A multi-laboratory study of diverse RSV neutralization assays indicates feasibility for harmonization with an international standard

Hosken, Nancy; Plikaytis, Brian D.; Trujillo, Carrie; Mahmood, Kutub; Higgins, Deborah

doi:10.1016/j.vaccine.2017.04.053

Cited by 20 publications

(14 citation statements)

References 16 publications

(18 reference statements)

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“…Reduced harmonization may have been due to multiple causes, including inexperience of a laboratory with a particular assay, variability of reagents used, or systematic differences between tests of the standard and the samples. Moreover, testing results exhibited similar patterns of inter-and intra-laboratory variability as seen with other standards [46,49].…”

Section: Discussionsupporting

confidence: 61%

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

et al. 2020

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The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1.

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Section: Discussionsupporting

confidence: 61%

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

et al. 2020

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“…The WHO 3rd International Standard (IS) 97/648 (WHO 3 IS, NIBSC) was reconstituted per instructions yielding assigned units of 3000 mIU/mL, and a portion further diluted 1:10 in DPBS. Human immune globulin to RSV (huIgG, 10% IgG, BEI #21973) was diluted to 1% IgG concentration in DPBS [6]. Human sera depleted of IgG, IgM, and IgA was obtained from SunnyLab (UK).…”

Section: Methodsmentioning

confidence: 99%

Development of a high-throughput assay to measure measles neutralizing antibodies

et al. 2019

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Measles virus is highly infectious and remains a leading cause of vaccine preventable deaths in children. Neutralizing antibody responses elicited by measles virus infection or immunization are a serological correlate of protection. We describe a high-throughput neutralization assay to improve surveillance for measles immunity. Measles virus-antibody mixtures were incubated on Vero cell monolayers and 24 hours later cell-lysates harvested and subjected to one-step SYBR green RT-qPCR to amplify a target sequence within the measles virus nucleoprotein gene. Neutralization endpoint titers were interpolated to determine the dilution that inhibited the relative amplicon copy number by at least 90% compared to the mean signal obtained in virus control wells in the absence of serum. Anti-measles virus and anti-measles hemagglutinin antisera specifically neutralized measles virus in the microneutralization RT-qPCR assay while pre-immune sera and sera raised against other viruses did not. The microneutralization RT-qPCR assay obeyed the Percentage Law for measles virus inputs ranging from 100–5000 TCID 50 /well. The linear range of the assay corresponds to measles antibody concentrations of 30 to 3000 mIU/mL. Bland-Altman analysis and two-way analysis of variance demonstrated that results obtained using the microneutralization RT-qPCR assay were comparable to those obtained using a plaque reduction neutralization test and correctly identified human serum samples that were seropositive (95% and 100%, sensitivity and specificity, respectively). Furthermore, these comparisons suggest that a concentration of 300 mIU/mL may be a conservative cut-point to use to identify individuals likely to be protected against severe measles disease when the endpoint is based on 90% inhibition of virus replication. Measles virus microneutralization RT-qPCR is a rapid, sensitive, specific, and robust assay for detecting measles neutralizing antibodies that may help to improve immunization strategies nationally and achieve measles elimination globally.

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“…International standardization of RSV neutralization assays and subsequent availability of International Standard reference sera is recommended to improve the comparison between studies. 19 Recently, a World Health Organization (WHO) International Standard antiserum has become available for RSV-A. 20 The neutralization assay used in this study was included in the collaborative study to establish this WHO International Standard.…”

Section: Discussionmentioning

confidence: 99%

Prevalent levels of RSV serum neutralizing antibodies in healthy adults outside the RSV-season

Plas

Verdijk

Brummelen

et al. 2019

Human Vaccines & Immunotherapeutics

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One of the main challenges in early clinical research with respiratory syncytial virus (RSV) live-attenuated vaccines (LAVs) is to assess immunogenicity in healthy adults. Healthy adults will have preexisting levels of serum neutralizing antibodies that could prematurely neutralize the LAV and underestimate the potential effect of the vaccine on the immune system. Data on prevalence and distribution of virus neutralizing titers (VNTs) in healthy adults is limited and there is no absolute threshold for protection against RSV-infection that can serve as an eligibility criterion in early phase trials. We assessed the RSV-specific serum VNT in healthy adults outside the Dutch RSV-Season in two clinical studies performed in 2017 (exploratory study, n = 100) and 2018 (first-in-human LAV-study, n = 190) using the same neutralizing assay. Our findings show that the prevalence and distribution of serum VNT was overall consistent in the two clinical studies. Log 2 VNTs were normally distributed, distributions of VNTs were similar and there was no statistical difference in mean log 2 VNT for both studies ( p = .3). Serum VNTs were comparable during the 6 months of screening in the FIH LAV-study. Our findings will help to determine a cutoff serum VNT to be used as an eligibility criterion in future early phase clinical trials.

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A multi-laboratory study of diverse RSV neutralization assays indicates feasibility for harmonization with an international standard

Cited by 20 publications

References 16 publications

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

Development of a high-throughput assay to measure measles neutralizing antibodies

Prevalent levels of RSV serum neutralizing antibodies in healthy adults outside the RSV-season

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