Safety and Immunogenicity of a ChAd155-Vectored Respiratory Syncytial Virus Vaccine in Infants 6-7 Months of age: A Phase 1/2 Randomized Trial.

BACKGROUND: Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants. This phase 1/2, observer-blind, randomized, controlled study assessed the safety and immunogenicity of an investigational chimpanzee-derived adenoviral vector RSV vaccine (ChAd155-RSV, expressing RSV F, N, and M2-1) in infants. METHODS: Healthy 6- to 7-month-olds were 1:1:1-randomized to receive 1 low ChAd155-RSV dose (1.5 × 1010 viral particles) followed by placebo (RSV_1D); 2 high ChAd155-RSV doses (5 × 1010 viral particles) (RSV_2D); or active comparator vaccines/placebo (comparator) on days 1 and 31. Follow-up lasted approximately 2 years. RESULTS: Two hundred one infants were vaccinated (RSV_1D: 65; RSV_2D: 71; comparator: 65); 159 were RSV-seronaive at baseline. Most solicited and unsolicited adverse events after ChAd155-RSV occurred at similar or lower rates than after active comparators. In infants who developed RSV infection, there was no evidence of vaccine-associated enhanced respiratory disease (VAERD). RSV-A neutralizing titers and RSV F-binding antibody concentrations were higher post-ChAd155-RSV than postcomparator at days 31, 61, and end of RSV season 1 (mean follow-up, 7 months). High-dose ChAd155-RSV induced stronger responses than low-dose, with further increases post-dose 2. CONCLUSIONS: ChAd155-RSV administered to 6- to 7-month-olds had a reactogenicity/safety profile like other childhood vaccines, showed no evidence of VAERD, and induced a humoral immune response. Clinical Trials Registration. NCT03636906.

Assessment of Mumps Virus-Specific Antibodies: Comparison of Plaque Reduction Neutralization Test and Enzyme-Linked Immunosorbent Assay Estimates.

BACKGROUND: The plaque reduction neutralization test (PRNT), which measures a subset of immunoglobulin antibodies (functional neutralizing antibodies), and the enzyme-linked immunosorbent assay (ELISA), which measures total immunoglobulin (neutralizing and nonneutralizing antibodies), characterize different aspects of the anti-mumps virus antibody response after vaccination. METHODS: Data from a recent phase 3 clinical trial (NCT01681992) of 2 measles-mumps-rubella vaccines were used to compare anti-mumps antibody responses measured using an unenhanced PRNT (GSK; seropositivity cutoff and threshold, 2.5 and 4 times the 50% end-point dilution, respectively) with those estimated using an ELISA (thresholds, 5 and 10 ELISA units/mL, respectively). RESULTS: Of 3990 initially seronegative samples, 3284 (82.3%) were seropositive after vaccination for anti-mumps antibodies in both assays. The Pearson correlation coefficient for double-positive samples was 0.57, indicative of a moderate correlation. Receiver operating characteristic curve analysis showed that an ELISA threshold of 51.7 ELISA units/mL best corresponded to the PRNT seroresponse threshold. There was no obvious vaccine brand effect on the correlation between assays. CONCLUSIONS: The moderate correlation between the anti-mumps antibody measurements obtained with PRNT and ELISA reflects different aspects of the serological response. In the absence of a well-defined protective serological threshold, PRNT provides complementary information on the antibody response, whereas ELISA remains a critically useful measurement of vaccine immunogenicity.

Sustained Immunogenicity of 2-dose Human Papillomavirus 16/18 AS04-adjuvanted Vaccine Schedules in Girls Aged 9-14 Years: A Randomized Trial.

Background: We previously reported the noninferiority 1 month after the last dose of 2-dose human papillomavirus 16/18 AS04-adjuvanted (AS04-HPV-16/18) vaccine schedules at months 0 and 6 (2D_M0,6) and months 0 and 12 (2D_M0,12) in girls aged 9-14 years compared with a 3-dose schedule at months 0, 1, and 6 (3D_M0,1,6) in women aged 15-25 years. Here, we report the results at study end (month 36 [M36]). Methods: Girls were randomized 1:1 and received 2 vaccine doses either 6 months (2D_M0,6) or 12 months apart (2D_M0,12); women received 3 doses at months 0, 1, and 6 (3D_M0,1,6). Endpoints included noninferiority of HPV-16/18 antibodies for 2D_M0,6 versus 3D_M0,1,6; 2D_M0,12 versus 3D_M0,1,6; and 2D_M0,12 versus 2D_M0,6; and assessment of neutralizing antibodies, T cells, B cells, and safety. Results: At M36, the 2D_M0,6 and 2D_M0,12 schedules remained noninferior to the 3D_M0,1,6 schedule in terms of seroconversion rates and 3D/2D geometric mean titers for anti-HPV-16 and anti-HPV-18. All schedules elicited sustained immune responses up to M36. Conclusions: Both 2-dose schedules in young girls remained noninferior to the 3-dose schedule in women up to study conclusion at M36. The AS04-HPV-16/18 vaccine administered as a 2-dose schedule was immunogenic and well tolerated in young girls.

Immunogenicity and Safety of an AS03-Adjuvanted H7N9 Pandemic Influenza Vaccine in a Randomized Trial in Healthy Adults.

BACKGROUND: Almost 700 cases of human infection with avian influenza A/H7N9 have been reported since 2013. Pandemic preparedness strategies include H7N9 vaccine development. METHODS: We evaluated an inactivated H7N9 vaccine in an observer-blind study in healthy adults aged 18-64 years. Participants (420) were randomized to receive 1 of 4 AS03-adjuvanted vaccines (low or medium dose of hemagglutinin with AS03A or AS03B), one nonadjuvanted vaccine, or placebo. The coprimary immunogenicity objective determined whether adjuvanted vaccines elicited an immune response against the vaccine-homologous virus, 21 days after the second vaccine dose per US and European licensure criteria in the per-protocol cohort (n = 389). RESULTS: All adjuvanted vaccines met regulatory acceptance criteria. In groups receiving adjuvanted formulations, seroconversion rates were ≥85.7%, seroprotection rates ≥91.1%, and geometric mean titers ≥92.9% versus 23.2%, 28.6%, and 17.2 for the nonadjuvanted vaccine. The AS03 adjuvant enhanced immune response at antigen-sparing doses. Injection site pain occurred more frequently with adjuvanted vaccines (in ≤98.3% of vaccinees) than with the nonadjuvanted vaccine (40.7%) or placebo (20.0%). None of the 20 serious adverse events reported were related to vaccination. CONCLUSIONS: Two doses of AS03-adjuvanted H7N9 vaccine were well tolerated and induced a robust antibody response at antigen-sparing doses in healthy adults. CLINICAL TRIALS REGISTRATION: NCT01999842.

Randomized Open Trial Comparing 2-Dose Regimens of the Human Papillomavirus 16/18 AS04-Adjuvanted Vaccine in Girls Aged 9-14 Years Versus a 3-Dose Regimen in Women Aged 15-25 Years.

BACKGROUND: This randomized, open trial compared regimens including 2 doses (2D) of human papillomavirus (HPV) 16/18 AS04-adjuvanted vaccine in girls aged 9-14 years with one including 3 doses (3D) in women aged 15-25 years. METHODS: Girls aged 9-14 years were randomized to receive 2D at months 0 and 6 (M0,6; (n = 550) or months 0 and 12 (M0,12; n = 415), and women aged 15-25 years received 3D at months 0, 1, and 6 (n = 482). End points included noninferiority of HPV-16/18 antibodies by enzyme-linked immunosorbent assay for 2D (M0,6) versus 3D (primary), 2D (M0,12) versus 3D, and 2D (M0,6) versus 2D (M0,12); neutralizing antibodies; cell-mediated immunity; reactogenicity; and safety. Limits of noninferiority were predefined as <5% difference in seroconversion rate and <2-fold difference in geometric mean antibody titer ratio. RESULTS: One month after the last dose, both 2D regimens in girls aged 9-14 years were noninferior to 3D in women aged 15-25 years and 2D (M0,12) was noninferior to 2D (M0,6). Geometric mean antibody titer ratios (3D/2D) for HPV-16 and HPV-18 were 1.09 (95% confidence interval, .97-1.22) and 0.85 (.76-.95) for 2D (M0,6) versus 3D and 0.89 (.79-1.01) and 0.75 (.67-.85) for 2D (M0,12) versus 3D. The safety profile was clinically acceptable in all groups. CONCLUSIONS: The 2D regimens for the HPV-16/18 AS04-adjuvanted vaccine in girls aged 9-14 years (M0,6 or M0,12) elicited HPV-16/18 immune responses that were noninferior to 3D in women aged 15-25 years. CLINICAL TRIALS REGISTRATION: NCT01381575.

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

Association Between Hemagglutination Inhibition Antibody Titers and Protection Against Reverse-Transcription Polymerase Chain Reaction-Confirmed Influenza Illness in Children 6-35 Months of Age: Statistical Evaluation of a Correlate of Protection.

BACKGROUND: Data from a randomized controlled efficacy trial of an inactivated quadrivalent influenza vaccine in children 6-35 months of age were used to determine whether hemagglutination inhibition (HI) antibody titer against A/H1N1 and A/H3N2 is a statistical correlate of protection (CoP) for the risk of reverse-transcription polymerase chain reaction (RT-PCR)-confirmed influenza associated with the corresponding strain. METHODS: The Prentice criteria were used to statistically validate strain-specific HI antibody titer as a CoP. The probability of protection was identified using the Dunning model corresponding to a prespecified probability of protection at an individual level. The group-level protective threshold was identified using the Siber approach, leading to unbiased predicted vaccine efficacy (VE). A case-cohort subsample was used for this exploratory analysis. RESULTS: Prentice criteria confirmed that HI titer is a statistical CoP for RT-PCR-confirmed influenza. The Dunning model predicted a probability of protection of 49.7% against A/H1N1 influenza and 54.7% against A/H3N2 influenza at an HI antibody titer of 1:40 for the corresponding strain. Higher titers of 1:320 were associated with >80% probability of protection. The Siber method predicted VE of 61.0% at a threshold of 1:80 for A/H1N1 and 46.6% at 1:113 for A/H3N2. CONCLUSIONS: The study validated HI antibody titer as a statistical CoP, by demonstrating that HI titer is correlated with clinical protection against RT-PCR-confirmed influenza associated with the corresponding influenza strain and is predictive of VE in children 6-35 months of age. CLINICAL TRIALS REGISTRATION: NCT01439360.

Reactogenicity, safety, and immunogenicity of chimeric haemagglutinin influenza split-virion vaccines, adjuvanted with AS01 or AS03 or non-adjuvanted: a phase 1-2 randomised controlled trial.

BACKGROUND: One strategy to develop a universal influenza virus vaccine is to redirect the immune system to the highly conserved haemagglutinin stalk domain by sequentially administering vaccines expressing chimeric (c) haemagglutinins with a conserved stalk domain and divergent head domain, to which humans are naive. We aimed to assess the reactogenicity, safety, and immunogenicity of adjuvanted and unadjuvanted investigational supra-seasonal universal influenza virus vaccines (SUIVs) in healthy young adults. METHODS: In this observer-masked, randomised, controlled, phase 1-2 trial, we recruited adults aged 18-39 years with no clinically significant conditions from six centres in Belgium and the USA. Participants were randomly assigned to ten equally sized groups via an online system with the MATerial Excellence programme. Vaccines contained heterosubtypic group 1 H8, H5, or H11 haemagglutinin heads, an H1 haemagglutinin stalk, and an N1 neuraminidase (cH8/1N1, cH5/1N1, and cH11/1N1; haemagglutinin dose 15 µg/0·5 mL), administered on days 1 and 57, with a month 14 booster. SUIVs were evaluated in the sequences: cH8/1N1-placebo-cH5/1N1, cH5/1N1-placebo-cH8/1N1, or cH8/1N1-cH5/1N1-cH11/1N1, adjuvanted with either AS03 or AS01, or not adjuvanted. The last group received inactivated quadrivalent influenza vaccine (IIV4)-placebo-IIV4. Primary outcomes were safety (analysed in the exposed population) and immunogenicity in terms of the anti-H1 stalk humoral response at 28 days after vaccination (analysed in the per-protocol population, defined as participants who received the study vaccines according to the protocol). This trial is registered with ClinicalTrials.gov, NCT03275389. FINDINGS: Between Sept 25, 2017, and March 26, 2020, 507 eligible participants were enrolled. 468 (92%) participants received at least one dose of study vaccine (exposed population), of whom 244 (52%) were included in the per-protocol population at final analysis at month 26. The safety profiles of all chimeric vaccines were clinically acceptable, with no safety concerns identified. Injection-site pain was the most common adverse event, occurring in 84-96% of participants receiving an adjuvanted SUIV or non-adjuvanted IIV4 and in 40-50% of participants receiving a non-adjuvanted SUIV. Spontaneously reported adverse events up to 28 days after vaccination occurred in 36-60% of participants, with no trends observed for any group. 17 participants had a serious adverse event, none of which were considered to be causally related to the vaccine. Anti-H1 stalk antibody titres were highest in AS03-adjuvanted groups, followed by AS01-adjuvanted and non-adjuvanted groups, and were higher after cH8/1N1 than after cH5/1N1 and after a two-dose primary schedule than after a one-dose schedule. Geometric mean concentrations by ELISA ranged from 21 938·1 ELISA units/mL (95% CI 18 037·8-26 681·8) in the IIV4-placebo-IIV4 group to 116 596·8 ELISA units/mL (93 869·6-144 826·6) in the AS03-adjuvanted cH8/1N1-cH5/1N1-cH11/1N1 group 28 days after the first dose and from 15 105·9 ELISA units/mL (12 007·7-19 003·6) in the non-adjuvanted cH5/1N1-placebo-cH8/1N1 group to 74 639·7 ELISA units/mL (59 986·3-92 872·6) in the AS03-adjuvanted cH8/1N1-cH5/1N1-cH11/1N1 group 28 days after the second dose. INTERPRETATION: The stalk domain seems to be a rational target for development of a universal influenza virus vaccine via administration of chimeric haemagglutinins with head domains to which humans are naive. FUNDING: GlaxoSmithKline Biologicals.

Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study.

Current vaccination strategies against influenza focus on generating an antibody response against the viral haemagglutination surface protein, however there is increasing interest in neuraminidase (NA) as a target for vaccine development. A critical tool for development of vaccines that target NA or include an NA component is available validated serology assays for quantifying anti-NA antibodies. Additionally serology assays have a critical role in defining correlates of protection in vaccine development and licensure. Standardisation of these assays is important for consistent and accurate results. In this study we first validated a harmonized enzyme-linked lectin assay (ELLA)- Neuraminidase Inhibition (NI) SOP for N1 influenza antigen and demonstrated the assay was precise, linear, specific and robust within classical acceptance criteria for neutralization assays for vaccine testing. Secondly we tested this SOP with NA from influenza B viruses and showed the assay performed consistently with both influenza A and B antigens. Third, we demonstrated that recombinant NA (rNA) could be used as a source of antigen in ELLA-NI. In addition to validating a harmonized SOP we finally demonstrated a clear improvement in inter-laboratory agreement across several studies by using a calibrator. Importantly we showed that the use of a calibrator significantly improved agreement when using different sources of antigen in ELLA-NI, namely reverse genetics viruses and recombinant NA. We provide a freely available and detailed harmonized SOP for ELLA-NI. Our results add to the growing body of evidence in support of developing biological standards for influenza serology.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 µg hemagglutinin (HA)/dose + AS03A; 2. inactivated vaccine with 5.75 µg HA/dose + AS03A; 3. inactivated vaccine with 11.5 µg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 µg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Non-neutralizing antibody responses following A(H1N1)pdm09 influenza vaccination with or without AS03 adjuvant system.

BACKGROUND: Non-neutralizing antibodies inducing complement-dependent lysis (CDL) and antibody-dependent cell-mediated cytotoxicity (ADCC) activity may contribute to protection against influenza infection. We investigated CDL and ADCC responses in healthy adults randomized to receive either non-adjuvanted or AS03-adjuvanted monovalent A(H1N1)pdm09 vaccine (containing 15 µg/3.75 µg of hemagglutinin, respectively) on a 2-dose schedule 21 days apart. METHODS: We conducted an exploratory analysis of a subset of 106 subjects having no prior history of A(H1N1)pdm09 infection or seasonal influenza vaccination enrolled in a previously reported study (NCT00985673). Antibody responses against the homologous A/California/7/2009 (H1N1) vaccine strain and a related A/Brisbane/59/2007 (H1N1) seasonal influenza strain were analyzed up to Day 42. RESULTS: Baseline seropositivity determined with hemagglutination inhibition (HI), CDL and ADCC antibody titers against viral strains was high; A/California/7/2009 (HI [40.4-48.1%]; CDL [34.6-36.0%]; ADCC [92.1-92.3%]); A/Brisbane/59/2007 (HI [73.1-88.9%]; CDL [38.0-42.0%]; ADCC [86.8-97.0%]). CDL seropositivity increased following vaccination with both adjuvanted and non-adjuvanted formulations (A/California/7/2009 [95.9-100%]; A/Brisbane/59/2007 [75.5-79.6%]). At Day 21, increases in CDL and ADCC antibody geometric mean titers against both strains were observed for both formulations. After 2 doses of AS03-adjuvanted vaccine, vaccine responses of 95.8% (≥9-fold increase from baseline in CDL titers) and 34.3% (≥16-fold increase from baseline in ADCC titers) were seen against A/California/7/2009; and 22.4% and 42.9%, respectively, against A/Brisbane/59/2007. Vaccine responses after 2 doses of the non-adjuvanted vaccine were broadly similar. CONCLUSIONS: Broadly comparable non-neutralizing immune responses were observed following vaccination with non-adjuvanted and AS03-adjuvanted A(H1N1)pdm09 formulations; including activity against a related vaccine strain.

Immunogenicity and Safety of AS03-adjuvanted H5N1 Influenza Vaccine in Children 6-35 Months of Age: Results From a Phase 2, Randomized, Observer-blind, Multicenter, Dose-ranging Study.

BACKGROUND: This phase 2 observer-blind, randomized, multicenter, dose-ranging study evaluated immunogenicity and safety of different formulations of an AS03-adjuvanted H5N1 influenza vaccine in children 6-35 months of age. METHODS: One hundred eighty-five children randomized into 5 groups [1.9 µg hemagglutinin (HA)/AS03B, 0.9 µg HA/AS03C, 1.9 µg HA/AS03C, 3.75 µg HA/AS03C or 3.75 µg HA/AS03D] were to receive 2 doses administered 21 days apart (primary vaccination). AS03 was classified by amount of DL-α-tocopherol, with AS03B the highest amount. One year later, all subjects were to receive unadjuvanted 3.75 µg HA as antigen challenge. Immunogenicity was assessed 21 days after primary vaccination (day 42) and 7 days after antigen challenge (day 392). Immunogenicity-fever index, based on hemagglutination inhibition and microneutralization antibody titers at day 42 and fever 7 days after each vaccination, was used to guide the selection of an acceptable formulation. RESULTS: After primary vaccination, formulations elicited strong homologous immune responses with all subjects' hemagglutination inhibition titers ≥1:40 post-vaccination. Immunogenicity-fever index based on hemagglutination inhibition and microneutralization assays showed that 1.9 µg HA/AS03B ranked the highest. Antibody levels persisted >4 times above baseline 12 months after primary vaccination with all formulations (day 385). Antibodies increased >4-fold after antigen challenge (day 392/day 385) with 1.9 µg HA/AS03B, 0.9 µg HA/AS03C and 1.9 µg HA/AS03C formulations. Overall per subject, the incidence of fever ranged from 28.6% (3.75 µg HA/AS03D) to 60.5% (1.9 µg HA/AS03B). CONCLUSIONS: All formulations were highly immunogenic and demonstrated acceptable safety profiles, with the 1.9 µg HA/AS03B providing the most favorable balance of immunogenicity versus reactogenicity for use in children 6-35 months of age.

Immunogenicity and safety of the AS04-HPV-16/18 and HPV-6/11/16/18 human papillomavirus vaccines in asymptomatic young women living with HIV aged 15-25 years: A phase IV randomized comparative study.

BACKGROUND: Women living with HIV (WLWH) are at higher risk of acquisition and progression of human papillomavirus (HPV) infection. Evidence on effect of HPV vaccination in this population is limited. METHODS: This phase IV randomized controlled observer-blind study assessed immunogenicity and safety of two HPV vaccines (AS04-HPV-16/18 vs. 4vHPV) given in WLWH (stage 1) and HIV- females aged 15-25 years. Co-primary endpoints were to demonstrate, in WLWH subjects, non-inferiority (and if demonstrated, superiority) of AS04-HPV-16/18 vs. 4vHPV for HPV-16 and HPV-18 by pseudovirion-based neutralization assay (PBNA) at month 7 and safety. Non-inferiority criteria was lower limit (LL) of the 95% confidence interval (CI) of the GMT ratio AS04-HPV-16/18/4vHPV above 0.5, in the according to protocol population. NCT01031069. FINDINGS: Among 873 subjects recruited between 26-Oct-2010 and 14-May-2015, 546 were randomized (1:1) and received at least one vaccine dose (total vaccinated cohort, TVC): 257 were WLWH (129 AS04-HPV-16/18; 128 4vHPV) and 289 were subjects without HIV (144 AS04-HPV-16/18; 145 4vHPV). Baseline CD4 cell count in WLWH was at least 350 cells/mm3.At month 7, AS04-HPV-16/18 showed immunological superiority to 4vHPV in WLWH. Neutralizing anti-HPV-16 and HPV-18 antibody GMTs were 2·74 (95% CI: 1·83; 4·11) and 7·44 (95% CI: 4·79; 11·54) fold higher in AS04-HPV-16/18 vs. 4vHPV (LL of the GMT ratio >1 in TVC, p<0·0001), respectively. Similar results were observed by ELISA up to month 24.Solicited local and general symptoms were in line with product labels. The number of reported serious adverse events (SAEs) was balanced throughout the study. INTERPRETATION: Both vaccines showed an acceptable safety profile in all subjects. Despite the absence of an immunological correlate of protection for HPV, differences in immune responses elicited by the vaccines especially for HPV-18 may translate into longer lasting or more robust protection against cervical cancer with the AS04-HPV-16/18 vaccine in WLWH.

Comparison between a new multiplex electrochemiluminescence assay and the WHO reference enzyme-linked immunosorbent assay to measure serum antibodies against pneumococcal serotype-specific polysaccharides.

BACKGROUND: Two electrochemiluminescence (ECL) assays were developed which, together, can simultaneously measure serum antibodies against pneumococcal capsular polysaccharides (PnPS) for 17 serotypes. The assays were validated for the 13 PnPS included in the 13-valent pneumococcal conjugate vaccine (PCV13). As recommended by the World Health Organization (WHO), we compared the ECL assays with the WHO reference enzyme-linked immunosorbent assay (ELISA) and derived a threshold corresponding to the 0.35 µg/mL threshold established for the WHO reference ELISA for the non-inferiority comparison and licensure of new PCVs against invasive pneumococcal disease. METHODS: A panel of 452 serum samples from children vaccinated with one of the three licensed PCVs was assessed with the ECL assays and the WHO reference ELISA. The ECL assay threshold for the aggregated seven PnPS included in the 7-valent PCV (PCV7) and serotype-specific thresholds were determined using a receiver operating characteristics (ROC) curve-based approach and Deming regression. To evaluate concordance between the ECL assays and the WHO reference ELISA, serostatus agreement rates between both assays and geometric means of the ratios (GMRs) of concentrations obtained with both assays were calculated. RESULTS: The thresholds for the seven aggregated PCV7 serotypes obtained with the ROC curve-based approach and Deming regression approximated 0.35 µg/mL (0.38 and 0.34 µg/mL, respectively). Individual thresholds for the PCV13 serotypes ranged between 0.24 and 0.51 µg/mL across both approaches. Serostatus agreement rates using a 0.35 µg/mL threshold for both assays were ≥86.9% for all PCV13 serotypes. GMRs ranged between 0.85 and 1.25 for 11/13 serotypes and were <1.29 for the two remaining serotypes. CONCLUSION: The ECL assays were comparable to the WHO reference ELISA and offer a sensitive, time- and serum volume-saving method to quantify serotype-specific anti-PnPS antibodies in pediatric sera. A 0.35 µg/mL threshold will be used for each PCV13 serotype to assess PCV immunogenicity in clinical trials.

Pandemic influenza virus vaccines boost hemagglutinin stalk-specific antibody responses in primed adult and pediatric cohorts.

Licensed influenza virus vaccines target the head domain of the hemagglutinin (HA) glycoprotein which undergoes constant antigenic drift. The highly conserved HA stalk domain is an attractive target to increase immunologic breadth required for universal influenza virus vaccines. We tested the hypothesis that immunization with a pandemic influenza virus vaccine boosts pre-existing anti-stalk antibodies. We used chimeric cH6/1, full length H2 and H18 HA antigens in an ELISA to measure anti-stalk antibodies in recipients participating in clinical trials of A/H1N1, A/H5N1 and A/H9N2 vaccines. The vaccines induced high titers of anti-H1 stalk antibodies in adults and children, with higher titers elicited by AS03-adjuvanted vaccines. We also observed cross-reactivity to H2 and H18 HAs. The A/H9N2 vaccine elicited plasmablast and memory B-cell responses. Post-vaccination serum from vaccinees protected mice against lethal challenge with cH6/1N5 and cH5/3N4 viruses. These findings support the concept of a chimeric HA stalk-based universal influenza virus vaccine. clinicaltrials.gov: NCT02415842.

Heterologous prime-boost with A(H5N1) pandemic influenza vaccines induces broader cross-clade antibody responses than homologous prime-boost.

Highly pathogenic avian influenza (HPAI) A(H5Nx) viruses continue to pose a pandemic threat. US national vaccine stockpiles are a cornerstone of the influenza pandemic preparedness plans. However, continual genetic and antigenic divergence of A(H5Nx) viruses requires the development of effective vaccination strategies using stockpiled vaccines and adjuvants for pandemic preparedness. Human sera collected from healthy adults who received either homologous (2 doses of a AS03A-adjuvanted A/turkey/Turkey/1/2005, A/Turkey), or heterologous (primed with AS03A-adjuvanted A/Indonesia/5/2005, A/Indo, followed by A/Turkey boost) prime-boost vaccination regimens were analyzed by hemagglutination inhibition and microneutralization assays against 8 wild-type HPAI A(H5Nx) viruses from 6 genetic clades. Molecular, structural and antigenic features of the A(H5Nx) viruses that could influence the cross-clade antibody responses were also explored. Compared with homologous prime-boost vaccinations, priming with a clade 2.1.3.2 antigen (A/Indo) followed by one booster dose of a clade 2.2.1 antigen (A/Turkey) administered 18 months apart did not compromise the antibody responses to the booster vaccine (A/Turkey), it also broadened the cross-clade antibody responses to several antigenically drifted variants from 6 heterologous clades, including an antigenically distant A(H5N8) virus (A/gyrfalcon/Washington/410886/2014, clade 2.3.4.4) that caused recent outbreaks in US poultry. The magnitude and breadth of the cross-clade antibody responses against emerging HPAI A(H5Nx) viruses are associated with genetic, structural and antigenic differences from the vaccine viruses and enhanced by the inclusion of an adjuvant. Heterologous prime-boost vaccination with AS03A adjuvanted vaccine offers a vaccination strategy to use existing stockpiled vaccines for pandemic preparedness against new emerging HPAI A(H5Nx) viruses.

Anamnestic Immune Response and Safety of an Inactivated Quadrivalent Influenza Vaccine in Primed Versus Vaccine-Naïve Children.

BACKGROUND: It has not yet been demonstrated whether 2 doses of inactivated quadrivalent influenza vaccine (IIV4) prime a booster response in infants. We evaluated the anamnestic immune response to an IIV4 in children 17-48 months of age. METHODS: Children were randomized to 2 doses of IIV4 or control in the primary phase III study (NCT01439360). One year later, in an open-label revaccination extension study (NCT01702454), a subset of children who received IIV4 in the primary study (primed group) received 1 IIV4 dose and children who received control in the primary study (unprimed) received 2 IIV4 doses 28 days apart. The primary objective was to evaluate hemagglutination inhibition (HI) antibody titers 7 days after first IIV4 vaccination in the per-protocol cohort (N = 224 primed; N = 209 unprimed). Neutralizing and antineuraminidase antibodies were also measured. Safety was analyzed in the total vaccinated cohort (N = 241 primed; N = 229 unprimed). RESULTS: An anamnestic response was observed in primed children relative to unprimed controls, measured by age-adjusted geometric mean HI titer ratios against strains homologous (A/H1N1: 9.0; B/Victoria: 3.9) and heterologous (A/H3N2: 2.7; B/Yamagata: 6.7) to those in the primary vaccination series. The anamnestic response in primed children included increases in neutralizing antibodies (mean geometric increase: 5.0-10.6) and antineuraminidase antibodies (4.9-8.8). No serious adverse events related to vaccination were reported. CONCLUSIONS: In this study, 2-dose priming with IIV4 induced immune memory that was recalled with 1-dose IIV4 the following year to boost HI, antineuraminidase and neutralizing antibodies, even though the IIV4 strain composition partially changed.

Prevention of vaccine-matched and mismatched influenza in children aged 6-35 months: a multinational randomised trial across five influenza seasons.

BACKGROUND: Despite the importance of vaccinating children younger than 5 years, few studies evaluating vaccine prevention of influenza have been reported in this age group. We evaluated efficacy of an inactivated quadrivalent influenza vaccine (IIV4) in children aged 6-35 months. METHODS: In this phase 3, observer-blinded, multinational trial, healthy children from 13 countries in Europe, Central America, and Asia were recruited in five independent cohorts, each in a different influenza season. Participants were randomly assigned (1:1) to either IIV4 (15 µg haemagglutinin antigen per strain per 0·5 mL dose; a single dose on day 0 for vaccine-primed children, and two doses, on days 0 and 28, for vaccine-unprimed children) or to one or two doses of a non-influenza control vaccine. Primary endpoints were moderate-to-severe influenza or all influenza (irrespective of disease severity) confirmed by RT-PCR on nasal swabs. Cultured isolates were further characterised as antigenically matched or mismatched to vaccine strains. Efficacy was assessed in the per-protocol cohort and total vaccinated cohort (time-to-event analysis), and safety was assessed in the total vaccinated cohort. FINDINGS: Between Oct 1, 2011, and Dec 31, 2014, 12 018 children were recruited into the total vaccinated cohort (6006 children in the IIV4 group and 6012 children in the control group). 356 (6%) children in the IIV4 group and 693 (12%) children in the control group had at least one case of RT-PCR-confirmed influenza. Of these 1049 influenza strains, 138 (13%) were A/H1N1, 529 (50%) were A/H3N2, 69 (7%) were B/Victoria, and 316 (30%) were B/Yamagata. Overall, 539 (64%) of 848 antigenically characterised isolates were vaccine-mismatched (16 [15%] of 105 for A/H1N1; 368 [97%] of 378 for A/H3N2; 54 [86%] of 63 for B/Victoria; 101 [33%] of 302 for B/Yamagata). Vaccine efficacy was 63% (97·5% CI 52-72) against moderate-to-severe influenza and 50% (42-57) against all influenza in the per-protocol cohort, and 64% (53-73) against moderate-to-severe influenza and 50% (42-57) against all influenza in the total vaccinated cohort. There were no clinically meaningful safety differences between IIV4 and control. INTERPRETATION: IIV4 prevented influenza A and B in children aged 6-35 months despite high levels of vaccine mismatch. Vaccine efficacy was highest against moderate-to-severe disease, which is the most clinically important endpoint associated with greatest burden. FUNDING: GlaxoSmithKline Biologicals SA.

Separate and combined disruptions of two exo-beta-1,3-glucanase genes decrease the efficiency of Pichia anomala (strain K) biocontrol against Botrytis cinerea on apple.

The modes of action of the antagonistic yeast Pichia anomala (strain K) have been studied; however, thus far, there has been no clear demonstration of the involvement of exo-beta-1,3-glucanase in determining the level of protection against Botrytis cinerea afforded by this biocontrol agent on apple. In the present study, the exo-beta-1,3-glucanase-encoding genes PAEXG1 and PAEXG2, previously sequenced from the strain K genome, were separately and sequentially disrupted. Transfer of the URA3-Blaster technique to strain K, allowing multiple use of URA3 marker gene, first was validated by efficient inactivation of the PaTRP1 gene and recovery of a double auxotrophic strain (uracil and tryptophan). The PAEXG1 and PAEXG2 genes then were inactivated separately and sequentially with the unique URA3 marker gene. The resulting mutant strains showed a significantly reduced efficiency of biocontrol of B. cinerea when applied to wounded apple fruit, the calculated protection level dropping from 71% (parental strain) to 8% (mutated strain) under some experimental conditions. This suggests that exo-beta-1,3-glucanases play a role in the biological control of B. cinerea on apple. Furthermore, biological control experiments carried out in this study underline the complexity of the host-antagonist-pathogen interaction. Two experimental parameters (yeast inoculum concentration and physiological stage of the fruit) were found to influence dramatically the protection level. Results also suggest that, under some conditions, the contribution of exo-beta-1,3-glucanase to biological control may be masked by other modes of action, such as competition.

Predictive modelling of temperature and water activity (solutes) on the in vitro radial growth of Botrytis cinerea Pers.

The objective of this work was to develop validated models predicting the 'in vitro' effect of a(w) and temperature on the radial growth of Botrytis cinerea. The growth rate (g, mm d(-1)) of B. cinerea was calculated at three incubation temperatures (25 degrees C, 15 degrees C, 5 degrees C) and six water activities (ranging from 0.995 to 0.890). The water activity was adjusted with glucose, NaCl, glycerol, or sorbitol. Statistical analysis showed a significant effect of temperature, solute, a(w), and their two- and three-way interactions on the growth rate. No growth was observed at a(w)=0.93 in the presence of NaCl or at 0.89 in the presence of a non-ionic solute. The maximum colony growth rate decreased when the incubation temperature and water activity was lowered. Secondary models, relating the colony growth rate with a(w) or a(w) and temperature were developed. Optimum a(w) values for growth ranged from 0.981 to 0.987 in glycerol-, sorbitol-, or glucose-modified medium and were close to 1 in NaCl-modified medium. A quadratic polynomial equation was used to describe the combined effects of temperature and a(w) on g (mm d(-1)) in the presence of each solute. The highest and lowest radial growth rates were observed in models based on glucose and NaCl respectively, whatever the incubation temperature. All models prove to be good predictors of the growth rates of B. cinerea within the limits of experiments. The quadratic polynomial equation has bias factors of 0.957, 1.036, 0.950, and 0.860 and accuracy factors of 1.089, 1.070, 1.120 and 1.260 in media supplemented with glucose, NaCl, glycerol and sorbitol respectively. The results from modelling confirm the general finding that a(w) has a greater influence on fungal growth than temperature.