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The urgency and importance of organizing a global effort to harmonize clinical assay validation specific to the vaccine industry was identified during the drafting of the 2020 White Paper in Bioanalysis due to the lack of clarity and regulatory guidance/guidelines in vaccine immunoassay validation. Indeed, the Workshop on Recent Issues in Bioanalysis (WRIB) issues the White Paper in Bioanalysis yearly, which is one of the high-profile articles of the Bioanalysis Journal focused on detailed discussions and recommendations on vaccine assay validation. Since 2017, participation in the WRIB working groups by vaccine assay validation experts and regulators has rapidly increased due to its unique format where industry leaders and regulators can meet and exchange ideas on topics of interest to both groups. In early 2021, Vaccine manufacturers approached WRIB for sponsoring/supporting the authorship and publication of an overarching vaccine assay validation document based on the 2017-2020 discussions and consensus starting from immunogenicity assays first and followed by future papers on molecular and cell-based assay validation. Using industry and WRIB vaccine network, a vaccine immunogenicity assay validation working group was assembled consisting of 16 companies. The work on the first white paper started officially in April 2021 focusing on Vaccine LBA Validation (Part 1), and the drafting of Vaccine LBA Development (Part 2) and Vaccine LBA Monitoring & Transfer (Part 3) are presently ongoing and expected to be published shortly after this paper. Moreover, recommendations on Vaccine Cell-Based Assays Validation (ELISpot and Flow cytometry) and Vaccine Molecular Assays Validation (PCR, NGS, NanoString) are also on the WRIB publications agenda and the drafting is planned to start in mid-2024. For too long, vaccine scientists have not had a clear validation guidance for clinical vaccine immunogenicity assays. We hope that this common effort will help close this regulatory gap.

The 14 th edition of the Workshop on Recent Issues in Bioanalysis (14 th 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 14 th 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.

Background: Maternal immunization against group B streptococcus (GBS) could protect infants from invasive GBS disease. Additional doses in subsequent pregnancies may be needed. We evaluated the safety and immunogenicity of a second dose of an investigational trivalent CRM197-glycoconjugate GBS vaccine (targeting serotypes Ia/Ib/III), administered to nonpregnant women 4-6 years postdose 1., Methods: Healthy women either previously vaccinated with 1 dose of trivalent GBS vaccine 4-6 years before enrollment (n = 53) or never GBS vaccinated (n = 27) received a single trivalent GBS vaccine injection. Adverse events (AEs) were recorded. Serotype-specific (Ia/Ib/III) anti-GBS antibodies were measured by multiplex immunoassay prevaccination and 30/60 days postvaccination., Results: AEs were reported with similar rates after a first or second dose; none were serious. Of previously GBS-vaccinated women, 92%-98% had anti-GBS concentrations that exceeded an arbitrary threshold (8 µg/mL) for each serotype 60 days postdose 2 vs 36%-56% postdose 1 in previously non-GBS-vaccinated women. Of previously GBS-vaccinated women with undetectable baseline (predose 1) anti-GBS levels, 90%-98% reached this threshold postdose 2. For each serotype, anti-GBS geometric mean concentrations (GMCs) 30/60 days postdose 2 in previously GBS-vaccinated women were ≥200-fold higher than baseline GMCs. Among women with undetectable baseline anti-GBS levels, postdose 2 GMCs in previously GBS-vaccinated women exceeded postdose 1 GMCs in previously non-GBS-vaccinated women (≥7-fold)., Conclusions: A second trivalent GBS vaccine dose administered 4-6 years postdose 1 was immunogenic with a favorable safety profile. Women with undetectable preexisting anti-GBS concentrations may benefit from a sufficiently spaced second vaccine dose., Clinical Trials Registration: NCT02690181., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: We evaluated the safety and immunogenicity of liquid and lyophilized formulations of an investigational trivalent group B streptococcus (GBS) vaccine in non-pregnant women and assessed the formulations' equivalence in terms of serotype-specific immune response., Methods: This phase II, randomized, comparative, observer-blind trial enrolled healthy non-pregnant women 18-40 years of age. Women received a single dose of fully liquid (n = 529) or lyophilized (n = 521) trivalent GBS vaccine on day 1. Safety assessments were performed up to day 181 (study termination). Serotype Ia/Ib/III-specific immunoglobulin G (IgG) antibodies were measured in sera from women on day 1 (pre-vaccination) and day 31. Equivalence between the two formulations was demonstrated if the two-sided 95% confidence interval (CI) for the ratio (liquid/lyophilized) of the geometric mean concentrations (GMCs) on day 31 was contained in a (0.5, 2.0) interval for each serotype., Results: Solicited and unsolicited adverse events were reported at similar rates for both formulations. Serious adverse events were reported for six (1.1%) liquid GBS and nine (1.7%) lyophilized GBS vaccinated women, none of which were considered related to vaccination or fatal. On day 31, serotype-specific IgG concentrations were 8-16-fold higher than on day 1 in both groups. Equivalence of the liquid to the lyophilized formulation 30 days post-vaccination was demonstrated as the 95% CIs of the GMC ratios were within the pre-specified interval for the three serotypes: GMC ratios were 1.02 (95% CI: 0.79, 1.32) for serotype Ia, 0.93 (0.71, 1.21) for serotype Ib and 0.99 (0.76, 1.30) for serotype III., Conclusions: Both formulations of the investigational trivalent GBS vaccine had favorable safety profiles and induced similar GBS serotype-specific antibody concentrations. This study demonstrated that the fully liquid formulation was equivalent to the lyophilized formulation in healthy non-pregnant women in terms of immunogenicity for all three serotypes., Clinical Trials Registration: NCT02270944., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ZB, ML, OH, BC, AK, AD and CS are employees of the GSK group of companies. ML, OH, BC and AK hold shares in the GSK group of companies. For the work under consideration, GLR, PVD, CV and MAI declare that their institutions have been compensated by the GSK group of companies for the costs involved in the conduct of the study, JdH declares funding from Novartis Vaccine for industry sponsored clinical trial and JP declares personal fees from the GSK group of companies for service as principal investigator. Outside the submitted work, GLR declares personal fees from the GSK group of companies, Osivax, Virometix and Ablynx for consulting activities, PVD declares that his institution has received grants from the GSK group of companies, Pfizer, Sanofi, Merck, Takeda, Baxter, CanSino China, Osivax, Themis, Johnson & Johnson, Abbott, the Bill & Melinda Gates foundation, the Flemish government and the European Union; SB declares that she is consultant statistician for the GSK group of companies. JB and CJ have nothing to disclose., (Copyright © 2020 GlaxoSmithKline Biologicals S.A. Published by Elsevier Ltd.. All rights reserved.)

Group B streptococcus (GBS) is a leading cause of young infant mortality and morbidity globally, with vaccines being developed for over four decades but none licensed to date. A serocorrelate of protection against invasive disease in young infants is being considered to facilitate vaccine early licensure, followed by demonstration of efficacy assessed postlicensure. In this Review, we synthesise the available scientific evidence to define an immune correlate associated with GBS disease risk reduction on the basis of studies of natural infection. We summarise studies that have investigated GBS serum anticapsular or anti-protein antibodies, and studies measuring the association between antibody function and disease risk reduction. We highlight how knowledge on the development of correlates of protection from existing vaccines could be harnessed to facilitate GBS vaccine development. These lessons include aggregation of serocorrelates of protection for individual serotypes, understanding the relationship between immunity derived from natural exposure of adults and vaccine-induced immunity, or using extrapolation of protection from in-vitro immunoassay results. We also highlight key considerations for the assessment of the role of antibodies to derive a serocorrelate of risk reduction in future seroepidemiological studies of GBS disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Prevnar [heptavalent pneumococcal conjugate vaccine (PCV7)] is licensed in the United States for routine administration in infants and may be coadministered with other infant vaccines. Safety and immunogenicity data on the coadministration of the fourth dose of PCV7 with measles-mumps-rubella (MMR), varicella and Haemophilus influenzae type b (Hib) vaccines are limited., Methods: Children 12-15 months of age received either MMR with PCV7 (group 1) or MMR without PCV7 (group 2). All subjects received Hib and varicella vaccines. Group 2 received PCV7 6-9 weeks after MMR vaccination. Sera for analysis of all non-PCV7 antibodies were collected just before administration of MMR vaccine and 6 weeks later. Optimal antigen responses were assessed with the use of predetermined antibody titers. The primary end point was >90% response rate (all antigens). Noninferiority was defined as <10% difference between groups. Local and systemic reactions and postvaccination adverse events were monitored and compared between groups., Results: A total of 694 subjects (347 per group) were enrolled. After immunization with MMR plus PCV7 concurrently, or MMR followed 6 weeks later by PCV7, the percentages of subjects seroconverting were significantly greater than 90% for all antigens. The difference between the 2 groups was significantly less than 10%., Conclusion: The immune response to MMR, Hib and varicella vaccines, when administered concurrently with a 4th (booster) dose of PCV7, was noninferior to that of these vaccines when given without PCV7. These results support concomitant administration of PCV7 with MMR, varicella and Hib as part of the recommended immunization schedule for children 12-15 months of age.

A new meningococcal group C-CRM(197) conjugate vaccine (MnCC; Meningitec) has been evaluated in multiple clinical trials in the United States and most recently has been approved for routine administration in the United Kingdom. Meningococcal serogroup C (MnC)-specific immunoglobulin G (IgG) antibodies in pre- and postimmunization sera obtained from healthy U.S. adults, toddlers, and infants were quantitated by enzyme-linked immunosorbent assay (ELISA) and by an antibody-dependent, complement-mediated serum bactericidal assay (SBA). Serogroup-specific IgG antibody (micrograms per milliliter) in adults immunized either with the quadrivalent polysaccharide (A, C, Y, and W-135) vaccine or with MnCC showed a strong correlation (r = 0.848 and 0.934, respectively) by linear regression analysis with SBA. Sera from infants immunized with the MnCC (n = 30) and an age-matched unimmunized control group (n = 15) were also analyzed. Linear regression analysis of serum bactericidal and IgG ELISA data from sera obtained at 2 months of age (preimmunization) showed no correlation; however, a high degree of correlation was observed at time points after two (r = 0.877) and three (r = 0.951) immunizations, where significant rises in anti-MnC polysaccharide antibodies occurred relative to the age-matched control group. Infants previously primed with 3 doses of MnCC were given a booster dose of conjugate vaccine at 12 to 15 months of age. The correlation coefficient of ELISA to SBA for combined pre- and postbooster data was r = 0.836 (n = 48 pairs). In conclusion, increases in serum bactericidal activity in immunized adult, toddler, and infant populations were found to correlate very well with increases in serogroup-specific IgG concentrations, whereas the correlation between these two assays in nonimmunized 2-month-old infants was poor. Characterizing the relationship between these methods is important for understanding the significance of antigen-specific antibody concentrations relative to vaccine performance and protection from disease.

When the three major structural proteins, VP2, VP6, and VP7, of rotavirus are co-expressed in insect cells infected with recombinant baculoviruses, they self-assemble into triple-layered virus-like particles (VLPs) that are similar in morphology to native rotavirus. In order to establish the most favorable conditions for the synthesis of rotavirus VLPs, we have compared the kinetics of 2/6/7-VLP synthesis in two different insect cell lines: High Five cells propagated in Excell 405 medium and Spodoptera frugiperda 9 cells in Excell 400 medium. The majority of VLPs produced in both cell lines were released into the culture medium, and these released VLPs were predominantly triple-layered and were found to be stable for the period of six or seven days examined. The optimal synthesis of VLPs depended upon the cell line and the culture medium used as well as the time of harvesting infected cell cultures. The highest yield of VLPs was obtained from High Five cultures in the late phase of infection when the yield was at least 5-fold higher than that from S. frugiperda 9 cultures on a per cell basis. Our results demonstrate the usefulness of High Five cells for the production of VLPs as potential rotavirus subunit vaccines., (Copyright 1998 John Wiley & Sons, Inc.)

A gene encoding an occluded virion structural protein was isolated from an expression library constructed from the Helicoverpa zea S-type nuclear polyhedrosis virus (NPV) isolate HzS-15 using both polyclonal and monoclonal antibody screening. The gene was located within a Pstl-Sall fragment of the HzS-15 genome spanning from 96.5 to 97.3 m.u. Sequencing analyses revealed a long open reading frame of 927 nucleotides that predicted a protein of 37 kDa in size. Immunoblot analyses using the monoclonal antibody ENV409 demonstrated that the gene corresponded to a 40-kDa protein (p40) in SDS-polyacrylamide gels that was present exclusively in enveloped occluded virions but not in extracellular budded virions or envelope-stripped nucleocapsids. A p40 protein-specific transcript was detected at 16 hr postinfection in HzS-15-infected Hz 1075/UND-K cells and remained until 22 hr. Primer extension analyses demonstrated that the p40 protein-specific transcript started at -49 nucleotides from the ATG start codon within an ATAAG consensus pentamer found in late protein genes of Baculoviruses. The deduced amino acid sequence of the HzS-15 p40 protein gene shared 44 and 45% sequence homology with the p40 proteins of Bombyx mori NPV and Autographa californica NPV, respectively.

The transposable IFP2 element of Trichoplusia ni was originally isolated as a host DNA insertion in spontaneous FP mutants of Galleria mellonella or Autographa californica nuclear polyhedrosis viruses (NPVs). The termini of IFP2 insertions from five independently isolated FP mutants were sequenced. In all cases IFP2 is flanked by 13-bp terminal inverted repeats and has additional inverted repeats of 19 bp in length located asymmetrically with respect to the ends of the element. Insertion of IFP2 into the viral genome always generated a duplication of the tetranucleotide target site, TTAA. There was an apparent preference for insertion within a 12-bp A + T-rich imperfect palindromic sequence surrounding the target site. Sequence analysis of three independent IFP2 elements revealed an internal domain of 2.475 kb containing an RNA polymerase II promoter region and two large open reading frames. Primer extension analysis of IFP2-specific mRNA positioned the 5' terminus of the transcript. The element is present in DNA isolated from T. ni cell lines TN-368 and TN-5B1, but is not apparent in DNAs isolated from the TN-R2 cell line or our laboratory colony of T. ni larvae, suggesting IFP2 was recently introduced into the T. ni genome.

Twenty plaque-purified strains of Heliothis zea S-type nuclear polyhedrosis virus (HzSNPV) were characterized based upon restriction endonuclease digests of viral DNAs, structural protein profiles, and larval melanization response. Each of the 20 strains had a unique genotype. Genomic heterogeneity between strains was localized to 4 regions of the HzSNPV genome. Differences in protein profiles of occluded virus were noted for each strain relative to the wild-type isolate. The strains were separated into 3 groups based upon differences in relative virulence and rate of larval melanization upon death. There was no correlation between differences in pathology and specific alterations in either genotype or structural proteins. The genotype of strain HzS-15 was extensively characterized. Physical maps were constructed for the HzS-15 strain and all of the plaque-purified strains.

Transfection conditions were optimized for the cloned UND-K derivative of the IPLB-HZ 1075 cell line using the calcium-phosphate co-precipitation technique and the DNA genome of the Heliothis zea S-type nuclear polyhedrosis virus. Optimal efficiencies were obtained using supercoiled viral DNA, and by extending the adsorption period for the diluted precipitate to 12 h. Transfection efficiencies ranging from 0.5 to 1.3 x 10(3) plaque forming units per microgram of supercoiled viral DNA were routinely obtained for UND-K cells and HzS-15 viral DNA. Transfection efficiencies were compared for 10 other cloned Heliothis cell strains and the uncloned parental IPLB-HZ 1075 cell line. The cloned cell strains UND-F, L, and U were incapable of transfection, while UND-I and G were 3 and 131 fold (respectively) less efficient than UND-K. The UND-K cells and the calcium phosphate transfection procedure permit relatively efficient in vitro manipulation of the Heliothis zea NPV virus genome.