 58 days (29-70) after dose 2. 121 symptomatic COVID-19 cases were reported at least 14 days after dose 2 (32 in the vaccine group and 89 in the placebo group), with an overall vaccine efficacy of 64·7% (95% CI 46·6 to 77·2). Vaccine efficacy against symptomatic COVID-19 was 75·1% (95% CI 56·3 to 86·6) in SARS-CoV-2 non-naive participants and 30·9% (-39·3 to 66·7) in SARS-CoV-2-naive participants. Viral genome sequencing identified the infecting strain in 68 (56·2%) of 121 cases (omicron [BA.1 and BA.2] in 63; delta in four; and both omicron and delta in one). Immediate unsolicited adverse events were reported by four (<0·1%) participants in the vaccine group and seven (0·1%) participants in the placebo group. Immediate unsolicited adverse reactions within 30 min after any injection were reported by four (<0·1%) participants in the vaccine group and six (<0·1%) participants in the placebo group. In the reactogenicity subset with available data, solicited reactions (solicited injection-site reactions and solicited systemic reactions) within 7 days after any injection occurred in 1398 (57·8%) of 2420 vaccine recipients and 983 (40·9%) of 2403 placebo recipients. Grade 3 solicited reactions were reported by 196 (8·1%; 95% CI 7·0 to 9·3) of 2420 vaccine recipients and 118 (4·9%; 4·1 to 5·9) of 2403 placebo recipients within 7 days after any injection, with comparable frequencies after dose 1 and dose 2 in the vaccine group. At least one serious adverse event occurred in 30 (0·5%) participants in the vaccine group and 26 (0·4%) in the placebo group. The proportion of adverse events of special interest and deaths was less than 0·1% in both study groups. No adverse event of special interest, serious adverse event, or death was deemed to be treatment related. There were no reported cases of thrombosis with thrombocytopenia syndrome, myocarditis, pericarditis, Bell's Palsy, or Guillain-Barré syndrome, or other immune-mediated diseases. INTERPRETATION: The bivalent variant vaccine conferred heterologous protection against symptomatic SARS-CoV-2 infection in the epidemiological context of the circulating contemporary omicron variant. These findings suggest that vaccines developed with an antigen from a non-predominant strain could confer cross-protection against newly emergent SARS-CoV-2 variants, although further investigation is warranted. FUNDING: Sanofi, US Biomedical Advanced Research and Development Authority, and the US National Institute of Allergy and Infectious Diseases.

Efficacy of a monovalent (D614) SARS-CoV-2 recombinant protein vaccine with AS03 adjuvant in adults: a phase 3, multi-country study.

Background: The literature on first generation COVID-19 vaccines show they were less effective against new SARS-CoV-2 variants of concern including Omicron (BA.1, BA.2, BA.4 and BA.5 subvariants). New vaccines developed against variant strains may provide cross-protection against emerging variants when used as boosters and facilitate vaccination across a range of countries, healthcare settings and populations. However, there are no data on such vaccines when used as a primary series. Methods: A global Phase 3, multi-stage efficacy study (NCT04904549) among adults (≥18 years) was conducted in 53 research centres in eight countries (United States, Honduras, Japan, Colombia, Kenya, India, Ghana, Nepal). Participants were randomized 1:1 to receive two intramuscular injections of a monovalent SARS-CoV-2 recombinant protein vaccine with AS03-adjuvant (10 µg of the spike (S) protein from the ancestral D614 strain) or placebo on Day 1 (D01) and Day 22 (D22). The primary efficacy endpoint was prevention of virologically confirmed SARS-CoV-2 infection with symptoms of COVID-19-like illness (CLI) ≥14 days after the second injection (post-dose 2 [PD2]) in participants who were SARS-CoV-2 naïve on D01 + D22. Safety and reactogenicity were also evaluated. Findings: Between May 26 and November 7, 2021, 10,114 participants received ≥1 study injection, and 9441 participants received both injections. 2108 (20.8%) participants were SARS-CoV-2 naïve at D01 and D22. The primary endpoint was analysed in a subset of the full analysis set (the modified full analysis set PD2 [mFAS-PD2], excluding participants who did not complete the vaccination schedule or received vaccination despite meeting one of the contraindication criteria, had onset of symptomatic COVID-19 between the first injection and before 14 days after the second injection, or participants who discontinued before 14 days after the second injection [n = 9377; vaccine, n = 4702; placebo, n = 4675]). Data were available for 2051 SARS-CoV-2 naïve and 7159 non-naïve participants. At the cut-off date (January 28, 2022), symptomatic COVID-19 was reported in 169 naïve participants (vaccine, n = 81; placebo, n = 88) ≥14 days PD2, with a vaccine efficacy (VE) of 15.3% (95% CI, -15.8; 38.2). VE regardless of D01/D22 serostatus was 32.9% (95% CI, 15.3; 47.0) and VE in non-naïve participants was 52.7% (95% CI, 31.2; 67.9). Viral genome sequencing was performed up to the data cut-off point and identified the infecting strain in 99/169 adjudicated cases in the PD2 naïve population (Delta [25], Omicron [72], other variants [3], one participant had infection with both Delta and Omicron variants and has been included in the totals for both Delta and Omicron). The vaccine was well-tolerated with an acceptable safety profile. Interpretation: In the context of changing circulating viral variants, it is challenging to induce protection in naïve individuals with a two-dose priming schedule based on the parental D614 strain. However, while the primary endpoint of this trial was not met, the results show that a monovalent D614 vaccine can still be of value in individuals previously exposed to SARS-CoV-2. Funding: This study was funded in whole or in part by Sanofi and by federal funds from the Biomedical Advanced Research and Development Authority, part of the office of the Administration for Strategic Preparedness and Response at the U.S. Department of Health and Human Services under contract number HHSO100201600005I, and in collaboration with the U.S. Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense under contract number W15QKN-16-9-1002. The views presented here are those of the authors and do not purport to represent those of the Department of the Army, the Department of Health and Human Services, or the U.S. government.

Accuracy and efficacy of pre-dengue vaccination screening for previous dengue infection with a new dengue rapid diagnostic test: a retrospective analysis of phase 3 efficacy trials.

BACKGROUND: A dengue pre-vaccination test that is convenient, highly specific, and highly sensitive is still needed. The OnSite Dengue IgG rapid diagnostic test (RDT) is a new rapid diagnostic test specifically designed for pre-vaccination screening. We aimed to retrospectively assess the efficacy of a tetravalent dengue vaccine (CYD-TDV) in participants determined to be dengue seropositive by the OnSite IgG RDT and to evaluate assay performances. METHODS: This was a complementary study using pre-vaccination samples from two CYD-TDV efficacy trials done in five countries in the Asia-Pacific region (NCT01373281) and five countries in Latin America (NCT01374516). Baseline dengue serostatus was determined by the OnSite IgG RDT on samples from the immunogenicity subsets of the two trials. In participants who were test positive, we calculated CYD-TDV vaccine efficacy against symptomatic virologically confirmed dengue (VCD) over 25 months, and against hospitalisation with VCD over 72 months of follow-up after the first vaccination. We used a reference algorithm to determine the reference dengue serostatus for each sample, and sensitivity and specificity of the OnSite IgG RDT were calculated. Analyses were done on the whole population (aged 2-16 years), and on those aged 6 years or older and those aged 9 years or older. FINDINGS: Of 3983 participants in the immunogenicity subsets of the efficacy trials CYD14 and CYD15, 3962 had complete dengue reference test results enabling baseline serostatus classification and 3833 had sufficient serum samples remaining for evaluation with the OnSite IgG RDT. Of the samples tested, 2486 (64·9%) of 3833 were OnSite IgG RDT-positive. In participants aged 2-16 years who were OnSite IgG RDT-positive, vaccine efficacy was 84·1% (95% CI 71·6-91·1) against symptomatic VCD, and 69·2% (38·8-84·5) against hospitalisation with VCD, with similar findings in those aged 6 years or older and those aged 9 years or older. The OnSite IgG RDT showed very high sensitivity (91·1%, 89·9-92·1) and high specificity (92·8%, 91·2-94·2) in participants aged 2-16 years, with significantly higher specificity in those aged 9 years or older (96·6%, 94·9-97·8). INTERPRETATION: The OnSite IgG RDT should provide a valuable tool for screening for previous dengue infection at the point of vaccination. In individuals who were OnSite IgG RDT-positive, the vaccine efficacy of CYD-TDV was high across all three age groups. FUNDING: Sanofi Pasteur.

Safety and immunogenicity of an AS03-adjuvanted SARS-CoV-2 recombinant protein vaccine (CoV2 preS dTM) in healthy adults: interim findings from a phase 2, randomised, dose-finding, multicentre study.

BACKGROUND: We evaluated our SARS-CoV-2 prefusion spike recombinant protein vaccine (CoV2 preS dTM) with different adjuvants, unadjuvanted, and in a one-injection and two-injection dosing schedule in a previous phase 1-2 study. Based on interim results from that study, we selected a two-injection schedule and the AS03 adjuvant for further clinical development. However, lower than expected antibody responses, particularly in older adults, and higher than expected reactogenicity after the second vaccination were observed. In the current study, we evaluated the safety and immunogenicity of an optimised formulation of CoV2 preS dTM adjuvanted with AS03 to inform progression to phase 3 clinical trial. METHODS: This phase 2, randomised, parallel-group, dose-ranging study was done in adults (≥18 years old), including those with pre-existing medical conditions, those who were immunocompromised (except those with recent organ transplant or chemotherapy) and those with a potentially increased risk for severe COVID-19, at 20 clinical research centres in the USA and Honduras. Women who were pregnant or lactating or, for those of childbearing potential, not using an effective method of contraception or abstinence, and those who had received a COVID-19 vaccine, were excluded. Participants were randomly assigned (1:1:1) using an interactive response technology system, with stratification by age (18-59 years and ≥60 years), rapid serodiagnostic test result (positive or negative), and high-risk medical conditions (yes or no), to receive two injections (day 1 and day 22) of 5 7mu;g (low dose), 10 7mu;g (medium dose), or 15 7mu;g (high dose) CoV2 preS dTM antigen with fixed AS03 content. All participants and outcome assessors were masked to group assignment; unmasked study staff involved in vaccine preparation were not involved in safety outcome assessments. All laboratory staff performing the assays were masked to treatment. The primary safety objective was to describe the safety profile in all participants, for each candidate vaccine formulation. Safety endpoints were evaluated for all randomised participants who received at least one dose of the study vaccine (safety analysis set), and are presented here for the interim study period (up to day 43). The primary immunogenicity objective was to describe the neutralising antibody titres to the D614G variant 14 days after the second vaccination (day 36) in participants who were SARS-CoV-2 naive who received both injections, provided samples at day 1 and day 36, did not have protocol deviations, and did not receive an authorised COVID-19 vaccine before day 36. Neutralising antibodies were measured using a pseudovirus neutralisation assay and are presented here up to 14 days after the second dose. As a secondary immunogenicity objective, we assessed neutralising antibodies in non-naive participants. This trial is registered with ClinicalTrials.gov (NCT04762680) and is closed to new participants for the cohort reported here. FINDINGS: Of 722 participants enrolled and randomly assigned between Feb 24, 2021, and March 8, 2021, 721 received at least one injection (low dose=240, medium dose=239, and high dose=242). The proportion of participants reporting at least one solicited adverse reaction (injection site or systemic) in the first 7 days after any vaccination was similar between treatment groups (217 [91%] of 238 in the low-dose group, 213 [90%] of 237 in the medium-dose group, and 218 [91%] of 239 in the high-dose group); these adverse reactions were transient, were mostly mild to moderate in intensity, and occurred at a higher frequency and intensity after the second vaccination. Four participants reported immediate unsolicited adverse events; two (one each in the low-dose group and medium-dose group) were considered by the investigators to be vaccine related and two (one each in the low-dose and high-dose groups) were considered unrelated. Five participants reported seven vaccine-related medically attended adverse events (two in the low-dose group, one in the medium-dose group, and four in the high-dose group). No vaccine-related serious adverse events and no adverse events of special interest were reported. Among participants naive to SARS-CoV-2 at day 36, 158 (98%) of 162 in the low-dose group, 166 (99%) of 168 in the medium-dose group, and 163 (98%) of 166 in the high-dose group had at least a two-fold increase in neutralising antibody titres to the D614G variant from baseline. Neutralising antibody geometric mean titres (GMTs) at day 36 for participants who were naive were 2189 (95% CI 1744-2746) for the low-dose group, 2269 (1792-2873) for the medium-dose group, and 2895 (2294-3654) for the high-dose group. GMT ratios (day 36: day 1) were 107 (95% CI 85-135) in the low-dose group, 110 (87-140) in the medium-dose group, and 141 (111-179) in the high-dose group. Neutralising antibody titres in non-naive adults 21 days after one injection tended to be higher than titres after two injections in adults who were naive, with GMTs 21 days after one injection for participants who were non-naive being 3143 (95% CI 836-11 815) in the low-dose group, 2338 (593-9226) in the medium-dose group, and 7069 (1361-36 725) in the high-dose group. INTERPRETATION: Two injections of CoV2 preS dTM-AS03 showed acceptable safety and reactogenicity, and robust immunogenicity in adults who were SARS-CoV-2 naive and non-naive. These results supported progression to phase 3 evaluation of the 10 7mu;g antigen dose for primary vaccination and a 5 7mu;g antigen dose for booster vaccination. FUNDING: Sanofi Pasteur and Biomedical Advanced Research and Development Authority.

Zika and Dengue Interactions in the Context of a Large Dengue Vaccine Clinical Trial in Latin America.

A phase III dengue vaccine trial including 9- to 16-year-olds in Latin America (NCT01374516) was ongoing at the time of a Zika outbreak. We explored interactions between dengue and Zika, in the context of dengue vaccination. Symptomatic virologically confirmed Zika (VCZ) was evaluated using acute-phase sera from febrile participants (January 2013-March 2018). Neutralizing antibody geometric mean titers (GMTs) were evaluated pre- and post-Zika outbreak (months 25 and 72) in 2,000 randomly selected participants. Baseline dengue serostatus was determined using the plaque reduction neutralization test or inferred post hoc using nonstructural protein 1 IgG ELISA at M13 (case-cohort analysis). Vaccine efficacy against VCZ and serologically suspected Zika (SSZ) was estimated. Overall, 239/10,157 (2.4%) acute-phase samples were VCZ positive during the study. Dengue vaccine efficacy against VCZ was 27.8% (95% CI: 0.3; 47.7) among baseline dengue-seropositive participants. No vaccine effect was evident against SSZ. Zika antibody GMTs increased from pre- to post-Zika epidemic, with smaller increases observed for participants who were dengue seropositive at baseline than for those who were dengue seronegative: post-/pre-Zika GMT ratios for baseline dengue-seropositive participants were 21.5 (vaccine group) and 30.8 (placebo); and for dengue seronegatives, 88.1 and 89.5, respectively. Dengue antibody GMTs post-Zika were higher in dengue vaccine and placebo recipients with SSZ than those without SSZ in both dengue seropositives and seronegatives. Dengue vaccine did not enhance symptomatic Zika illness in dengue-seropositive individuals, rather it reduced the risk of VCZ. Zika infection boosted preexisting vaccine-induced or naturally occurring dengue-neutralizing antibodies.

Immunobridging efficacy of a tetravalent dengue vaccine against dengue and against hospitalized dengue from children/adolescents to adults in highly endemic countries.

BACKGROUND: The recombinant tetravalent live-attenuated dengue vaccine based on the YF 17D vaccine virus backbone (CYD-TDV) demonstrated vaccine efficacy (VE) against symptomatic, virologically confirmed dengue of any serotype from month 13 to month 25 (VCD-DENV-AnyM13→M25) in the CYD14 (2-14-y-olds) and CYD15 (9-16-y-olds) phase 3 trials. Fifty percent plaque reduction neutralization test (PRNT50) titers are a potential surrogate for immunobridging VE to adults. METHODS: Using PRNT50 calibration datasets, we applied immunobridging approaches using baseline and/or M13 PRNT50 titers to estimate VE against VCD-DENV-AnyM0→M25 and against hospitalized VCD (HVCD)-DENV-AnyM0→M72 in hypothetical 18-45-y-old and 46-50-y-old CYD14 and CYD15 cohorts. RESULTS: Baseline and M13 geometric mean PRNT50 titers were greater in 18-45-y-olds and in 46-50-y-olds vs 9-16-y-olds for most comparisons. Estimated VE (95% CIs against VCD-DENV-AnyM0→M25 ranged from 75.3% to 90.9% (52.5% to 100%) for 18-45-y-olds and 74.8% to 92.0% (53.4% to 100%) for 46-50-y-olds. Estimated VE (95% CIs) against HVCD-DENV-AnyM0→M72 ranged from 58.8% to 78.1% (40.9 to 98.9%) for 18-45-y-olds and 57.2% to 78.4% (40.5 to 97.6%) for 46-50-y-olds. Corresponding predictions among baseline-seropositive individuals yielded comparable or higher VE estimates. CONCLUSIONS: VE M0→M25 against DENV-Any and VE against HVCD-DENV-AnyM0→M72 are both expected to be higher in 18-45 and 46-50-y-olds vs CYD14 and CYD15 9-16-y-olds.