2023 White Paper on Recent Issues in Bioanalysis: EU IVDR 2017/746 Implementation/Impact, IVD/CDx/CLIA Approved Assays, High Dimensional Cytometry, Multiplexing Technologies, LBA Tissue Analysis, Vaccine Study Endpoints, Cell-Based Assays for Biomarkers, Cell Therapy and Vaccines (PART 2 - Recommendations on Development & Validation of Biomarkers, IVD, CDx, Cell-Based, Flow Cytometry, Ligand-Binding and Enzyme Assays; Advanced Critical Reagents Strategies).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation (Part 3 - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Comparative humoral and cellular immunogenicity and safety of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine and HPV-6/11/16/18 vaccine in healthy women aged 18-45 years: follow-up through Month 48 in a Phase III randomized study.

We previously reported higher anti-HPV-16 and -18 immune responses induced by HPV-16/18 vaccine compared with HPV-6/11/16/18 vaccine at Month 7 (one month after completion of full vaccination series) in women aged 18-45 y in an observer-blind study NCT00423046; the differences of immune response magnitudes were maintained up to Month 24. Here we report follow-up data through Month 48. At Month 48, in according-to-protocol cohort for immunogenicity (seronegative and DNA-negative for HPV type analyzed at baseline), geometric mean titers of serum neutralizing antibodies were 2.0- to 5.2-fold higher (HPV-16) and 8.6- to 12.8-fold higher (HPV-18) in HPV-16/18 vaccine group than in HPV-6/11/16/18 vaccine group. The majority of women in both vaccine groups remained seropositive for HPV-16. The same trend was observed for HPV-18 in HPV-16/18 vaccine group; however, seropositivity rates in HPV-6/11/16/18 vaccine group decreased considerably, particularly in the older age groups. In the total vaccinated cohort (regardless of baseline serological and HPV-DNA status), anti-HPV-16 and -18 neutralizing antibody levels induced by HPV-16/18 vaccine were higher than those induced by HPV-6/11/16/18 vaccine. CD4+ T-cell response for HPV-16 and HPV-18 was higher in HPV-16/18 vaccine group than in HPV-6/11/16/18 vaccine group. Memory B-cell responses appeared similar between vaccine groups. Both vaccines were generally well tolerated. Overall, the higher immune response observed with the HPV-16/18 vaccine was maintained up to Month 48. A head-to-head study incorporating clinical endpoints would be required to confirm whether the observed differences in immune response between the vaccines influence the duration of protection they provided.

Safety and immunogenicity of a rederived, live-attenuated dengue virus vaccine in healthy adults living in Thailand: a randomized trial.

Safety and immunogenicity of two formulations of a live-attenuated tetravalent dengue virus (TDEN) vaccine produced using rederived master seeds from a precursor vaccine were tested against a placebo control in a phase II, randomized, double blind trial (NCT00370682). Two doses were administered 6 months apart to 120 healthy, predominantly flavivirus-primed adults (87.5% and 97.5% in the two vaccine groups and 92.5% in the placebo group). Symptoms and signs reported after vaccination were mild to moderate and transient. There were no vaccine-related serious adverse events or dengue cases reported. Asymptomatic, low-level viremia (dengue virus type 2 [DENV-2], DENV-3, or DENV-4) was detected in 5 of 80 vaccine recipients. One placebo recipient developed a subclinical natural DENV-1 infection. All flavivirus-unprimed subjects and at least 97.1% of flavivirus-primed subjects were seropositive to antibodies against all four DENV types 1 and 3 months post-TDEN dose 2. The TDEN vaccine was immunogenic with an acceptable safety profile in flavivirus-primed adults.

Effects of varying antigens and adjuvant systems on the immunogenicity and safety of investigational tetravalent human oncogenic papillomavirus vaccines: results from two randomized trials.

BACKGROUND: A prophylactic human papillomavirus (HPV) vaccine targeting oncogenic HPV types in addition to HPV-16 and -18 may broaden protection against cervical cancer. Two Phase I/II, randomized, controlled studies were conducted to compare the immunogenicity and safety of investigational tetravalent HPV L1 virus-like particle (VLP) vaccines, containing VLPs from two additional oncogenic genotypes, with the licensed HPV-16/18 AS04-adjuvanted vaccine (control) in healthy 18-25 year-old women. METHODS: In one trial (NCT00231413), subjects received control or one of 6 tetravalent HPV-16/18/31/45 AS04 vaccine formulations at months (M) 0,1,6. In a second trial (NCT00478621), subjects received control or one of 5 tetravalent HPV-16/18/33/58 vaccines formulated with different adjuvant systems (AS04, AS01 or AS02), administered on different schedules (M0,1,6 or M0,3 or M0,6). RESULTS: One month after the third injection (Month 7), there was a consistent trend for lower anti-HPV-16 and -18 geometric mean antibody titers (GMTs) for tetravalent AS04-adjuvanted vaccines compared with control. GMTs were statistically significantly lower for an HPV-16/18/31/45 AS04 vaccine containing 20/20/10/10 µg VLPs for both anti-HPV-16 and anti-HPV-18 antibodies, and for an HPV-16/18/33/58 AS04 vaccine containing 20/20/20/20 µg VLPs for anti-HPV-16 antibodies. There was also a trend for lower HPV-16 and -18-specific memory B-cell responses for tetravalent AS04 vaccines versus control. No such trends were observed for CD4(+) T-cell responses. Immune interference could not always be overcome by increasing the dose of HPV-16/18 L1 VLPs or by using a different adjuvant system. All formulations had acceptable reactogenicity and safety profiles. Reactogenicity in the 7-day post-vaccination period tended to increase with the introduction of additional VLPs, especially for formulations containing AS01. CONCLUSIONS: HPV-16 and -18 antibody responses were lower when additional HPV L1 VLPs were added to the HPV-16/18 AS04-adjuvanted vaccine. Immune interference is a complex phenomenon that cannot always be overcome by changing the antigen dose or adjuvant system.

A phase II, randomized, safety and immunogenicity study of a re-derived, live-attenuated dengue virus vaccine in healthy adults.

Two formulations of a new live tetravalent dengue virus (DENV) vaccine produced using re-derived master seeds from a precursor vaccine and that same precursor vaccine as a control were compared in a placebo-controlled, randomized, observer-blind, phase II trial of 86 healthy adults. Two vaccine doses were administered 6 months apart; a third dose was offered to a subset. Symptoms and signs of dengue-like illness reported after vaccination were mild to moderate, transient, and occurred with similar frequency among recipients of the new DENV vaccine and placebo, except for rash. Neither dengue nor vaccine-related serious adverse events were reported. The first DENV vaccine dose was moderately immunogenic; the second dose increased the potency and breadth of the neutralizing antibody response. Tetravalent response rates to the new formulations were 60% and 66.7% in unprimed subjects. A third dose did not increase tetravalent antibody rates. The new DENV vaccine candidates merit additional evaluation.

Comparative immunogenicity and safety of human papillomavirus (HPV)-16/18 vaccine and HPV-6/11/16/18 vaccine: follow-up from months 12-24 in a Phase III randomized study of healthy women aged 18-45 years.

In this observer-blind study (NCT00423046), women (N=1,106), stratified by age (18-26, 27-35, 36-45 y), were randomized (1:1) to receive the HPV-16/18 vaccine (Cervarix®, GlaxoSmithKline Biologicals, Months 0, 1, 6) or the HPV-6/11/16/18 vaccine (Gardasil® Merck & Co., Inc., Months 0, 2, 6). Month 7 results were previously reported; we now report Month 24 results. In the according-to-protocol cohort for immunogenicity (seronegative and DNA-negative at baseline for HPV type analyzed), seropositivity rates of neutralizing antibodies (nAbs) [pseudovirion-based neutralization assay] were, across all age strata, 100% (HPV-16/18 vaccine) and 97.5-100% (HPV-6/11/16/18 vaccine) for HPV-16, and 99.0-100% (HPV-16/18 vaccine) and 72.3-84.4% (HPV-6/11/16/18 vaccine) for HPV-18. Corresponding geometric mean titers (GMTs) were 2.4-5.8-fold higher for HPV-16 and 7.7-9.4-fold higher for HPV-18 with the HPV-16/18 vaccine versus the HPV-6/11/16/18 vaccine; HPV-16 and HPV-18 GMTs were significantly higher with the HPV-16/18 vaccine than the HPV-6/11/16/18 vaccine (p< 0.0001) in the total vaccinated cohort (received ≥1 vaccine dose, irrespective of baseline sero/DNA-status). Similar results were obtained using enzyme-linked immunosorbent assay (ELISA). Positivity rates and GMTs of antigen-specific IgG antibodies in cervicovaginal secretions (ELISA) were not significantly different between vaccines. At Month 24, CD4⁺ T-cell responses for HPV-16 and HPV-18 were higher with the HPV-16/18 vaccine; memory B-cell response was higher for HPV-18 with the HPV-16/18 vaccine and similar between vaccines for HPV-16. Both vaccines were generally well tolerated. Although an immunological correlate of protection has not been defined, differences in the magnitude of immune response between vaccines may represent determinants of duration of protection.

Comparison of the immunogenicity and safety of Cervarix and Gardasil human papillomavirus (HPV) cervical cancer vaccines in healthy women aged 18-45 years.

This observer-blind study compared the prophylactic human papillomavirus (HPV) vaccines, Cervarix (GlaxoSmithKline) and Gardasil (Merck), by assessing immunogenicity and safety through one month after completion of the three-dose vaccination course. Women (n = 1106) were stratified by age (18-26, 27-35, 36-45 years) and randomized (1:1) to receive Cervarix (Months 0, 1, 6) or Gardasil (Months 0, 2, 6). At Month 7 after first vaccination, all women in the according-to-protocol cohort who were seronegative/DNA negative before vaccination for the HPV type analyzed had seroconverted for HPV-16 and HPV-18 serum neutralizing antibodies, as measured by pseudovirion-based neutralization assay (PBNA), except for two women aged 27-35 years in the Gardasil group who did not seroconvert for HPV-18 (98%). Geometric mean titers of serum neutralizing antibodies ranged from 2.3-4.8-fold higher for HPV-16 and 6.8-9.1-fold higher for HPV-18 after vaccination with Cervarix compared with Gardasil, across all age strata. In the total vaccinated cohort (all women who received at least one vaccine dose, regardless of their serological and DNA status prior to vaccination), Cervarix induced significantly higher serum neutralizing antibody titers in all age strata (p < 0.0001). Positivity rates for anti-HPV-16 and -18 neutralizing antibodies in cervicovaginal secretions and circulating HPV-16 and -18 specific memory B-cell frequencies were also higher after vaccination with Cervarix compared with Gardasil. Both vaccines were generally well tolerated. The incidence of unsolicited adverse events was comparable between vaccinated groups. The incidence of solicited symptoms was generally higher after Cervarix, injection site reactions being most common. However, compliance rates with the three-dose schedules were similarly high (>or= 84%) for both vaccines. Although the importance of differences in magnitude of immune response between these vaccines is unknown, they may represent determinants of duration of protection against HPV-16/18. Long-term studies evaluating duration of efficacy after vaccination are needed for both vaccines.

Correlation between direct ELISA, single epitope-based inhibition ELISA and pseudovirion-based neutralization assay for measuring anti-HPV-16 and anti-HPV-18 antibody response after vaccination with the AS04-adjuvanted HPV-16/18 cervical cancer vaccine.

To monitor immune status during clinical trials and after vaccine registration, several assays have been developed to measure type-specific human papillomavirus (HPV) serum antibody levels. These include neutralization assays, single epitope-based inhibition immunoassays, and direct enzyme-linked immunosorbent assays (ELISAs). Neutralization assays based on multiple epitopes and independent of vaccine material are considered the 'gold standard' for unbiased assessment of the protective potential of vaccine-induced antibodies. However, their use in large clinical trials is challenging. Here, we compare both the direct ELISA and the single epitope-based inhibition ELISA with the pseudovirion-based neutralization assay (PBNA) for HPV-16/18 antibody responses in vaccinated women enrolled in trials of Cervarix, GSK's cervical cancer vaccine. The direct ELISA, which is based on multiple epitopes, was shown to have a higher degree of sensitivity and correlation with the PBNA when compared with the single epitope-based inhibition ELISA. Among double-positive results, high correlations were observed between the PBNA and the direct ELISA (0.70-0.88 for HPV-16 and 0.82-0.94 for HPV-18) and also with the single epitope-based inhibition ELISA (0.60-0.89 for HPV-16 and 0.57-0.96 for HPV-18) in women aged 15-25 years. The correlation persisted up to 6.4 years after primary vaccination. Similar levels of correlation were observed for adolescents aged 10-14 years and women aged 46-55 years. Therefore, the direct ELISA appears to be an excellent surrogate for neutralizing activity and can be used to evaluate antibody response induced by L1 virus-like particle-based cervical cancer vaccines, regardless of time elapsed after vaccination (up to 6.4 years) and the age of the vaccine recipient.

Comparative evaluation of three assays for measurement of dengue virus neutralizing antibodies.

Plaque reduction neutralization tests (PRNTs) are commonly used for measuring levels of dengue virus (DENV) neutralizing antibodies. However, these assays lack a standardized format, generally have a low sample throughput, and are labor-intensive. The objective of the present study was to evaluate two alternative DENV neutralizing antibody assays: an enzyme-linked immunosorbent assay-based microneutralization (MN) assay, and a fluorescent antibody cell sorter-based, DC-SIGN expresser dendritic cell (DC) assay. False-positive rates, serotype specificity, reproducibility, sensitivity, and agreement among the assay methods were assessed using well-characterized but limited numbers of coded test sera. Results showed that all three assays had false-positive rates of less than 10% with titers near the cut-off and generally below the estimated limits of detection. All three methods demonstrated a high degree of specificity and good agreement when used to assay sera and serum mixtures from monovalent vaccinees and sera from patients after primary natural infection, with the only notable exception being moderate-to-high neutralizing antibody titers against DENV 2 measured by PRNT in a mixture containing only DENV 3 and DENV 4 sera. The MN and DC assays demonstrated good reproducibility. All three assays were comparable in their sensitivity, except that the PRNT was less sensitive for measuring DENV 4 antibody, and the MN and DC assays were less sensitive for measuring DENV 2 antibody. However, when used to test sera from persons after tetravalent DENV vaccination or secondary DENV infection, there was poor specificity and poor agreement among the different assays.

Evaluation of systemic and mucosal anti-HPV16 and anti-HPV18 antibody responses from vaccinated women.

Ideal methods to monitor HPV neutralizing antibodies induced by vaccination have not been established yet. Here, we evaluated systemic and cervical antibody levels induced by HPV16/18 AS04-adjuvanted vaccine (GlaxoSmithKline Biologicals) using a secreted alkaline phosphatase neutralization assay (SEAP-NA) and enzyme-linked immunosorbent assay (ELISA). Serum and cervical secretions from 50 vaccinated women were used to assess (1) overall assay reproducibility; (2) inter-assay and inter-specimen correlation; (3) correlations between month 1 and month 12 titers. Strong correlations between SEAP-NA and ELISA were observed (serum anti-HPV16/18, rho=0.91/0.85; cervix anti-HPV16/18, rho=0.84/0.89). Systemic and cervical antibody measures also correlated well (rho range: 0.64-0.75); except at mid-cycle (rho range: 0.28-0.65). Correlations between antibody levels at 1 and 12 months following the start of vaccination were poor (rho range: 0.16-0.38). In conclusion, HPV16/18 VLP-based ELISA is a reliable and valid method to monitor anti-HPV16/18 neutralizing potential for the first year following vaccination; however, additional studies will be required to better define the effects of the time on cycle and patterns of antibody response over time following vaccination.

Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only.

An effective virus-like particle (VLP) based prophylactic vaccine designed to protect against persistent infection with human papillomavirus (HPV) types 16 and 18 and subsequent lesion development will need to induce a strong humoral and cellular immune response capable of providing long-term protection. Our objective was to evaluate the ability of an HPV16/18 L1 VLP vaccine formulated with the AS04 adjuvant system (3-O-desacyl-4'-monophosphoryl lipid A (MPL) and aluminium salt) to induce an immune response of higher magnitude and persistence compared to a vaccine formulated with aluminium salt only. We demonstrated that MPL adsorbed onto aluminium salt retains its capacity to activate an innate immune response as assessed by the production of TNFalpha by human monocytes (U937). In addition, vaccination of mice, monkeys or human subjects with AS04 formulations induced higher total anti-L1 VLP16 and L1 VLP18 antibody responses (1.6-8.5-fold) than the aluminium salt only formulations. The enhanced antibody response induced by the AS04 vaccine formulation (1.6-4.1-fold) in monkeys and humans was shown to be targeted to functional neutralising L1 VLP16 and L1 VLP18 epitopes as assessed by V5/J4 specific ELISAs or HPV16 and HPV18 pseudo-neutralization assays. The enhanced immune profile observed with the AS04 formulation in terms of both total, V5/J4 specific and neutralizing antibodies was shown to persist for at least 3.5-year post-vaccination in human subjects. Finally, using the newly developed B cell ELISPOT assay we also demonstrated that the AS04 formulation elicited an increased frequency (2.2-5.2-fold) of HPV L1 VLP specific memory B cells when compared with the aluminium salt only formulations. These data strongly support the role of the AS04 adjuvant, which includes the immunostimulant MPL, in triggering a persistent vaccine-induced immune response of high quality.