Quantifying the Vaccine-Induced Humoral Immune Response to Spike-Receptor Binding Domain as a Surrogate for Neutralization Testing Following mRNA-1273 (Spikevax) Vaccination Against COVID-19.

INTRODUCTION: There is a need for automated, high-throughput assays to quantify immune response after SARS-CoV-2 vaccination. This study assessed the combined utility of the Elecsys® Anti-SARS-CoV-2 S (ACOV2S) and the Elecsys Anti-SARS-CoV-2 (ACOV2N) assays using samples from the mRNA-1273 (Spikevax™) phase 2 trial (NCT04405076). METHODS: Samples from 593 healthy participants in two age cohorts (18-54 and ≥ 55 years), who received two injections with placebo (n = 198) or mRNA-1273 (50 µg [n = 197] or 100 µg [n = 198]), were collected at days 1 (first vaccination), 15, 29 (second vaccination), 43, and 57. ACOV2S results were used to assess humoral response to vaccination in different subgroups and were compared to live virus microneutralization assay. Samples from patients with either previous or concomitant infection (identified per ACOV2N) were analyzed separately. RESULTS: Receptor-binding domain-specific antibodies were readily detectable by ACOV2S for the vast majority of participants (174/189, 92.1% [50 µg dose] and 178/192, 92.7% [100 µg dose]) at the first post-vaccination assessment, with non-converters predominantly older in age. Seroconversion for all participants was observed at day 29 (before the second vaccine dose). Two weeks after the first dose, geometric mean concentration (GMC) of antibody levels was 1.37-fold higher in the 100 versus 50 µg group (p = 0.0098), reducing to 1.09-fold 2 weeks after the second dose (p = 0.0539, n.s.). In both dose groups, a more pronounced response was observed in the younger versus older age group on day 15 (50 µg, 2.49-fold [p < 0.0001]; 100 µg, 3.94-fold [p < 0.0001] higher GMC, respectively), and day 29 (1.93-fold, p = 0.0002, and 2.44-fold, p < 0.0001). Eight subjects had previous or concomitant SARS-CoV-2 infection; vaccination boosted their humoral response to very high ACOV2S results compared to infection-naïve recipients. ACOV2S strongly correlated with microneutralization (Pearson's r = 0.779; p < 0.0001), including good qualitative agreement. CONCLUSION: These results confirmed that ACOV2S is a highly valuable assay for tracking vaccine-related immune responses. Combined application with ACOV2N enables monitoring for breakthrough infection or stratification of previous natively infected individuals. The adaptive measuring range and high resolution of ACOV2S allow for early identification of seroconversion and resolution of very high titers and longitudinal differences between subgroups. Additionally, good correlation with live virus microneutralization suggests that ACOV2S is a reliable estimate of neutralization capacity in routine diagnostic settings.

Immune response to SARS-CoV-2 after a booster of mRNA-1273: an open-label phase 2 trial.

Rising breakthrough infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in previously immunized individuals have raised concerns for the need for a booster vaccine dose to combat waning antibody levels and new variants. Here we report the results of the open-label, non-randomized part B of a phase 2 trial in which we evaluated the safety and immunogenicity of a booster injection of 50 µg of the coronavirus disease 2019 (COVID-19) vaccine mRNA-1273 in 344 adult participants immunized 6-8 months earlier with a primary series of two doses of 50 µg or 100 µg of mRNA-1273 ( NCT04405076 ). Neutralizing antibody (nAb) titers against wild-type SARS-CoV-2 at 1 month after the booster were 1.7-fold (95% confidence interval (CI): 1.5, 1.9) higher than those at 28 days after the second injection of the primary series, which met the pre-specified non-inferiority criterion (primary immunogenicity objective) and might indicate a memory B cell response. The nAb titers against the Delta variant (B.1.617.2) (exploratory objective) at 1 month after the booster were 2.1-fold (95% CI: 1.8, 2.4) higher than those at 28 days after the second injection of the primary series. The seroresponse rate (95% CI (four-fold rise from baseline)) was 100% (98.7, 100.0) at 28 days after the booster compared to 98.3% (96.0, 99.4) after the primary series. The higher antibody titers at 28 days after the booster dose compared to 28 days after the second dose in the phase 3 COVE study were also observed in two assays for anti-spike IgG antibody measured by ELISA and by Meso Scale Discovery (MSD) Multiplex. The frequency of solicited local and systemic adverse reactions after the booster dose was similar to that after the second dose in the primary two-dose series of mRNA-1273 (50 µg or 100 µg); no new signals were observed in the unsolicited adverse events; and no serious adverse events were reported in the 1-month follow-up period. These results show that a booster injection of mRNA-1273 more than 6 months after completing the primary two-dose series is safe and elicited nAb titers that were statistically significantly higher than the peak titers detected after the primary vaccination series, suggesting that a booster dose of mRNA-1273 might result in increased vaccine effectiveness against infection and disease caused by SARS-CoV-2.

Clinical utility of Elecsys Anti-SARS-CoV-2 S assay in COVID-19 vaccination: An exploratory analysis of the mRNA-1273 phase 1 trial.

BACKGROUND: The ability to quantify an immune response after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential. This study assessed the clinical utility of the quantitative Roche Elecsys® Anti-SARS-CoV-2 S assay (ACOV2S) using samples from the 2019-nCoV vaccine (mRNA-1273) phase 1 trial (NCT04283461). METHODS: Samples from 30 healthy participants, aged 18-55 years, who received two injections with mRNA-1273 at a dose of 25 µg (n=15) or 100 µg (n=15), were collected at Days 1 (first vaccination), 15, 29 (second vaccination), 43 and 57. ACOV2S results (shown in U/mL - equivalent to BAU/mL per the first WHO international standard) were compared with results from ELISAs specific to antibodies against the Spike protein (S-2P) and the receptor binding domain (RBD) as well as neutralization tests including nanoluciferase (nLUC80), live-virus (PRNT80), and a pseudovirus neutralizing antibody assay (PsVNA50). RESULTS: RBD-specific antibodies were already detectable by ACOV2S at the first time point of assessment (d15 after first vaccination), with seroconversion before in all but 2 participants (25 µg dose group); all had seroconverted by Day 29. Across all post-baseline visits, geometric mean concentration of antibody levels were 3.27-7.48-fold higher in the 100 µg compared with the 25 µg dose group. ACOV2S measurements were highly correlated with those from RBD ELISA (Pearson's r=0.938; p<0.0001) and S-2P ELISA (r=0.918; p<0.0001). For both ELISAs, heterogeneous baseline results and smaller increases in antibody levels following the second vs first vaccination compared with ACOV2S were observed. ACOV2S showed absence of any baseline noise indicating high specificity detecting vaccine-induced antibody response. Moderate-strong correlations were observed between ACOV2S and neutralization tests (nLUC80 r=0.933; PsVNA50, r=0.771; PRNT80, r=0.672; all p≤0.0001). CONCLUSION: The Elecsys Anti-SARS-CoV-2 S assay (ACOV2S) can be regarded as a highly valuable method to assess and quantify the presence of RBD-directed antibodies against SARS-CoV-2 following vaccination, and may indicate the presence of neutralizing antibodies. As a fully automated and standardized method, ACOV2S could qualify as the method of choice for consistent quantification of vaccine-induced humoral response.

Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to growing concerns over increased transmissibility and the ability of some variants to partially escape immunity. Sera from participants immunized on a prime-boost schedule with the mRNA-1273 COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants, including variants of concern (VOCs) and variants of interest (VOIs), compared to neutralization of the wild-type SARS-CoV-2 virus (designated D614G). Results showed minimal, statistically nonsignificant effects on neutralization titers against the B.1.1.7 (Alpha) variant (1.2-fold reduction compared with D614G); other VOCs, such as B.1.351 (Beta, including B.1.351-v1, B.1.351-v2, and B.1.351-v3), P.1 (Gamma), and B.1.617.2 (Delta), showed significantly decreased neutralization titers ranging from 2.1-fold to 8.4-fold reductions compared with D614G, although all remained susceptible to mRNA-1273-elicited serum neutralization. IMPORTANCE In light of multiple variants of SARS-CoV-2 that have been documented globally during the COVID-19 pandemic, it remains important to continually assess the ability of currently available vaccines to confer protection against newly emerging variants. Data presented herein indicate that immunization with the mRNA-1273 COVID-19 vaccine produces neutralizing antibodies against key emerging variants tested, including variants of concern and variants of interest. While the serum neutralization elicited by mRNA-1273 against most variants tested was reduced compared with that against the wild-type virus, the level of neutralization is still expected to be protective. Such data are crucial to inform ongoing and future vaccination strategies to combat COVID-19.

Safety and immunogenicity of SARS-CoV-2 variant mRNA vaccine boosters in healthy adults: an interim analysis.

The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) with decreased susceptibility to neutralization has generated interest in assessments of booster doses and variant-specific vaccines. Clinical trial participants who received a two-dose primary series of the COVID-19 vaccine mRNA-1273 approximately 6 months earlier entered an open-label phase 2a study ( NCT04405076 ) to evaluate the primary objectives of safety and immunogenicity of a single booster dose of mRNA-1273 or variant-modified mRNAs, including multivalent mRNA-1273.211. As the trial is currently ongoing, this exploratory interim analysis includes preliminary descriptive results only of four booster groups (n = 20 per group). Immediately before the booster dose, neutralizing antibodies against wild-type D614G virus had waned (P < 0.0001) relative to peak titers against wild-type D614G measured 1 month after the primary series, and neutralization titers against B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) VOCs were either low or undetectable. Both the mRNA-1273 booster and variant-modified boosters were safe and well-tolerated. All boosters, including mRNA-1273, numerically increased neutralization titers against the wild-type D614G virus compared to peak titers against wild-type D614G measured 1 month after the primary series; significant increases were observed for mRNA-1273 and mRNA-1273.211 (P < 0.0001). In addition, all boosters increased neutralization titers against key VOCs and VOIs, including B.1.351, P.1. and B.1.617.2, that were statistically equivalent to peak titers measured after the primary vaccine series against wild-type D614G virus, with superior titers against some VOIs. This trial is ongoing.

Clinical Utility of Elecsys Anti-SARS-CoV-2 S Assay in COVID-19 Vaccination: An Exploratory Analysis of the mRNA-1273 Phase 1 Trial.

Background: The ability to quantify an immune response after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential. This study assessed the clinical utility of the quantitative Roche Elecsys® Anti-SARS-CoV-2 S assay (ACOV2S) using samples from the 2019-nCoV vaccine (mRNA-1273) phase 1 trial (NCT04283461). Methods: Samples from 30 healthy participants, aged 18-55 years, who received two injections with mRNA-1273 at a dose of 25 µg (n=15) or 100 µg (n=15), were collected at Days 1 (first vaccination), 15, 29 (second vaccination), 43 and 57. ACOV2S results (shown in U/mL - equivalent to BAU/mL per the first WHO international standard) were compared with results from ELISAs specific to antibodies against the Spike protein (S-2P) and the receptor binding domain (RBD) as well as neutralization tests including nanoluciferase (nLUC80), live-virus (PRNT80), and a pseudovirus neutralizing antibody assay (PsVNA50). Results: RBD-specific antibodies were already detectable by ACOV2S at the first time point of assessment (d15 after first vaccination), with seroconversion before in all but two participants (25 µg dose group); all had seroconverted by Day 29. Across all post-baseline visits, geometric mean concentration of antibody levels was 3.27-7.48-fold higher in the 100 µg compared with the 25 µg dose group. ACOV2S measurements were highly correlated with those from RBD ELISA (Pearson's r=0.938; p<0.0001) and S-2P ELISA (r=0.918; p<0.0001). For both ELISAs, heterogeneous baseline results and smaller increases in antibody levels following the second vs first vaccination compared with ACOV2S were observed. ACOV2S showed absence of any baseline noise indicating high specificity detecting vaccine-induced antibody response. Moderate-strong correlations were observed between ACOV2S and neutralization tests (nLUC80 r=0.933; PsVNA50, r=0.771; PRNT80, r=0.672; all p ≤ 0.0001). Conclusion: The Elecsys Anti-SARS-CoV-2 S assay (ACOV2S) can be regarded as a highly valuable method to assess and quantify the presence of RBD-directed antibodies against SARS-CoV-2 following vaccination and may indicate the presence of neutralizing antibodies. As a fully automated and standardized method, ACOV2S could qualify as the method of choice for consistent quantification of vaccine-induced humoral response.

Pristane-Accelerated Autoimmune Disease in (SWR X NZB) F1 Mice Leads to Prominent Tubulointerstitial Inflammation and Human Lupus Nephritis-Like Fibrosis.

Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.

Stromal transcriptional profiles reveal hierarchies of anatomical site, serum response and disease and identify disease specific pathways.

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.

Pathways activated during human asthma exacerbation as revealed by gene expression patterns in blood.

BACKGROUND: Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations. METHODOLOGY/PRINCIPAL FINDINGS: Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations. CONCLUSIONS/SIGNIFICANCE: This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.

Mapping similarities in mTOR pathway perturbations in mouse lupus nephritis models and human lupus nephritis.

INTRODUCTION: Treatment with sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to be efficacious in the MRL/lpr and NZB x NZW F1 mouse models of lupus nephritis, indicating a critical role for the mTOR pathway in both models. This type of demonstration of efficacy in animal models is usually a pre-requisite for advancement into clinical development. However, efficacy in an animal model often has not translated to the desired activity in the clinic. Therefore, a more profound understanding of the mechanistic similarities and differences between various animal models and human diseases is highly desirable. METHODS: Transcriptional profiling was performed on kidneys from mice with lupus nephritis; from mice who had efficacious drug treatment; and from mice before they developed nephritis. Analysis of variance with false discovery rate adjusted to p < 0.05 and an average fold change of two or more was used to identify transcripts significantly associated with disease and response to therapy. Pathway analyses (using various bioinformatics tools) were carried out to understand the basis for drug efficacy in the mouse model. The relevance in human lupus of the pathways identified in the mouse model was explored using information from several databases derived from the published literature. RESULTS: We identified a set of nephritis-associated genes in mouse kidney. Expression of the majority of these returned to asymptomatic levels on sirolimus treatment, confirming the correlation between expression levels and symptoms of nephritis. Network analysis showed that many of these nephritis genes are known to interact with the mTOR pathway. This led us to ask what human diseases are linked to the mTOR pathway. We constructed the mTOR pathway interactome consisting of proteins that interact with members of the mTOR pathway and identified a strong association between mTOR pathway genes and genes reported in the literature as being involved in human lupus. CONCLUSIONS: Our findings implicate the mTOR pathway as a critical contributor to human lupus. This broad pathway-based approach to understanding the similarities in, and differences between, animal models and human diseases may have broader utility.

Risk factors associated with beta-amyloid(1-42) immunotherapy in preimmunization gene expression patterns of blood cells.

BACKGROUND: A phase 2a, double-blind, placebo-controlled, multicenter study was conducted to evaluate safety, tolerability, and pilot efficacy of immunization with beta-amyloid((1-42)) in patients with Alzheimer disease. Six immunizations were planned but were halted when meningoencephalitis was recognized as an adverse event in 6% of immunized patients. OBJECTIVE: To identify biomarkers associated with both the risk of meningoencephalitis and antibody responsiveness. PARTICIPANTS: One hundred fifty-three patients with mild to moderate Alzheimer disease.Main Outcome Measure Association between response to immunization and preimmunization expression levels of 8239 messenger RNA transcripts expressed in peripheral blood mononuclear cells that had been collected at the screening visit. RESULTS: Expression patterns of genes related to apoptosis and proinflammatory pathways (tumor necrosis factor pathway in particular) were identified as biomarkers of risk for the development of meningoencephalitis. Expression patterns of genes related to protein synthesis, protein trafficking, DNA recombination, DNA repair, and cell cycle were strongly associated with IgG response to immunization. CONCLUSIONS: Candidate biomarkers associated with risk of immunotherapy-related meningoencephalitis were detected in blood collected prior to treatment. In addition, a different set of biomarkers were identified that were associated with the desired outcome of IgG response.