SCB-2019 protein vaccine as heterologous booster of neutralizing activity against SARS-CoV-2 Omicron variants after immunization with other COVID-19 vaccines.

We assessed the non-inferiority of homologous boosting compared with heterologous boosting with the recombinant protein vaccine, SCB-2019, in adults previously immunized with different COVID-19 vaccines. Three equal cohorts (N ~ 420) of Philippino adults (18-80 years) previously immunized with Comirnaty, CoronaVac or Vaxzevria COVID-19 vaccines were randomized 1:1 to receive homologous or heterologous (SCB-2019) boosters. Neutralizing antibodies against prototype SARS-CoV-2 (Wuhan-Hu-1) were measured in all participants and against Delta variant and Omicron sub-lineages in subsets (30‒50 per arm) 15 days after boosting. Participants recorded solicited adverse events for 7 days and unsolicited and serious adverse events until Day 60. Prototype SARS-CoV-2 neutralizing responses on Day 15 after SCB-2019 were statistically non-inferior to homologous Vaxzevria boosters, superior to CoronaVac, but lower than homologous Comirnaty. Neutralizing responses against Delta and Omicron BA.1, BA.2, BA.4 and BA.5 variants after heterologous SCB-2019 were higher than homologous CoronaVac or Vaxzevria, but lower than homologous Comirnaty. Responses against Omicron BF.7, BQ.1.1.3, and XBB1.5 following heterologous SCB-2019 were lower than after homologous Comirnaty booster but significantly higher than after Vaxzevria booster. SCB-2019 reactogenicity was similar to CoronaVac or Vaxzevria, but lower than Comirnaty; most frequent events were mild/moderate injection site pain, headache and fatigue. No vaccine-related serious adverse events were reported. Heterologous SCB-2019 boosting was well tolerated and elicited neutralizing responses against all tested SARS-COV-2 viruses including Omicron BA.1, BA.2, BA.4, BA.5, BF.7, BQ.1.1.3, and XBB1.5 sub-lineages that were non-inferior to homologous boosting with CoronaVac or Vaxzevria, but not homologous Comirnaty booster.

Superior Boosting of Neutralizing Titers Against Omicron SARS-CoV-2 Variants by Heterologous SCB-2019 Vaccine vs a Homologous Booster in CoronaVac-Primed Adults.

BACKGROUND: We compared homologous and heterologous boosting in adults in the Philippines primed with 2 or 3 doses of CoronaVac, with recombinant protein vaccine, SCB-2019. METHODS: CoronaVac-immunized adults (18-72 years) received a homologous or heterologous full or half dose SCB-2019 booster. We assessed all neutralizing antibody (NAb) responses against prototype SARS-CoV-2 after 15 days and NAb against SARS-CoV-2 Delta and Omicron variants in subsets (30‒50 per arm). Participants recorded adverse events. RESULTS: In 2-dose CoronaVac-primed adults prototype NAb geometric mean titers (GMT) were 203 IU/mL (95% confidence interval [CI], 182-227) and 939 IU/mL (95% CI, 841-1049) after CoronaVac and SCB-2019 boosters; the GMT ratio (4.63; 95% CI, 3.95-5.41) met predefined noninferiority and post-hoc superiority criteria. After 3-dose CoronaVac-priming prototype NAb GMTs were 279 IU/mL (95% CI, 240-325), 1044 IU/mL (95% CI, 898-1213), and 668 IU/mL (95% CI, 520-829) following CoronaVac, full and half-dose SCB-2019 boosters, respectively. NAb GMT ratios against Delta and Omicron comparing SCB-2019 with CoronaVac were all greater than 2. Mild to moderate reactogenicity was evenly balanced between groups. No vaccine-related serious adverse events were reported. CONCLUSIONS: Full or half dose SCB-2019 boosters were well tolerated with superior immunogenicity than homologous CoronaVac, particularly against newly emerged variants. Clinical Trials Registration. NCT05188677.

Safety and immunogenicity of SCB-2019, an adjuvanted, recombinant SARS-CoV-2 trimeric S-protein subunit COVID-19 vaccine in healthy 12-17 year-old adolescents.

We previously demonstrated the efficacy of the COVID-19 vaccine candidate, SCB-2019, in adults in the SPECTRA phase 2/3 efficacy study. We extended the study to include 1278 healthy 12-17-year-old adolescents in Belgium, Colombia, and the Philippines who received either two doses of SCB-2019 or placebo 21 days apart, to assess immunogenicity as neutralizing antibodies against prototype SARS-CoV-2 and variants of concern, and safety and reactogenicity as solicited and unsolicited adverse events with a comparator group of young adults (18-25 years). In participants with no evidence of prior SARS-CoV-2 infection SCB-2019 immunogenicity in adolescents was non-inferior to that in young adults; respective geometric mean neutralizing titers (GMT) against prototype SARS-CoV-2 14 days after the second vaccination were 271 IU/mL (95% CI: 211-348) and 144 IU/mL (116-178). Most adolescents (1077, 84.3%) had serologic evidence of prior SAR-CoV-2 exposure at baseline; in these seropositive adolescents neutralizing GMTs increased from 173 IU/mL (135-122) to 982 IU/mL (881-1094) after the second dose. Neutralizing titers against Delta and Omicron BA SARS-CoV-2 variants were also increased, most notably in those with prior exposure. SCB-2019 vaccine was well tolerated with generally mild or moderate, transient solicited and unsolicited adverse events that were comparable in adolescent vaccine and placebo groups except for injection site pain - reported after 20% of SCB-2019 and 7.3% of placebo injections. SCB-2019 vaccine was highly immunogenic against SARS-CoV-2 prototype and variants in adolescents, especially in those with evidence of prior exposure, with comparable immunogenicity to young adults. Clinical trial registration: EudraCT 2020-004272-17; ClinicalTrials.gov NCT04672395.

Six-month safety follow-up of an adjuvanted SARS-CoV-2 trimeric S-protein subunit vaccine (SCB-2019) in adults: A phase 2/3, double-blind, randomized study.

BACKGROUND: We evaluated the safety of SCB-2019, a protein subunit vaccine candidate containing a recombinant SARS-CoV-2 spike (S) trimer fusion protein, combined with CpG-1018/alum adjuvants. METHODS: This ongoing phase 2/3, double-blind, placebo-controlled, randomized trial is being conducted in Belgium, Brazil, Colombia, the Philippines, and South Africa in participants ≥ 12 years of age. Participants were randomly assigned to receive 2 doses of SCB-2019 or placebo administered intramuscularly 21 days apart. Here, we present the safety results of SCB-2019 over the 6-month period following 2-dose primary vaccination series in all adult participants (≥18 years of age). RESULTS: A total of 30,137 adult participants received at least one dose of study vaccine (n = 15,070) or placebo (n = 15,067) between 24 March 2021 and 01 December 2021. Unsolicited adverse events, medically-attended adverse events, adverse events of special interest, and serious adverse events were reported in similar frequencies in both study arms over the 6-month follow-up period. Vaccine-related SAEs were reported by 4 of 15,070 SCB-2019 recipients (hypersensitivity reactions in two participants, Bell's palsy, and spontaneous abortion) and 2 of 15,067 placebo recipients (COVID-19, pneumonia, and acute respiratory distress syndrome in one participant and spontaneous abortion in the other one). No signs of vaccine-associated enhanced disease were observed. CONCLUSIONS: SCB-2019 administered as a 2-dose series has an acceptable safety profile. No safety concerns were identified during the 6-month follow-up after the primary vaccination. CLINICAL TRIALS REGISTRATION: NCT04672395; EudraCT: 2020-004272-17.

Immunogenicity of an adjuvanted SARS-CoV-2 trimeric S-protein subunit vaccine (SCB-2019) in SARS-CoV-2-naïve and exposed individuals in a phase 2/3, double-blind, randomized study.

BACKGROUND: We evaluated immunogenicity of SCB-2019, a subunit vaccine candidate containing a pre-fusion trimeric form of the SARS-CoV-2 spike (S)-protein adjuvanted with CpG-1018/alum. METHODS: The phase 2/3, double-blind, randomized SPECTRA trial was conducted in five countries in participants aged ≥ 18 years, either SARS-CoV-2-naïve or previously exposed. Participants were randomly assigned to receive two doses of SCB-2019 or placebo administered intramuscularly 21 days apart. In the phase 2 part of the study, on days 1, 22, and 36, neutralizing antibodies were measured by pseudovirus and wild-type virus neutralization assays to SARS-CoV-2 prototype and variants, and ACE2-receptor-binding antibodies and SCB-2019-binding antibodies were measured by ELISA. Cell-mediated immunity was measured by intracellular cytokine staining via flow cytometry. RESULTS: 1601 individuals were enrolled between 24 March and 13 September 2021 and received at least one vaccine dose. Immunogenicity analysis was conducted in a phase 2 subset of 691 participants, including 428 SARS-CoV-2-naïve (381 vaccine and 47 placebo recipients) and 263 SARS-CoV-2-exposed (235 vaccine and 28 placebo recipients). In SARS-CoV-2-naïve participants, GMTs of neutralizing antibodies against prototype virus increased 2 weeks post-second dose (day 36) compared to baseline (224 vs 12.7 IU/mL). Seroconversion rate was 82.5 %. In SARS-CoV-2-exposed participants, one SCB-2019 dose increased GMT of neutralizing antibodies by 48.3-fold (1276.1 IU/mL on day 22) compared to baseline. Seroconversion rate was 92.4 %. Increase was marginal post-second dose. SCB-2019 also showed cross-neutralization capability against nine variants, including Omicron, in SARS-CoV-2-exposed participants at day 36. SCB-2019 stimulated Th1-biased cell-mediated immunity to the S-protein in both naïve and exposed participants. The vaccine was well tolerated, no safety concerns were raised from the study. CONCLUSIONS: A single dose of SCB-2019 was immunogenic in SARS-CoV-2-exposed individuals, whereas two doses were required to induce immune response in SARS-CoV-2-naïve individuals. SCB-2019 elicited a cross-neutralizing response against emergent SARS-CoV-2 variants at antibody levels associated with clinical protection, underlining its potential as a booster. CLINICALTRIALS: gov: NCT04672395; EudraCT: 2020-004272-17.

Efficacy of the adjuvanted subunit protein COVID-19 vaccine, SCB-2019: a phase 2 and 3 multicentre, double-blind, randomised, placebo-controlled trial.

BACKGROUND: A range of safe and effective vaccines against SARS CoV 2 are needed to address the COVID 19 pandemic. We aimed to assess the safety and efficacy of the COVID-19 vaccine SCB-2019. METHODS: This ongoing phase 2 and 3 double-blind, placebo-controlled trial was done in adults aged 18 years and older who were in good health or with a stable chronic health condition, at 31 sites in five countries (Belgium, Brazil, Colombia, Philippines, and South Africa). The participants were randomly assigned 1:1 using a centralised internet randomisation system to receive two 0·5 mL intramuscular doses of SCB-2019 (30 µg, adjuvanted with 1·50 mg CpG-1018 and 0·75 mg alum) or placebo (0·9% sodium chloride for injection supplied in 10 mL ampoules) 21 days apart. All study staff and participants were masked, but vaccine administrators were not. Primary endpoints were vaccine efficacy, measured by RT-PCR-confirmed COVID-19 of any severity with onset from 14 days after the second dose in baseline SARS-CoV-2 seronegative participants (the per-protocol population), and the safety and solicited local and systemic adverse events in the phase 2 subset. This study is registered on EudraCT (2020-004272-17) and ClinicalTrials.gov (NCT04672395). FINDINGS: 30 174 participants were enrolled from March 24, 2021, until the cutoff date of Aug 10, 2021, of whom 30 128 received their first assigned vaccine (n=15 064) or a placebo injection (n=15 064). The per-protocol population consisted of 12 355 baseline SARS-CoV-2-naive participants (6251 vaccinees and 6104 placebo recipients). Most exclusions (13 389 [44·4%]) were because of seropositivity at baseline. There were 207 confirmed per-protocol cases of COVID-19 at 14 days after the second dose, 52 vaccinees versus 155 placebo recipients, and an overall vaccine efficacy against any severity COVID-19 of 67·2% (95·72% CI 54·3-76·8), 83·7% (97·86% CI 55·9-95·4) against moderate-to-severe COVID-19, and 100% (97·86% CI 25·3-100·0) against severe COVID-19. All COVID-19 cases were due to virus variants; vaccine efficacy against any severity COVID-19 due to the three predominant variants was 78·7% (95% CI 57·3-90·4) for delta, 91·8% (44·9-99·8) for gamma, and 58·6% (13·3-81·5) for mu. No safety issues emerged in the follow-up period for the efficacy analysis (median of 82 days [IQR 63-103]). The vaccine elicited higher rates of mainly mild-to-moderate injection site pain than the placebo after the first (35·7% [287 of 803] vs 10·3% [81 of 786]) and second (26·9% [189 of 702] vs 7·4% [52 of 699]) doses, but the rates of other solicited local and systemic adverse events were similar between the groups. INTERPRETATION: Two doses of SCB-2019 vaccine plus CpG and alum provides notable protection against the entire severity spectrum of COVID-19 caused by circulating SAR-CoV-2 viruses, including the predominating delta variant. FUNDING: Clover Biopharmaceuticals and the Coalition for Epidemic Preparedness Innovations.

Immunogenicity of SCB-2019 Coronavirus Disease 2019 Vaccine Compared With 4 Approved Vaccines.

A significant correlation has been shown between the binding antibody responses against original severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and vaccine efficacy of 4 approved coronavirus disease 2019 vaccines. We therefore assessed the immune response against original SARS-CoV-2 elicited by the adjuvanted S-Trimer vaccine, SCB-2019 + CpG/alum, in the same assay and laboratory. Responses to SCB-2019 were comparable or superior for antibody to original and Alpha variant when compared with 4 approved vaccines. The comparison accurately predicted success of the recently reported efficacy trial of SCB-2019 vaccine. Immunogenicity comparisons to original strain and variants of concern should be considered as a basis for authorization of vaccines.

Persistence of the Immune Responses and Cross-Neutralizing Activity With Variants of Concern Following 2 Doses of Adjuvanted SCB-2019 Coronavirus Disease 2019 Vaccine.

BACKGROUND: We have previously reported the safety and immunogenicity 4 weeks after 2 doses of the Clover coronavirus disease 2019 (COVID-19) vaccine candidate, SCB-2019, a stabilized prefusion form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S-trimer). We now report persistence of antibodies up to 6 months after vaccination, and cross-neutralization titers against 3 variants of concern (VoCs). METHODS: In a phase 1 study, adult (18-54 years of age) and elderly (55-75 years of age) volunteers received 2 vaccinations 21 days apart with placebo or 3-, 9-, or 30-µg. We measured immunoglobulin G (IgG) antibodies against SCB-2019, angiotensin-converting enzyme 2 (ACE2) competitive binding antibodies, and neutralizing antibodies against wild-type SARS-CoV-2 (Wuhan-Hu-1) at days 101 and 184, and neutralizing antibodies against 3 VoCs, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1), in day 36 sera. RESULTS: Titers waned from their peak at days 36-50, but SCB-2019 IgG antibodies, ACE2 competitive binding antibodies, and neutralizing antibodies against wild-type SARS-CoV-2 persisted at 25%-35% of their observed peak levels at day 184. Day 36 sera also demonstrated dose-dependent increases in neutralizing titers against the 3 VoCs. CONCLUSIONS: SCB-2019 dose-dependently induced immune responses against wild-type SARS-CoV-2, which persisted up to day 184. Neutralizing antibodies were cross-reactive against 3 of the most prevalent VoCs.

Assay Harmonization and Use of Biological Standards To Improve the Reproducibility of the Hemagglutination Inhibition Assay: a FLUCOP Collaborative Study.

The hemagglutination inhibition (HAI) assay is an established technique for assessing influenza immunity, through measurement of antihemagglutinin antibodies. Improved reproducibility of this assay is required to provide meaningful data across different testing laboratories. This study assessed the impact of harmonizing the HAI assay protocol/reagents and using standards on interlaboratory variability. Human pre- and postvaccination sera from individuals (n = 30) vaccinated against influenza were tested across six laboratories. We used a design of experiment (DOE) method to evaluate the impact of assay parameters on interlaboratory HAI assay variability. Statistical and mathematical approaches were used for data analysis. We developed a consensus protocol and assessed its performance against in-house HAI testing. We additionally tested the performance of several potential biological standards. In-house testing with four reassortant viruses showed considerable interlaboratory variation (geometric coefficient of variation [GCV] range of 50% to 117%). The age, concentration of turkey red blood cells, incubation duration, and temperature were key assay parameters affecting variability. Use of a consensus protocol with common reagents, including viruses, significantly reduced GCV between laboratories to 22% to 54%. Pooled postvaccination human sera from different vaccination campaigns were effective as biological standards. Our results demonstrate that the harmonization of protocols and critical reagents is effective in reducing interlaboratory variability in HAI assay results and that pools of postvaccination human sera have potential as biological standards that can be used over multiple vaccination campaigns. Moreover, the use of standards together with in-house protocols is as potent as the use of common protocols and reagents in reducing interlaboratory variability. IMPORTANCE The hemagglutination inhibition (HAI) assay is the most commonly used serology assay to detect antibodies from influenza vaccination or influenza virus infection. This assay has been used for decades but requires improved standardization of procedures to provide meaningful data. We designed a large study to assess selected parameters for their contribution to assay variability and developed a standard protocol to promote consistent HAI testing methods across laboratories. The use of this protocol and common reagents resulted in lower levels of variability in results between participating laboratories than achieved using in-house HAI testing. Human sera sourced from vaccination campaigns over several years, and thus including antibody to different influenza vaccine strains, served as effective assay standards. Based on our findings, we recommend the use of a common protocol and/or human serum standards, if available, for testing human sera for the presence of antibodies against seasonal influenza using turkey red blood cells.

Safety and immunogenicity of S-Trimer (SCB-2019), a protein subunit vaccine candidate for COVID-19 in healthy adults: a phase 1, randomised, double-blind, placebo-controlled trial.

BACKGROUND: As part of the accelerated development of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we report a dose-finding and adjuvant justification study of SCB-2019, a protein subunit vaccine candidate containing a stabilised trimeric form of the spike (S)-protein (S-Trimer) combined with two different adjuvants. METHODS: Our study is a phase 1, randomised, double-blind placebo-controlled trial at a specialised clinical trials centre in Australia. We enrolled healthy adult volunteers in two age groups: younger adults (aged 18-54 years) and older adults (aged 55-75 years). Participants were randomly allocated either vaccine or placebo using a list prepared by the study funder. Participants were to receive two doses of SCB-2019 (either 3 µg, 9 µg, or 30 µg) or a placebo (0·9% NaCl) 21 days apart. SCB-2019 either had no adjuvant (S-Trimer protein alone) or was adjuvanted with AS03 or CpG/Alum. The assigned treatment was administered in opaque syringes to maintain masking of assignments. Reactogenicity was assessed for 7 days after each vaccination. Humoral responses were measured as SCB-2019 binding IgG antibodies and ACE2-competitive blocking IgG antibodies by ELISA and as neutralising antibodies by wild-type SARS-CoV-2 microneutralisation assay. Cellular responses to pooled S-protein peptides were measured by flow-cytometric intracellular cytokine staining. This trial is registered with ClinicalTrials.gov, NCT04405908; this is an interim analysis and the study is continuing. FINDINGS: Between June 19 and Sept 23, 2020, 151 volunteers were enrolled; three people withdrew, two for personal reasons and one with an unrelated serious adverse event (pituitary adenoma). 148 participants had at least 4 weeks of follow-up after dose two and were included in this analysis (database lock, Oct 23, 2020). Vaccination was well tolerated, with two grade 3 solicited adverse events (pain in 9 µg AS03-adjuvanted and 9 µg CpG/Alum-adjuvanted groups). Most local adverse events were mild injection-site pain, and local events were more frequent with SCB-2019 formulations containing AS03 adjuvant (44-69%) than with those containing CpG/Alum adjuvant (6-44%) or no adjuvant (3-13%). Systemic adverse events were more frequent in younger adults (38%) than in older adults (17%) after the first dose but increased to similar levels in both age groups after the second dose (30% in older and 34% in younger adults). SCB-2019 with no adjuvant elicited minimal immune responses (three seroconversions by day 50), but SCB-2019 with fixed doses of either AS03 or CpG/Alum adjuvants induced high titres and seroconversion rates of binding and neutralising antibodies in both younger and older adults (anti-SCB-2019 IgG antibody geometric mean titres at day 36 were 1567-4452 with AS03 and 174-2440 with CpG/Alum). Titres in all AS03 dose groups and the CpG/Alum 30 µg group were higher than were those recorded in a panel of convalescent serum samples from patients with COVID-19. Both adjuvanted SCB-2019 formulations elicited T-helper-1-biased CD4+ T-cell responses. INTERPRETATION: The SCB-2019 vaccine, comprising S-Trimer protein formulated with either AS03 or CpG/Alum adjuvants, elicited robust humoral and cellular immune responses against SARS-CoV-2, with high viral neutralising activity. Both adjuvanted vaccine formulations were well tolerated and are suitable for further clinical development. FUNDING: Clover Biopharmaceuticals and the Coalition for Epidemic Preparedness Innovations.

Analysis of the vaccine-induced influenza B virus hemagglutinin-specific antibody dependent cellular cytotoxicity response.

Influenza A virus (IAV) and influenza B virus (IBV) cause substantial morbidity and mortality during seasonal epidemics. On basis of variation in the surface glycoprotein hemagglutinin, two antigenically distinct lineages of IBV are distinguished: B/Victoria/2/87-like (B/Vic) and B/Yamagata/16/88-like (B/Yam). To prevent IAV and IBV infections, both trivalent (containing IBV of one lineage) and quadrivalent (containing IBV of both lineages) influenza vaccines are used. In addition to virus-neutralizing antibodies, inactivated influenza vaccines induce antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC). Here, we determine whether vaccination with trivalent or quadrivalent inactivated influenza vaccine induces ADCC mediating antibodies directed to IBV of the two different lineages, and whether these antibodies cross-react with IBV of the opposing lineage. A robust ADCC assay based on the use of recombinant hemagglutinin and a continuous natural killer cell line that expresses FcγRIII (CD16) was used to detect the presence of ADCC mediating antibodies. Paired pre- and post-vaccination serum samples from 26 and 15 study subjects that received a trivalent or quadrivalent inactivated influenza vaccine, respectively, were assessed for the presence of ADCC mediating antibodies specific for HA derived from viruses of the B/Vic or B/Yam-lineage. Furthermore, the relative contribution of HA1- and HA2-subunit-specific antibodies to the ADCC response was determined. We found that seasonal inactivated influenza vaccines induce HA-head- and HA-stalk-specific antibodies that mediate ADCC. As expected, the quadrivalent vaccine induced antibodies to HA from both IBV lineages. Notably, a trivalent vaccine containing HA from the B/Vic lineage induced antibodies that cross-react with the B/Yam lineage.

Use of a Blockade-of-Binding ELISA and Microneutralization Assay to Evaluate Zika Virus Serostatus in Dengue-Endemic Areas.

Zika virus (ZIKV) serological diagnostics are compromised in areas where dengue viruses (DENV) co-circulate because of their high levels of protein sequence homology. Here, we describe the characterization of a Zika blockade-of-binding ELISA (Zika BOB) and a Zika microneutralization assay (Zika MN) for the detection of ZIKV nonstructural protein 1 (NS1)-specific antibodies and ZIKV neutralizing antibodies, respectively. Zika BOB and Zika MN cutoffs were established as 10 and 100 endpoint titers, respectively, using samples collected pre- and post-virologically confirmed ZIKV infection from subjects living in DENV-endemic areas. Specificity of the assays was equally high, whereas sensitivity of Zika BOB was lower than that of Zika MN, especially in samples collected > 6 months post-infection. Immunosurveillance analysis, using combined results from both Zika BOB and Zika MN, carried out also in DENV-endemic regions in Colombia, Honduras, Mexico, and Puerto Rico before (2013-2014) and after (2017-2018) ZIKV introduction in the Americas suggests unapparent ZIKV seroprevalence rates ranged from 25% to 80% over the specified period of time in the regions investigated.

Rapid fluorescent focus inhibition test optimization and validation: Improved detection of neutralizing antibodies to rabies virus.

The rabies rapid fluorescent focus inhibition test (RFFIT) is the most widely used cell-based assay for detecting and quantitating rabies virus neutralizing antibodies (RVNA) in human serum. However, it is a complex, labor intensive, and somewhat subjective manual assay, the performance of which may be affected by a number of factors including the quality of cells and virus, variability of assay reagents and the skill and expertise of analysts. This study sought to identify and evaluate conditions that may impact RFFIT performance and RVNA detection by evaluating assay parameters including: different serial dilution scheme of serum samples in a 96-well microplate using semi-automated pipetting systems, the range of dose of challenge virus standard (CVS-11) strain of rabies virus, the effect of complement (C'), the effect of cell seeding density and passage number, the effect of diethylaminoethyl (DEAE) dextran concentration on virus infectivity, and the assay incubation period prior to immunostaining. In addition the evaluation of counting fluorescent foci using a microscope versus using scanned images from a cell imaging reader was performed in an effort to ease the reading of slides and have permanent records of the raw data. The results from optimization of each parameter are presented along with subsequent assay validation in accordance with the International Conference on Harmonization (ICH) guidelines. The improved and optimized RFFIT accuracy, linearity and sensitivity was demonstrated by testing World Health Organization (WHO)-1 and WHO-2 Standard Rabies Immune Globulins (SRIGs) and complete assay development and validation was performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines.

Integrated immunogenicity analysis of a tetravalent dengue vaccine up to 4 y after vaccination.

Two large pivotal phase III studies demonstrated the efficacy of the tetravalent dengue vaccine (CYD-TDV; Dengvaxia®, Sanofi Pasteur) against all dengue serotypes. Here we present an unprecedented integrated summary of the immunogenicity of CYD-TDV to identify the parameters driving the neutralizing humoral immune response and evolution over time. We summarized the immunogenicity profiles of a 3-dose schedule of CYD-TDV administered 6 months apart across 10 phase II and 6 phase III trials undertaken in dengue endemic and non-endemic countries. Dengue neutralizing antibody titers in sera were determined at centralized laboratories using the 50% plaque reduction neutralization test (PRNT50) at baseline, 28 d after the third dose, and annually thereafter for up to 4 y after the third dose in some studies. CYD-TDV elicits neutralizing antibody responses against all 4 dengue serotypes; geometric mean titers (GMTs) increased from baseline to post-dose 3. GMTs were influenced by several parameters including age, baseline dengue seropositivity and region. In the 2 pivotal studies, GMTs decreased initially during the first 2 y post-dose 3 but appear to stabilize or slightly increase again in the third year. GMTs persisted 1.2-3.2-fold higher than baseline levels for up to 4 y post-dose 3 in other studies undertaken in dengue endemic countries. Our integrated analysis captures the fullness of the CYD-TDV immunogenicity profile across studies, age groups and regions; by presenting the available data in this way general trends and substantial outliers within each grouping can be easily identified. CYD-TDV elicits neutralizing antibody responses against all dengue serotypes, with differences by age and endemicity, which persist above baseline levels in endemic countries.

Correlates of Protection against Influenza in the Elderly: Results from an Influenza Vaccine Efficacy Trial.

Although a number of studies have investigated and quantified immune correlates of protection against influenza in adults and children, data on immune protection in the elderly are sparse. A recent vaccine efficacy trial comparing standard-dose with high-dose inactivated influenza vaccine in persons 65 years of age and older provided the opportunity to examine the relationship between values of three immunologic assays and protection against community-acquired A/H3N2 influenza illness. The high-dose vaccine induced significantly higher antibody titers than the standard-dose vaccine for all assays. For the hemagglutination inhibition assay, a titer of 40 was found to correspond with 50% protection when the assay virus was antigenically well matched to the circulating virus--the same titer as is generally recognized for 50% protection in younger adults. A dramatically higher titer was required for 50% protection when the assay virus was a poor match to the circulating virus. With the well-matched virus, some protection was seen at the lowest titers; with the poorly matched virus, high levels of protection were not achieved even at the highest titers. Strong associations were also seen between virus neutralization test titers and protection, but reliable estimates for 50% protection were not obtained. An association was seen between titers of an enzyme-linked lectin assay for antineuraminidase N2 antibodies and protection; in particular, the proportion of treatment effect explained by assay titer in models that included both this assay and one of the other assays was consistently higher than in models that included either assay alone. (This study has been registered at ClinicalTrials.gov under registration no. NCT01427309.).

Automated Imaging and Analysis of the Hemagglutination Inhibition Assay.

The hemagglutination inhibition (HAI) assay quantifies the level of strain-specific influenza virus antibody present in serum and is the standard by which influenza vaccine immunogenicity is measured. The HAI assay endpoint requires real-time monitoring of rapidly evolving red blood cell (RBC) patterns for signs of agglutination at a rate of potentially thousands of patterns per day to meet the throughput needs for clinical testing. This analysis is typically performed manually through visual inspection by highly trained individuals. However, concordant HAI results across different labs are challenging to demonstrate due to analyst bias and variability in analysis methods. To address these issues, we have developed a bench-top, standalone, high-throughput imaging solution that automatically determines the agglutination states of up to 9600 HAI assay wells per hour and assigns HAI titers to 400 samples in a single unattended 30-min run. Images of the tilted plates are acquired as a function of time and analyzed using algorithms that were developed through comprehensive examination of manual classifications. Concordance testing of the imaging system with eight different influenza antigens demonstrates 100% agreement between automated and manual titer determination with a percent difference of ≤3.4% for all cases.

Influence of FcγRIIa-Expressing Cells on the Assessment of Neutralizing and Enhancing Serum Antibodies Elicited by a Live-Attenuated Tetravalent Dengue Vaccine.

Background. Recent trials of recombinant, live-attenuated chimeric yellow fever-dengue tetravalent dengue vaccine (CYD-TDV) demonstrated efficacy against symptomatic, virologically confirmed dengue disease with higher point estimates of efficacy toward dengue virus (DENV)3 and DENV4 and moderate levels toward DENV1 and DENV2. It is interesting to note that serotype-specific efficacy did not correlate with absolute neutralizing antibody (nAb) geometric mean titer (GMT) values measured in a Vero-based plaque reduction neutralization test assay. The absence of Fcγ receptors on Vero cells may explain this observation. Methods. We performed parallel seroneutralization assays in Vero cells and CV-1 cells that express FcγRIIa (CV-1-Fc) to determine the neutralizing and enhancing capacity of serotype-specific DENV Abs present in CYD-TDV clinical trial sera. Results. Enhancement of DENV infection was observed in CV-1-Fc cells in naturally exposed nonvaccine sera, mostly for DENV3 and DENV4, at high dilutions. The CYD-TDV-vaccinated sera showed similar enhancement patterns. The CV-1-Fc nAb GMT values were 2- to 9-fold lower than Vero for all serotypes in both naturally infected individuals and CYD-TDV-vaccinated subjects with and without previous dengue immunity. The relative (CV-1-Fc/Vero) GMT decrease for anti-DENV1 and anti-DENV2 responses was not greater than for the other serotypes. Conclusions. In vitro neutralization assays utilizing FcγRIIa-expressing cells provide evidence that serotype-specific Ab enhancement may not be a primary factor in the serotype-specific efficacy differences exhibited in the CYD-TDV trials.

Immune response to live-attenuated Japanese encephalitis vaccine (JE-CV) neutralizes Japanese encephalitis virus isolates from south-east Asia and India.

BACKGROUND: During clinical development of the licensed Japanese encephalitis chimeric virus vaccine (JE-CV), the neutralization capacity of vaccine-induced antibodies was assessed against the vaccine virus and against well characterized wild-type (wt) viruses isolated between 1949-1991. We assessed whether JE-CV-induced antibodies can also neutralize more recent wt Japanese encephalitis virus (JEV) isolates including a genotype 1 isolate. METHODS: Sera from 12-18 month-old children who received a single dose of JE-CV in a phase III study in Thailand and the Philippines (ClinicalTrials.gov NCT00735644) were randomly selected and pooled according to neutralization titer against JE-CV into eight samples. Neutralization was assessed by plaque reduction neutralization tests (PRNT50) against three recent isolates from JEV genotypes 1 and 3 in addition to four JEV previously tested. RESULTS: Neutralization titers against the three recent JEV strains were comparable to those observed previously against other strains and the vaccine virus. The observed differences between responses to genotype 1 and 3 viruses were within assay variability for the PRNT50. CONCLUSIONS: The results were consistent with previously generated data on the neutralization of wt JEV isolates, immune responses induced by JE-CV neutralize recently isolated virus from southeast (SE) Asia and India.

Optimization and validation of a plaque reduction neutralization test for the detection of neutralizing antibodies to four serotypes of dengue virus used in support of dengue vaccine development.

A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype-specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT(50) for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay. The PRNT(50) was then validated and demonstrated to be suitable to detect and measure dengue serotype-specific neutralizing antibodies in human serum samples with acceptable intra-assay and inter-assay precision, accuracy/dilutability, specificity, and with a lower limit of quantitation of 10.

Safety and immunogenicity of the pneumococcal pneumolysin derivative PlyD1 in a single-antigen protein vaccine candidate in adults.

BACKGROUND: Pneumococcal vaccines based on conserved protein antigens have the potential to offer expanded protection against Streptococcus pneumoniae. OBJECTIVE: This study examined the safety and immunogenicity in adults of three doses of a pneumococcal single-antigen protein vaccine candidate formulated with aluminum hydroxide adjuvant and recombinantly derived, highly detoxified, genetically mutated pneumolysin protein (PlyD1). METHODS: This phase I, randomized, placebo-controlled, observer-blinded, dose-escalating study enrolled adults (18-50 years). In a pilot safety study, participants received a single injection of 10 µg PlyD1 and were observed for 24 h. Following review of the pilot safety data, participants were randomized (2:1) to receive two injections of PlyD1 at one of three doses or placebo 30 days apart. Assignment of second injection and successive dose cohorts was made after blinded safety reviews after each injection at each dose level. Safety endpoints included rates of solicited injection site reactions, solicited systemic reactions, unsolicited adverse events (AEs), serious AEs (SAEs), and safety laboratory tests. Immunogenicity endpoints included geometric mean concentrations of anti-PlyD1 IgG as determined by ELISA and functional assessment in an in vitro toxin neutralization assay. RESULTS: The study included a total of 100 participants, including 10 in the pilot study and 90 in the randomized study. None of the participants in the pilot study had SAEs, allergic reactions, or other safety concerns. Ninety participants received two doses of or placebo (n=30) or active vaccine candidate at 10 (n=20), 25 (n=20), or 50 µg (n=20). No vaccine-related SAE or discontinuation due to an AE occurred. Most solicited reactions were mild and transient. The most frequently reported solicited reactions were pain at the injection site and myalgia. Antigen-specific IgG levels and functional activity showed dose-related increases. When comparing the three dose levels, a plateau effect was observed at the 25 µg dose. CONCLUSIONS: All dose levels were safe and immunogenic. Repeat vaccination significantly increased the level of anti-PlyD1 antibodies. Functional antibody activity was demonstrated in sera from vaccinated individuals (ClinicalTrials.gov no. NCT01444352).