Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01.

A new efficacious tuberculosis vaccine targeting adolescents/adults represents an urgent medical need. The M72/AS01E vaccine candidate protected half of the latently-infected adults against progression to pulmonary tuberculosis in a Phase IIb trial (NCT01755598). We report that three immunizations of mice, two weeks apart, with AS01-adjuvanted M72 induced polyfunctional, Th1-cytokine-expressing M72-specific CD4+/CD8+ T cells in blood and lungs, with the highest frequencies in lungs. Antigen-dose reductions across the three vaccinations skewed pulmonary CD4+ T-cell profiles towards IL-17 expression. In blood, reducing antigen and adjuvant doses of only the third injection (to 1/5th or 1/25th of those of the first injections) did not significantly alter CD4+ T-cell/antibody responses; applying a 10-week delay for the fractional third dose enhanced antibody titers. Delaying a full-dose booster enhanced systemic CD4+ T-cell and antibody responses. Cross-reactivity with PPE and non-PPE proteins was assessed, as Mycobacterium tuberculosis (Mtb) virulence factors and evasion mechanisms are often associated with PE/PPE proteins, to which Mtb39a (contained in M72) belongs. In silico/in vivo analyses revealed that M72/AS01 induced cross-reactive systemic CD4+ T-cell responses to epitopes in a non-vaccine antigen (putative latency-associated Mtb protein PPE24/Rv1753c). These preclinical data describing novel mechanisms of M72/AS01-induced immunity could guide future clinical development of the vaccine.

Subsequent AS01-adjuvanted vaccinations induce similar transcriptional responses in populations with different disease statuses.

Transcriptional responses to adjuvanted vaccines can vary substantially among populations. Interindividual diversity in levels of pathogen exposure, and thus of cell-mediated immunological memory at baseline, may be an important determinant of population differences in vaccine responses. Adjuvant System AS01 is used in licensed or candidate vaccines for several diseases and populations, yet the impact of pre-existing immunity on its adjuvanticity remains to be elucidated. In this exploratory post-hoc analysis of clinical trial samples (clinicalTrials.gov: NCT01424501), we compared gene expression patterns elicited by two immunizations with the candidate tuberculosis (TB) vaccine M72/AS01, between three groups of individuals with different levels of memory responses to TB antigens before vaccination. Analyzed were one group of TB-disease-treated individuals, and two groups of TB-disease-naïve individuals who were (based on purified protein derivative [PPD] skin-test results) stratified into PPD-positive and PPD-negative groups. Although TB-disease-treated individuals displayed slightly stronger transcriptional responses after each vaccine dose, functional gene signatures were overall not distinctly different between groups. Considering the similarities with the signatures found previously for other AS01-adjuvanted vaccines, many features of the response appeared to be adjuvant-driven. Across groups, cell proliferation-related signals at 7 days post-dose 1 were associated with increased anti-M72 antibody response magnitudes. These early signals were stronger in the TB-disease-treated group as compared to both TB-disease-naïve groups. Interindividual homogeneity in gene expression levels was also higher for TB-disease-treated individuals post-dose 1, but increased in all groups post-dose 2 to attain similar levels between the three groups. Altogether, strong cell-mediated memory responses at baseline accelerated and amplified transcriptional responses to a single dose of this AS01-adjuvanted vaccine, resulting in more homogenous gene expression levels among the highly-primed individuals as compared to the disease-naïve individuals. However, after a second vaccination, response heterogeneity decreased and was similar across groups, irrespective of the degree of immune memory acquired at baseline. This information can support the design and analysis of future clinical trials evaluating AS01-adjuvanted vaccines.

Final Analysis of a Trial of M72/AS01E Vaccine to Prevent Tuberculosis.

BACKGROUND: Results of an earlier analysis of a trial of the M72/AS01E candidate vaccine against Mycobacterium tuberculosis showed that in infected adults, the vaccine provided 54.0% protection against active pulmonary tuberculosis disease, without evident safety concerns. We now report the results of the 3-year final analysis of efficacy, safety, and immunogenicity. METHODS: From August 2014 through November 2015, we enrolled adults 18 to 50 years of age with M. tuberculosis infection (defined by positive results on interferon-γ release assay) without evidence of active tuberculosis disease at centers in Kenya, South Africa, and Zambia. Participants were randomly assigned in a 1:1 ratio to receive two doses of either M72/AS01E or placebo, administered 1 month apart. The primary objective was to evaluate the efficacy of M72/AS01E to prevent active pulmonary tuberculosis disease according to the first case definition (bacteriologically confirmed pulmonary tuberculosis not associated with human immunodeficiency virus infection). Participants were followed for 3 years after the second dose. Participants with clinical suspicion of tuberculosis provided sputum samples for polymerase-chain-reaction assay, mycobacterial culture, or both. Humoral and cell-mediated immune responses were evaluated until month 36 in a subgroup of 300 participants. Safety was assessed in all participants who received at least one dose of M72/AS01E or placebo. RESULTS: A total of 3575 participants underwent randomization, of whom 3573 received at least one dose of M72/AS01E or placebo, and 3330 received both planned doses. Among the 3289 participants in the according-to-protocol efficacy cohort, 13 of the 1626 participants in the M72/AS01E group, as compared with 26 of the 1663 participants in the placebo group, had cases of tuberculosis that met the first case definition (incidence, 0.3 vs. 0.6 cases per 100 person-years). The vaccine efficacy at month 36 was 49.7% (90% confidence interval [CI], 12.1 to 71.2; 95% CI, 2.1 to 74.2). Among participants in the M72/AS01E group, the concentrations of M72-specific antibodies and the frequencies of M72-specific CD4+ T cells increased after the first dose and were sustained throughout the follow-up period. Serious adverse events, potential immune-mediated diseases, and deaths occurred with similar frequencies in the two groups. CONCLUSIONS: Among adults infected with M. tuberculosis, vaccination with M72/AS01E elicited an immune response and provided protection against progression to pulmonary tuberculosis disease for at least 3 years. (Funded by GlaxoSmithKline Biologicals and Aeras; ClinicalTrials.gov number, NCT01755598.).

Long-term safety and immunogenicity of the M72/AS01E candidate tuberculosis vaccine in HIV-positive and -negative Indian adults: Results from a phase II randomized controlled trial.

OBJECTIVES: To assess the long-term safety and immunogenicity of the M72/ Adjuvant System (AS01E) candidate tuberculosis (TB) vaccine up to 3 years post-dose 2 (Y3) in human immunodeficiency virus (HIV)-positive (HIV+) and HIV-negative (HIV-) Indian adults. METHODS: This phase II, double-blind, randomised, controlled clinical trial (NCT01262976) was conducted at YRG CARE Medical Centre, in Chennai, India, between January 2011 and June 2015.Three cohorts (HIV+ participants stable on antiretroviral therapy [ART; HIV+ART+], HIV+ ART-naïve [HIV+ART-], and HIV- participants) were randomised (1:1) to receive 2 doses of M72/AS01E (M72/AS01E groups) or saline (control groups) 1 month apart and were followed up toY3. Latent TB infection was assessed at screening using an interferon-gamma (IFN-γ) release assay (IGRA). Safety and immunogenicity results up to Y1 post-vaccination were reported elsewhere. Here, we report serious adverse events (SAEs), humoral and cell-mediated immune (CMI) responses to M72 recorded at Y2 and Y3. RESULTS: Of 240 enrolled and vaccinated participants, 214 completed the long-term follow-up part of the study.In addition to SAEs previously described, between Y1 and Y2 1 M72/AS01E recipient in the HIV+ART+ cohort reported 2 SAEs (sinus cavernous thrombosis and gastroenteritis) that were not considered as causally related to the study vaccine.Vaccination elicited persistent humoral immune responses against M72. At Y3, seropositivity rates were 97.1%, 66.7%, and 97.3% and geometric mean concentrations (GMCs) were 22.0  ELISA units (EU)/mL, 4.9 EU/mL, and 24.3 EU/mL in the HIV+ART+, HIV+ART-, and HIV- cohorts, respectively. Humoral immune response was lowest in the HIV+ART- cohort.In M72/AS01E recipients, no notable decrease in the frequency of M72-specific CD4 T-cells expressing ≥2 immune markers among interleukin-2 (IL-2), IFN-γ, tumour necrosis factor alpha (TNF-α) and CD40 ligand (CD40L) was observed at Y3 post-vaccination. Median values (interquartile range) of 0.35% (0.13-0.49), 0.05% (0.01-0.10), and 0.15% (0.09-0.22) were recorded in the HIV+ART+, HIV+ART- and HIV- cohorts, respectively. CD4 T-cell response was lowest in the HIV+ART- cohort.No CD8 T-cell response was observed. CONCLUSION: The cellular and humoral immune responses induced by M72/AS01E in HIV+ and HIV- adults persisted up to Y3 post-vaccination. No safety concerns were raised regarding administration of M72/AS01E to HIV+ adults. CLINICAL TRIAL REGISTRATION: NCT01262976 (www.clinicaltrials.gov).

Phase 2b Controlled Trial of M72/AS01E Vaccine to Prevent Tuberculosis.

BACKGROUND: A vaccine to interrupt the transmission of tuberculosis is needed. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 2b trial of the M72/AS01E tuberculosis vaccine in Kenya, South Africa, and Zambia. Human immunodeficiency virus (HIV)-negative adults 18 to 50 years of age with latent M. tuberculosis infection (by interferon-γ release assay) were randomly assigned (in a 1:1 ratio) to receive two doses of either M72/AS01E or placebo intramuscularly 1 month apart. Most participants had previously received the bacille Calmette-Guérin vaccine. We assessed the safety of M72/AS01E and its efficacy against progression to bacteriologically confirmed active pulmonary tuberculosis disease. Clinical suspicion of tuberculosis was confirmed with sputum by means of a polymerase-chain-reaction test, mycobacterial culture, or both. RESULTS: We report the primary analysis (conducted after a mean of 2.3 years of follow-up) of the ongoing trial. A total of 1786 participants received M72/AS01E and 1787 received placebo, and 1623 and 1660 participants in the respective groups were included in the according-to-protocol efficacy cohort. A total of 10 participants in the M72/AS01E group met the primary case definition (bacteriologically confirmed active pulmonary tuberculosis, with confirmation before treatment), as compared with 22 participants in the placebo group (incidence, 0.3 cases vs. 0.6 cases per 100 person-years). The vaccine efficacy was 54.0% (90% confidence interval [CI], 13.9 to 75.4; 95% CI, 2.9 to 78.2; P=0.04). Results for the total vaccinated efficacy cohort were similar (vaccine efficacy, 57.0%; 90% CI, 19.9 to 76.9; 95% CI, 9.7 to 79.5; P=0.03). There were more unsolicited reports of adverse events in the M72/AS01E group (67.4%) than in the placebo group (45.4%) within 30 days after injection, with the difference attributed mainly to injection-site reactions and influenza-like symptoms. Serious adverse events, potential immune-mediated diseases, and deaths occurred with similar frequencies in the two groups. CONCLUSIONS: M72/AS01E provided 54.0% protection for M. tuberculosis-infected adults against active pulmonary tuberculosis disease, without evident safety concerns. (Funded by GlaxoSmithKline Biologicals and Aeras; ClinicalTrials.gov number, NCT01755598 .).

Safety and immunogenicity of the M72/AS01E candidate tuberculosis vaccine in adults with tuberculosis: A phase II randomised study.

Previous studies have shown that the M72/AS01E candidate tuberculosis vaccine is immunogenic with a clinically acceptable safety profile in healthy and Mycobacterium tuberculosis-infected adults. This phase II, observer-blind, randomised study compared the safety, reactogenicity, and immunogenicity of M72/AS01E in 3 cohorts: tuberculosis-naïve adults (n = 80), adults previously treated for tuberculosis (n = 49), and adults who have completed the intensive phase of tuberculosis treatment (n = 13). In each cohort, 18-59-year-old adults were randomised (1:1) to receive two doses of M72/AS01E (n = 71) or placebo (n = 71) and followed-up until six months post-dose 2. Safety and reactogenicity were assessed as primary objective. Recruitment in the study ended prematurely because of a high incidence of large injection site redness/swelling reactions in M72/AS01E-vaccinated adults undergoing tuberculosis treatment. No additional clinically relevant adverse events were observed, except one possibly vaccine-related serious adverse event (hypersensitivity in a tuberculosis-treated-M72/AS01E participant). Robust and persistent M72-specific humoral and polyfunctional CD4(+) T-cell-mediated immune responses were observed post-M72/AS01E vaccination in each cohort. In conclusion, the M72/AS01E vaccine was immunogenic in adults previously or currently treated for tuberculosis, but further analyses are needed to explain the high local reactogenicity in adults undergoing tuberculosis treatment. ClinicalTrials.gov: NCT01424501.

A Randomized, Controlled Safety, and Immunogenicity Trial of the M72/AS01 Candidate Tuberculosis Vaccine in HIV-Positive Indian Adults.

Human immunodeficiency virus (HIV)-associated tuberculosis is a major public health threat. We evaluated the safety and immunogenicity of the candidate tuberculosis vaccine M72/AS01 in HIV-positive and HIV-negative Indian adults.Randomized, controlled observer-blind trial (NCT01262976).We assigned 240 adults (1:1:1) to antiretroviral therapy (ART)-stable, ART-naive, or HIV-negative cohorts. Cohorts were randomized 1:1 to receive M72/AS01 or placebo following a 0, 1-month schedule and followed for 12 months (time-point M13). HIV-specific and laboratory safety parameters, adverse events (AEs), and M72-specific T-cell-mediated and humoral responses were evaluated.Subjects were predominantly QuantiFERON-negative (60%) and Bacille Calmette-Guérin-vaccinated (73%). Seventy ART-stable, 73 ART-naive, and 60 HIV-negative subjects completed year 1. No vaccine-related serious AEs or ART-regimen adjustments, or clinically relevant effects on laboratory parameters, HIV-1 viral loads or CD4 counts were recorded. Two ART-naive vaccinees died of vaccine-unrelated diseases. M72/AS01 induced polyfunctional M72-specific CD4 T-cell responses (median [interquartile range] at 7 days postdose 2: ART-stable, 0.9% [0.7-1.5]; ART-naive, 0.5% [0.2-1.0]; and HIV-negative, 0.6% [0.4-1.1]), persisting at M13 (0.4% [0.2-0.5], 0.09% [0.04-0.2], and 0.1% [0.09-0.2], respectively). Median responses were higher in the ART-stable cohort versus ART-naive cohort from day 30 onwards (P ≤ 0.015). Among HIV-positive subjects (irrespective of ART-status), median responses were higher in QuantiFERON-positive versus QuantiFERON-negative subjects up to day 30 (P ≤ 0.040), but comparable thereafter. Cytokine-expression profiles were comparable between cohorts after dose 2. At M13, M72-specific IgG responses were higher in ART-stable and HIV-negative vaccinees versus ART-naive vaccinees (P ≤ 0.001).M72/AS01 was well-tolerated and immunogenic in this population of ART-stable and ART-naive HIV-positive adults and HIV-negative adults, supporting further clinical evaluation.

Safety and immunogenicity of candidate vaccine M72/AS01E in adolescents in a TB endemic setting.

BACKGROUND: Vaccination that prevents tuberculosis (TB) disease, particularly in adolescents, would have the greatest impact on the global TB epidemic. Safety, reactogenicity and immunogenicity of the vaccine candidate M72/AS01E was evaluated in healthy, HIV-negative adolescents in a TB endemic region, regardless of Mycobacterium tuberculosis (M.tb) infection status. METHODS: In a phase II, double-blind randomized, controlled study (NCT00950612), two doses of M72/AS01E or placebo were administered intramuscularly, one month apart. Participants were followed-up post-vaccination, for 6 months. M72-specific immunogenicity was evaluated by intracellular cytokine staining analysis of T cells and NK cells by flow cytometry. RESULTS: No serious adverse events were recorded. M72/AS01E induced robust T cell and antibody responses, including antigen-dependent NK cell IFN-γ production. CD4 and CD8 T cell responses were sustained at 6 months post vaccination. Irrespective of M.tb infection status, vaccination induced a high frequency of M72-specific CD4 T cells expressing multiple combinations of Th1 cytokines, and low level IL-17. We observed rapid boosting of immune responses in M.tb-infected participants, suggesting natural infection acts as a prime to vaccination. CONCLUSIONS: The clinically acceptable safety and immunogenicity profile of M72/AS01E in adolescents living in an area with high TB burden support the move to efficacy trials.

Safety and immunogenicity of the M72/AS01 candidate tuberculosis vaccine when given as a booster to BCG in Gambian infants: an open-label randomized controlled trial.

UNLABELLED: We evaluated the candidate tuberculosis vaccine M72/AS01 in Bacille-Calmette-Guérin (BCG)-vaccinated infants after or concomitantly with Expanded-Programme-on-Immunization (EPI) vaccines. METHODS: In a Phase-II study in The Gambia (NCT01098474), 2 cohorts of 150 BCG-vaccinated infants each were randomized 1:1:1. The 'Outside-EPI' cohort received one or two M72/AS01 doses, or meningitis vaccine, 1-2 months after primary EPI vaccination. The 'Within-EPI' cohort received one or two M72/AS01 doses concomitantly with the third or last two doses of their primary EPI-regimen, respectively, or EPI vaccines alone. Safety, M72-specific humoral (ELISA) and cell-mediated (whole-blood ICS) responses, and humoral responses to EPI vaccines were assessed. RESULTS: M72/AS01 was acceptably tolerated with no vaccine-related serious adverse events reported. Seropositivity/seroprotection rates against EPI antigens in the Within-EPI cohort were comparable between groups, irrespective of M72/AS01 co-administration. Up to one year post M72/AS01 vaccination, M72-specific humoral and CD4(+) T-cell responses were higher after 2 doses versus 1 dose in both cohorts (p < 0.0001), and comparable between cohorts after either 1 or 2 doses (p > 0.05). CONCLUSION: M72/AS01 given to infants after or concomitantly with EPI vaccines had an acceptable safety profile. Our results suggest no interference of immunogenicity profiles occurred following co-administration of M72/AS01 and EPI vaccines. Two M72/AS01 doses elicited higher immune responses than one dose.

Safety and immunogenicity of the M72/AS01 candidate tuberculosis vaccine in HIV-infected adults on combination antiretroviral therapy: a phase I/II, randomized trial.

OBJECTIVE: Tuberculosis (TB) is highly prevalent among HIV-infected people, including those receiving combination antiretroviral therapy (cART), necessitating a well tolerated and efficacious TB vaccine for these populations. We evaluated the safety and immunogenicity of the candidate TB vaccine M72/AS01 in adults with well controlled HIV infection on cART. DESIGN: A randomized, observer-blind, controlled trial (NCT00707967). METHODS: HIV-infected adults on cART in Switzerland were randomized 3 : 1 : 1 to receive two doses, 1 month apart, of M72/AS01, AS01 or 0.9% physiological saline (N = 22, N = 8 and N = 7, respectively) and were followed up to 6 months postdose 2 (D210). Individuals with CD4⁺ cell counts below 200 cells/µl were excluded. Adverse events (AEs) including HIV-specific and laboratory safety parameters were recorded. Cell-mediated (ICS) and humoral (ELISA) responses were evaluated before vaccination, 1 month after each dose (D30, D60) and D210. RESULTS: Thirty-seven individuals [interquartile range (IQR) CD4⁺ cell counts at screening: 438-872 cells/µl; undetectable HIV-1 viremia] were enrolled; 73% of individuals reported previous BCG vaccination, 97.3% tested negative for the QuantiFERON-TB assay. For M72/AS01 recipients, no vaccine-related serious AEs or cART-regimen adjustments were recorded, and there were no clinically relevant effects on laboratory safety parameters, HIV-1 viral loads or CD4⁺ cell counts. M72/AS01 was immunogenic, inducing persistent and polyfunctional M72-specific CD4⁺ T-cell responses [medians 0.70% (IQR 0.37-1.07) at D60] and 0.42% (0.24-0.61) at D210, predominantly CD40L⁺IL-2⁺TNF-α⁺, CD40L⁺IL-2⁺ and CD40L⁺IL-2⁺TNF-α⁺IFN-γ⁺]. All M72/AS01 vaccines were seropositive for anti-M72 IgG after second vaccination until study end. CONCLUSION: M72/AS01 was clinically well tolerated and immunogenic in this population, supporting further clinical evaluation in HIV-infected individuals in TB-endemic settings.

A randomized, controlled dose-finding Phase II study of the M72/AS01 candidate tuberculosis vaccine in healthy PPD-positive adults.

PURPOSE: In this dose-finding Phase II study (NCT00621322), we evaluated the safety and immunogenicity of different formulations of the candidate tuberculosis vaccine containing the M72 antigen (10/20/40 µg doses) and the liposome-based AS01 Adjuvant System. We aimed to select the lowest-dose combination of M72 and AS01 that was clinically well tolerated with immunogenicity comparable to that of the previously tested M72/AS01B (40 µg) candidate vaccine. METHODS: Healthy PPD-positive (induration 3-10 mm) adults (18-45 years) in The Philippines were randomized (4:4:4:4:1:1) to receive 2 injections, 1 month apart, of M72/AS01B (40 µg), M72/AS01E (10 µg), M72/AS01E (20 µg), M72/AS02D (10 µg), M72/Saline (40 µg) or AS01B alone, and were followed up for 6 months. AS01E and AS02D contain half the quantities of the immunostimulants present in AS01B. AS02D is an oil-in-water emulsion. Vaccine selection was based on the CD4(+) T-cell responses at 1 month post vaccination. RESULTS: All formulations had a clinically acceptable safety profile with no vaccine-related serious adverse events reported. Two vaccinations of each adjuvanted M72 vaccine induced M72-specific CD4(+) T-cell and humoral responses persisting at 6 months post vaccination. No responses were observed with AS01B alone. One month post second vaccination, CD4(+) T-cell responses induced by each of the three M72/AS01 vaccine formulations were of comparable magnitudes, and all were significantly higher than those induced by M72/AS02D (10 µg) and M72/Saline. CONCLUSIONS: The formulation with the lowest antigen and adjuvant dose, M72/AS01E (10 µg), fulfilled our pre-defined selection criteria and has been selected for further clinical development.

Induction and regulation of T-cell immunity by the novel tuberculosis vaccine M72/AS01 in South African adults.

RATIONALE: Tuberculosis (TB) is a major cause of morbidity and mortality worldwide, thus there is an urgent need for novel TB vaccines. OBJECTIVES: We investigated a novel TB vaccine candidate, M72/AS01, in a phase IIa trial of bacille Calmette-Guérin-vaccinated, HIV-uninfected, and Mycobacterium tuberculosis (Mtb)-infected and -uninfected adults in South Africa. METHODS: Two doses of M72/AS01 were administered to healthy adults, with and without latent Mtb infection. Participants were monitored for 7 months after the first dose; cytokine production profiles, cell cycling, and regulatory phenotypes of vaccine-induced T cells were measured by flow cytometry. MEASUREMENTS AND MAIN RESULTS: The vaccine had a clinically acceptable safety profile, and induced robust, long-lived M72-specific T-cell and antibody responses. M72-specific CD4 T cells produced multiple combinations of Th1 cytokines. Analysis of T-cell Ki67 expression showed that most vaccination-induced T cells did not express Th1 cytokines or IL-17; these cytokine-negative Ki67(+) T cells included subsets of CD4 T cells with regulatory phenotypes. PD-1, a negative regulator of activated T cells, was transiently expressed on M72-specific CD4 T cells after vaccination. Specific T-cell subsets were present at significantly higher frequencies after vaccination of Mtb-infected versus -uninfected participants. CONCLUSIONS: M72/AS01 is clinically well tolerated in Mtb-infected and -uninfected adults, induces high frequencies of multifunctional T cells, and boosts distinct T-cell responses primed by natural Mtb infection. Moreover, these results provide important novel insights into how this immunity may be appropriately regulated after novel TB vaccination of Mtb-infected and -uninfected individuals.Clinical trial registered with www.clinicaltrials.gov (NCT 00600782).

Improved CD4⁺ T cell responses to Mycobacterium tuberculosis in PPD-negative adults by M72/AS01 as compared to the M72/AS02 and Mtb72F/AS02 tuberculosis candidate vaccine formulations: a randomized trial.

BACKGROUND: The Bacille Calmette-Guérin (BCG) tuberculosis (TB) vaccine provides incomplete protection, necessitating development of an effective vaccine against TB disease. The Mtb72F/AS02 candidate vaccine was previously shown to be clinically well tolerated and immunogenic in Purified Protein Derivative (PPD)-negative adults. To improve the stability of Mtb72F, a point mutation was introduced into a putative serine protease site to give the final M72 construct. AS01 is an Adjuvant System that can potentially improve both humoral and cellular immune responses compared to the AS02 Adjuvant System or unadjuvanted vaccine. This study evaluated the safety and immunogenicity in Mtb-naïve adults of vaccines containing 40 µg of the M72 antigen with AS02 or AS01 and compared the results with Mtb72F/AS02 vaccine (40 µg dose), M72 in saline (40 µg dose) and AS01 alone. METHODS: In this Phase I/II observer-blind controlled trial, 110 participants were randomized (4:4:1:1:1) to receive M72/AS01, M72/AS02, Mtb72F/AS02, M72/saline or AS01, following a 0, 1-month schedule. Subjects receiving the adjuvanted M72 vaccines were followed up until 3 years post vaccination. Evaluation of the immune response and safety/reactogenicity was performed. RESULTS: For all vaccines, solicited adverse events (AEs) were predominantly mild to moderate and transient. No vaccine-related serious AEs occurred and no subject withdrew due to an AE. Immune responses induced by Mtb72F and M72 antigens combined with AS02 were similar. M72/AS01 and M72/AS02 induced robust polyfunctional M72-specific CD4(+) T cell and antibody responses persisting at 3 years, with the highest CD4(+) T cell responses found with M72/AS01. CONCLUSION: This first clinical study with M72/AS01 and M72/AS02 showed that both vaccines were clinically well tolerated and induced high magnitude and persistent cell-mediated and humoral immune responses. The Mtb72F/AS02 and M72/AS02 vaccines were comparably immunogenic with significantly higher immune responses compared to the M72/saline control. Of the formulations tested, M72/AS01 demonstrated significantly higher vaccine specific Th1 CD4(+) T cell responses supporting its further clinical evaluation.

The candidate tuberculosis vaccine Mtb72F/AS02 in PPD positive adults: a randomized controlled phase I/II study.

UNLABELLED: Prevention of tuberculosis (TB) through vaccination would substantially reduce the global TB burden. Mtb72F/AS02 is a candidate TB vaccine shown to be immunogenic and well tolerated in PPD-negative adults. We evaluated the safety and immunogenicity of Mtb72F/AS02 in Mycobacterium-primed adults (BCG-vaccinated, or infected adults who had received post-exposure chemoprophylaxis or treatment for pulmonary TB disease). In this observer-blind controlled trial, 20 BCG-vaccinated adults and 18 adults previously infected with Mycobacterium tuberculosis (Mtb), were randomized 3:1 to receive three doses of Mtb72F/AS02 or AS02 at one-month intervals, and followed for 6 months post third vaccination. Mtb72F/AS02 was well tolerated in BCG-vaccinated adults, and tended to be more reactogenic in Mtb-infected adults. Adverse events were mainly self-limiting, resolving without sequelae. No serious adverse events were reported. The adverse events in Mtb72F/AS02 vaccinees were not clearly associated with vaccine-induced responses (as assessed by proinflammatory cytokines, total IgE and C-reactive protein levels). No Th2 T-cell responses, or vaccine-induced T-cell responses to Mtb antigens (CFP-10/PPD/ESAT-6) were detected by ICS. In both cohorts, Mtb72F/AS02 induced persistent polyfunctional Mtb72F-specific CD4(+) T-cell responses and anti-Mtb72F humoral responses. IFN-γ was detectable in serum one day post each vaccination. Further evaluation of the candidate vaccine, Mtb72F/AS02, is warranted. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00146744.

Validation of an enzyme-linked immunosorbent assay for the quantification of human IgG directed against the repeat region of the circumsporozoite protein of the parasite Plasmodium falciparum.

BACKGROUND: Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines. METHODS: The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum. RESULTS: The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time. CONCLUSIONS: This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine.

Induction of strong HIV-1-specific CD4+ T-cell responses using an HIV-1 gp120/NefTat vaccine adjuvanted with AS02A in antiretroviral-treated HIV-1-infected individuals.

BACKGROUND: Induction of HIV-1-specific CD4(+) T-cell responses by therapeutic vaccination represents an attractive intervention to potentially increase immune control of HIV-1. METHODS: We performed a double-blinded, randomized, placebo-controlled clinical trial to determine the safety and immunogenicity of GlaxoSmithKline Biologicals' HIV-1 gp120/NefTat subunit protein vaccine formulated with the AS02(A) Adjuvant System in subjects with well-controlled chronic HIV-1 infection on highly active antiretroviral therapy. Ten individuals received the vaccine; whereas adjuvant alone or placebo was given to 5 subjects each. Immunogenicity was monitored by intracellular cytokine flow cytometry and carboxyfluorescein succinimidyl ester-based proliferation assays. RESULTS: The vaccine was well tolerated with no related serious adverse events. Vaccine recipients had significantly stronger gp120-specific CD4(+) T-cell responses which persisted until week 48 and greater gp120-specific CD4(+) T-cell proliferation activity as compared with controls. In the vaccine group, the number of participants who demonstrated positive responses for both gp120-specific CD4(+) T-cell interleukin-2 production and gp120-specific CD8(+) T-cell proliferation were significantly higher at week 6. CONCLUSIONS: The gp120/NefTat/AS02(A) vaccine induced strong gp120-specific CD4(+) T-cell responses and a higher number of vaccinees developed both HIV-1-specific CD4(+) T-cell responses and CD8(+) T-cell proliferation. The induction of these responses may be important in enhancing immune-mediated viral control.

Induction of Plasmodium falciparum-specific CD4+ T cells and memory B cells in Gabonese children vaccinated with RTS,S/AS01(E) and RTS,S/AS02(D).

UNLABELLED: The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01(E) and RTS,S/AS02(D). Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2(+) CD4(+) T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01(E) and RTS,S/AS02(D) induced adaptive immune responses including antibodies, circulating memory B cells and CD4(+) T cells directed against P. falciparum CS protein. TRIAL REGISTRATION: ClinicalTrials.gov NCT00307021.

Four year immunogenicity of the RTS,S/AS02(A) malaria vaccine in Mozambican children during a phase IIb trial.

Previous studies with the malaria vaccine RTS,S/AS02(A) in young children in a malaria endemic area of Mozambique have shown it to have a promising safety profile and to reduce the risk of Plasmodium falciparum infection and disease. In this study, we assessed the antibody responses to the P. falciparum and hepatitis B components of the RTS,S/AS02(A) vaccine over a 45 months surveillance period in a large phase IIb trial which included 2022 children aged 1-4 years at recruitment. The RTS,S/AS02(A) vaccine induced high anti-circumsporozoite antibody levels with at least 96% of children remaining seropositive during the entire follow-up period. IgG titers decayed over the first 6 months of follow-up to about 25% of the initial level, but still remained 30-fold higher until month 45 compared to controls. Children with higher levels of naturally acquired immunity at baseline, assessed by blood stage indirect fluorescent antibody test, had slightly higher anti-circumsporozoite levels, after adjusting for the effect of age. The RTS,S/AS02(A) vaccine also induced high levels of anti-hepatitis B surface antigen antibodies (seroprotection >97%). RTS,S/AS02(A) vaccine is immunogenic and induces long-lasting anti-circumsporozoite antibodies, persisting at least 42 months after immunization. These antibodies may play a role in protection against malaria.

An adjuvanted polyprotein HIV-1 vaccine induces polyfunctional cross-reactive CD4+ T cell responses in seronegative volunteers.

BACKGROUND: This phase I/II partially blinded, randomized, dose-ranging study assessed the safety and immunogenicity of a novel human immunodeficiency virus type 1 (HIV-1) vaccine candidate consisting of a recombinant fusion protein (F4) containing 4 HIV-1 clade B antigens (Gag p24, Pol reverse transcriptase, Nef, and Gag p17) adjuvanted with AS01 in HIV-seronegative volunteers. Methods. Two doses of the recombinant F4 protein (10, 30, or 90 µg/dose), adjuvanted with AS01 or reconstituted with water for injection, were administered 1 month apart to 180 healthy volunteers aged 18-40 years. F4-specific CD4(+) T cell responses were measured using intracellular cytokine staining after in vitro stimulation by overlapping peptide pools covering the 4 individual antigens. Results. Reactogenicity was higher during the 7-day period after each vaccine dose in the adjuvanted than in the nonadjuvanted groups. In the adjuvanted groups, the overall immune response rate was high after the second vaccine dose, with highest responder rates seen in the 10-µg F4/AS01 group (100% to 3 HIV-1 antigens and 80% to all 4 HIV-1 antigens). High and long-lasting CD4(+) T cell frequencies were observed (up to a median value of 1.2% F4-specific CD4(+) T cells at day 44), with strongest responses directed against reverse transcriptase. Antigen-specific CD4(+) T cells exhibited a polyfunctional phenotype, expressing at least CD40 ligand and interleukin 2, often in combination with tumor necrosis factor α and/or interferon γ. Vaccine-induced CD4(+) T cell responses were broadly cross-reactive to all 4 antigens derived from HIV-1 clades A and C. Conclusions. These results support further clinical investigation of this HIV-1 vaccine candidate both in a prophylactic setting (alone, in conjunction with an envelope-based antigen or in combination with other vaccine approaches in a heterologous prime-boost regimen) and as a potentially disease-modifying therapeutic vaccine in HIV-1-infected subjects. CLINICAL TRIALS REGISTRATION: NCT00434512.

Evaluation of the safety and immunogenicity of two antigen concentrations of the Mtb72F/AS02(A) candidate tuberculosis vaccine in purified protein derivative-negative adults.

Tuberculosis (TB) remains a major cause of illness and death worldwide, making a new TB vaccine an urgent public health priority. Purified protein derivative (PPD)-negative adults (n = 50) were equally randomized to receive 3 doses at 1-month intervals (at 0, 1, and 2 months) of one of the following vaccines: Mtb72F/AS02(A) (10 or 40 µg antigen), Mtb72F/saline (10 or 40 µg antigen), or AS02(A). Mtb72F/AS02(A) recipients received an additional dose 1 year after the first dose to evaluate if the elicited immune response could be boosted. Mtb72F/AS02(A) vaccines were locally reactogenic but clinically well tolerated, with transient adverse events (usually lasting between 1 and 4 days) that resolved without sequelae being observed. No vaccine-related serious adverse events were reported. Vaccination with Mtb72F/AS02(A) induced a strong Mtb72F-specific humoral response and a robust Mtb72F-specific CD4(+) T-cell response, both of which persisted at 9 months after primary immunization and for 1 year after the booster immunization. There was no significant difference between the magnitude of the CD4(+) T-cell response induced by the 10-µg and 40-µg Mtb72F/AS02(A) vaccines. The Mtb72F-specific CD4(+) T cells predominantly expressed CD40L; CD40L and interleukin-2 (IL-2); CD40L and tumor necrosis factor alpha (TNF-α); CD40L, IL-2, and TNF-α; and CD40L, IL-2, TNF-α, and gamma interferon (IFN-γ). Serum IFN-γ, but not TNF-α, was detected 1 day after doses 2 and 3 for the Mtb72F/AS02(A) vaccine but did not persist. Vaccine-induced CD8(+) T-cell responses were not detected, and no immune responses were elicited with AS02(A) alone. In conclusion, Mtb72F/AS02(A) is clinically well tolerated and is highly immunogenic in TB-naïve adults. The 10- and 40-µg Mtb72F/AS02(A) vaccines show comparable safety and immunogenicity profiles.