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This study aimed to evaluate and compare the performance of three anti-S and one anti-N assays that were available to the project in detecting antibody levels after three commonly used SARS-CoV-2 vaccines (Pfizer, Moderna, and Johnson & Johnson). It also aimed to assess the association of age, sex, race, ethnicity, vaccine timing, and vaccine side effects on antibody levels in a cohort of 827 individuals. In September 2021, 698 vaccinated individuals donated blood samples as part of the Association for Diagnostics & Laboratory Medicine (ADLM) COVID-19 Immunity Study. These individuals also participated in a comprehensive survey covering demographic information, vaccination status, and associated side effects. Additionally, 305 age- and gender-matched samples were obtained from the ADLM 2015 sample bank as pre-COVID-19-negative samples. All these samples underwent antibody level analysis using three anti-S assays, namely Beckman Access SARS-CoV-2 IgG (Beckman assay), Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG (Ortho assay), Siemens ADVIA Centaur SARS-CoV-2 IgG (Siemens assay), and one anti-N antibody assay: Bio-Rad Platelia SARS-CoV-2 Total Ab assay (BioRad assay). A total of 827 samples (580 COVID-19 samples and 247 pre-COVID-19 samples) received results for all four assays and underwent further analysis. Beckman, Ortho, and Siemens anti-S assays showed an overall sensitivity of 99.5%, 97.6%, and 96.9%, and specificity of 90%, 100%, and 99.6%, respectively. All three assays indicated 100% sensitivity for individuals who received the Moderna vaccine and boosters, and over 99% sensitivity for the Pfizer vaccine. Sensitivities varied from 70.4% (Siemens), 81.5% (Ortho), and 96.3% (Beckman) for individuals who received the Johnson & Johnson vaccine. BioRad anti-N assays demonstrated 46.2% sensitivity and 99.25% specificity based on results from individuals with self-reported infection. The highest median anti-S antibody levels were measured in individuals who received the Moderna vaccine, followed by Pfizer and then Johnson & Johnson vaccines. Higher anti-S antibody levels were significantly associated with younger age and closer proximity to the last vaccine dose but were not associated with gender, race, or ethnicity. Participants with higher anti-S levels experienced significantly more side effects as well as more severe side effects (e.g., muscle pain, chills, fever, and moderate limitations) (p < 0.05). Anti-N antibody levels only indicated a significant correlation with headache. This study indicated performance variations among different anti-S assays, both among themselves and when analyzing individuals with different SARS-CoV-2 vaccines. Caution should be exercised when conducting large-scale studies to ensure that the same platform and/or assays are used for the most effective interpretation of the data.

ObjectivesThe objective of this prospective study was to investigate the role of adaptive immunity in response to SARS-CoV-2 vaccines.Design and methodsA cohort of 677 vaccinated individuals participated in a comprehensive survey of their vaccination status and associated side effects, and donated blood to evaluate their adaptive immune responses by neutralizing antibody (NAb) and T cell responses. The cohort then completed a follow-up survey to investigate the occurrence of breakthrough infections.ResultsNAb levels were the highest in participants vaccinated with Moderna, followed by Pfizer and Johnson & Johnson. NAb levels decreased with time after vaccination with Pfizer and Johnson & Johnson. T cell responses showed no significant difference among the different vaccines and remained stable up to 10 months after the study period for all vaccine types. In multivariate analyses, NAb responses (

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

ImportanceMost healthcare institutions require employees to be vaccinated against SARS-CoV-2 and many also require at least one booster.ObjectiveWe determine the impact of vaccine type, demographics, and health conditions on COVID-19 vaccine side effects in healthcare professionals.DesignA COVID-19 immunity study was performed at the 2021 American Association for Clinical Chemistry Annual Scientific meeting. As part of this study, a REDCap survey with cascading questions was administered from September 9, 2021 to October 20, 2021. General questions included participant demographics, past and present health conditions, smoking, exercise, and medications. COVID-19 specific questions asked about SARS-CoV-2 vaccine status and type, vaccine-associated side effects after each dose including any boosters, previous infection with COVID-19, diagnostic testing performed, and type and severity symptoms of COVID-19.ResultsThere were 975 participants (47.1% male, median age of 50 years) who completed the survey. Pfizer was the most commonly administered vaccine (56.4%) followed by Moderna (32.0%) and Johnson & Johnson (7.1%). There were no significant differences in vaccine type received by age, health conditions, smoking, exercise, or type or number of prescription medications. Side effects were reported more frequently after second dose (e.g., Moderna or Pfizer) (54.1%) or single/only dose of Johnson & Johnson (47.8%). Males were significantly more likely to report no side effects (p < 0.001), while females were significantly more likely to report injection site reactions (p < 0.001), fatigue (p < 0.001), headache (p < 0.001), muscle pain (p < 0.001), chills (p = 0.001), fever (p = 0.007), and nausea (p < 0.001). There was a significant upward trend in participants reporting no side effects with increasing age (p < 0.001). There were no significant trends in side effects among different races, ethnicities, health conditions, medications, smoking status or exercise. In multivariate logistic regressions analyses, the second dose of Moderna was associated with a significantly higher risk of side effects than both the second dose of Pfizer and the single dose of Johnson & Johnson.Conclusions and relevanceYounger people, females, and those receiving the second dose of Moderna had more COVID-19 vaccine side effects that per self-report led to moderate to severe limitations. As reported in other studies, the increase in side effects from Moderna may be explained by higher viral mRNA concentrations but be associated with additional protective immunity.

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

In 2021, the Association for Diagnostics & Laboratory Medicine (ADLM) (formerly the American Association for Clinical Chemistry [AACC]) developed a scientific study that aimed to contribute to the understanding of SARS-CoV-2 immunity during the evolving course of the pandemic. This study was led by a group of expert member volunteers and resulted in survey data from 975 individuals and blood collection from 698 of those participants. This paper describes the formulation and execution of this large-scale scientific study, encompassing best practices and insights gained throughout the endeavor. [ABSTRACT FROM AUTHOR]

Research describes two families of major histocompatibility complex class lb genes, M10 and M1, which are expressed in the basal zone of the vomeronasal organ, the site of pheromone receptor expression. Data indicate that M10 and M1 express V2R pheromone receptor expressing neurons.

We have assembled a contig of 81 yeast artificial chromosome clones that spans 8 Mb and contains the entire major histocompatibility complex (Mhc) from mouse strain C57BL/6 (H2[supb), and we are in the process of assembling an Mhc contig of bacterial artificial chromosome (BAC) clones from strain 129 (H2[supbc], which differs from C57BL/6 in the H2-Q and H2-T regions. The current BAC contig extends from Tapasin to D17Leh89 with gaps in the class II. H2-Q, and distal H2-H regions. Only four BAC clones were required to link the class I genes of the H2-Q and H2-T regions, and no new class I gene was found in the previous gap. The proximal 1 Mb of the H2-M region has been analyzed in detail and is ready for sequencing; it includes 21 class I genes or fragments, at least 14 olfactory receptor-like genes, and a number of non-class I genes that clearly establish a conserved synteny with the class I regions of the human and rat Mhc. [ABSTRACT FROM AUTHOR]

Aim We have previously considered transposon-and sonication-based library preparation methods for next generation sequencing of HLA-A, B, C, DRB1 and DQB on the Illumina MiSeq, and found that they are not suitable for routine clinical work. Here, we evaluated reagents from NEB, NEBNext dsDNA Fragmentase and NEBNext DNA Library Prep Master Mix, to generate sequencing libraries for clinical testing. Methods We performed long-range PCR for HLA-A, B, C, DRB1 and DQB1 on two IHWG samples using a combination of in-house and commercial primers. The amplified products ranged from 3900 to 4800 bp in length. The class I primers were designed to cover all exons, introns and UTRs whereas the class II primers covered exon 2 to the 3′UTR. Samples were enzymatically fragmented using NEBNext dsDNA Fragmentase which is composed of two different endonucleases isolated from V. vulnificus and T7. Length of incubation time of the amplicon and Fragmentase mixture determines general fragment length. We compared the ability of the endonucleases to generate fragments of two lengths, 100–300 bp and 300–600 bp, respectively. Following fragmentation, we prepared libraries for sequencing with the NEBNext Library Prep kit. Unique indices were ligated following end repair and dA-tailing of the fragments. Fragment size was determined by the Agilent 2100 Bioanalyzer instrument. Samples were equimolarly pooled and ran on the Illumina MiSeq using Reagent kit v2 for both 300 and 500 cycle runs. Results The two Fragmentase incubation times suggested by the manufacturer produced two very different average fragment sizes. Including the adaptor and index, the average size of the 100–300 bp incubation time was 273 bp and the 300–600 bp incubation time yielded a product of 619 bp. Conclusions Use of the NEBNext dsDNA Fragmentase allows the generation of fragments of different sizes. Although we easily generated our desired fragment sizes, we found the NEBNext DNA Library Prep Master Kit to be extremely time- and labor intensive requiring numerous transfer steps. Additionally, this kit requires a large number of AMPure XP Beads for sample clean-up. Analyses are in progress to evaluate the effect that size and library preparation method has on HLA typing assignments. [ABSTRACT FROM AUTHOR]