An omicron-specific neutralizing antibody test predicts neutralizing activity against XBB 1.5.

Introduction: Understanding the immune status of an individual using neutralizing antibody testing is complicated by the continued evolution of the SARS-CoV-2 virus. Previous work showed that assays developed against the wildtype strain of SARS-CoV-2 were insufficient predictors of neutralization of omicron variants, thus we developed an omicron-specific flow cytometry-based neutralizing antibody test and performed experiments to assess how well it compared to an omicron-specific PRNT assay (gold standard) and whether it could predict neutralizing activity to more recent omicron subvariants such as XBB.1.5. Methods: Accuracy of a novel flow cytometry-based neutralizing antibody (FC-NAb) assay was determined by comparison with an omicron-specific PRNT assay. A series of samples were evaluated in both the omicron FC-NAb assay and a second test was designed to assess neutralization of XBB.1.5. Results: Good concordance between the omicron FC-NAb test and the omicron PRNT was demonstrated (AUC = 0.97, p <0.001; sensitivity = 94%, specificity = 100%, PPV = 100%, and NPV = 97%). A strong linear relationship between the omicron FC-NAb and neutralization of XBB1.5 was observed (r = 0.83, p<0.001). Additionally, the omicron FC-NAb test was a very strong predictor of positive XBB1.5 NAb activity (AUC = 0.96, p<0.001; sensitivity = 94%, specificity = 90%, positive predictive value = 90%, and negative predictive values = 94%). Discussion: Our data suggest that despite continued evolution of the SARS-CoV-2 spike protein, the omicron FC-NAb assay described here is a good predictor of XBB1.5 neutralizing activity, as evidenced by a strong correlation and good predictive performance characteristics.

Simultaneous measurement of multiple variant-specific SARS-CoV-2 neutralizing antibodies with a multiplexed flow cytometric assay.

Introduction: Neutralizing antibodies (NAbs) have been recognized as surrogates of protection against SARS-CoV-2; however, the emergence of variants/subvariants escaping neutralization suggests that laboratory assessments of NAbs against the ancestral/wild type (WT) antigens likely overestimate the degree of protection. Methods: A novel flow cytometry-based multiplex test system was developed for the simultaneous detection of NAbs of multiple SARS-CoV-2 variants. SARS-CoV-2 antibodies (Abs) including IgG, IgM, IgA isotypes were measured in the same system. Samples from negative, convalesced, vaccinated, boosted, and breakthrough infection (BTI) populations were tested for both NAbs and Abs. Results: NAbs induced by WT showed neutralization activity that correlated strongly to all variants (R2 > 0.85) except omicron BA.1/BA.2 (R2 <0.50). Two doses of vaccine elicited very little protective immunity against BA.1/BA.2, though a booster dose significantly improved NAbs for all variants. NAbs/Abs increased more following BTI than after a booster, suggesting that hybrid immunity (vaccination + natural immunity) was more robust to all variants including BA.1/BA.2. BTIs occurring in the omicron era led to stronger NAb responses against BA.1/BA.2 than did older BTIs. In all comparisons, the RBD antigens demonstrated greater differences between WT and BA.1/BA.2 than the spike antigens. Discussion: Taken together, we demonstrated that both Ab and NAb against multiple SARS-CoV-2 variants/subvariants can be reliably detected on the same multiplex platform. Distinguishing NAbs to the appropriate antigenic target of prevalent variants offers the best correlate of protection and aids individual decisions about the appropriateness and cadence of vaccine boosters and other exposure mitigation strategies.

Evaluation of Transport Media and Specimen Transport Conditions for the Detection of SARS-CoV-2 by Use of Real-Time Reverse Transcription-PCR.

The global coronavirus (CoV) disease 2019 (COVID-19) pandemic has resulted in a worldwide shortage of viral transport media and raised questions about specimen stability. The objective of this study was to determine the stability of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) RNA in specimen transport media under various storage conditions. Transport media tested included UTM, UTM-RT, ESwab, M4, and saline (0.9% NaCl). Specimen types tested included nasopharyngeal/oropharyngeal swabs in the above-named transport media, bronchoalveolar lavage (BAL) fluid, and sputum. A high-titer SARS-CoV-2 remnant patient specimen was spiked into pooled SARS-CoV-2 RNA-negative specimen remnants for the various medium types. Aliquots of samples were stored at 18°C to 26°C, 2°C to 8°C, and -10°C to -30°C and then tested at time points up to 14 days. Specimens consistently yielded amplifiable RNA with mean cycle threshold differences of <3 over the various conditions assayed, thus supporting the use and transport of alternative collection media and specimen types under a variety of temperature storage conditions.

DNA Methylation Signatures of Early Childhood Malnutrition Associated With Impairments in Attention and Cognition.

BACKGROUND: Early childhood malnutrition affects 113 million children worldwide, impacting health and increasing vulnerability for cognitive and behavioral disorders later in life. Molecular signatures after childhood malnutrition, including the potential for intergenerational transmission, remain unexplored. METHODS: We surveyed blood DNA methylomes (~483,000 individual CpG sites) in 168 subjects across two generations, including 50 generation 1 individuals hospitalized during the first year of life for moderate to severe protein-energy malnutrition, then followed up to 48 years in the Barbados Nutrition Study. Attention deficits and cognitive performance were evaluated with the Connors Adult Attention Rating Scale and Wechsler Abbreviated Scale of Intelligence. Expression of nutrition-sensitive genes was explored by quantitative reverse transcriptase polymerase chain reaction in rat prefrontal cortex. RESULTS: We identified 134 nutrition-sensitive, differentially methylated genomic regions, with most (87%) specific for generation 1. Multiple neuropsychiatric risk genes, including COMT, IFNG, MIR200B, SYNGAP1, and VIPR2 showed associations of specific methyl-CpGs with attention and IQ. IFNG expression was decreased in prefrontal cortex of rats showing attention deficits after developmental malnutrition. CONCLUSIONS: Early childhood malnutrition entails long-lasting epigenetic signatures associated with liability for attention and cognition, and limited potential for intergenerational transmission.

Rare missense neuronal cadherin gene (CDH2) variants in specific obsessive-compulsive disorder and Tourette disorder phenotypes.

The recent finding that the neuronal cadherin gene CDH2 confers a highly significant risk for canine compulsive disorder led us to investigate whether missense variants within the human ortholog CDH2 are associated with altered susceptibility to obsessive-compulsive disorder (OCD), Tourette disorder (TD) and related disorders. Exon resequencing of CDH2 in 320 individuals identified four non-synonymous single-nucleotide variants, which were subsequently genotyped in OCD probands, Tourette disorder probands and relatives, and healthy controls (total N=1161). None of the four variants was significantly associated with either OCD or TD. One variant, N706S, was found only in the OCD/TD groups, but not in controls. By examining clinical data, we found there were significant TD-related phenotype differences between those OCD probands with and without the N845S variant with regard to the co-occurrence of TD (Fisher's exact test P=0.014, OR=6.03). Both N706S and N845S variants conferred reduced CDH2 protein expression in transfected cells. Although our data provide no overall support for association of CDH2 rare variants in these disorders considered as single entities, the clinical features and severity of probands carrying the uncommon non-synonymous variants suggest that CDH2, along with other cadherin and cell adhesion genes, is an interesting gene to pursue as a plausible contributor to OCD, TD and related disorders with repetitive behaviors, including autism spectrum disorders.

Gender-specific reduction of estrogen-sensitive small RNA, miR-30b, in subjects with schizophrenia.

Estrogen signaling pathways affect cortical function and metabolism, are thought to play a role in the pathophysiology of schizophrenia, and exert neuroprotective effects in female subjects at risk. However, the molecular signatures of estrogen signaling in normal and diseased cerebral cortex remain largely unexplored. Expression of the estrogen-sensitive small RNA, microRNA-30b (miR-30b), was studied in 30 controls and 30 matched samples from subjects diagnosed with schizophrenia from prefrontal cortex (PFC), as well as in 23 samples from parietal cortex (12 controls and 11 schizophrenia cases). The majority of case and control samples were genotyped for an estrogen receptor α (Esr1) sequence variant (rs2234693) previously associated with genetic risk, and a subset of them were subjected to further analysis to determine expression of mature and precursor forms of miR-30b (pre/pri-miR-30b). Gender-dimorphic expression was also explored in mouse frontal cortex and hippocampus. A significant interaction between gender and diagnosis was discovered for changes in mature miR-30b levels, so that miR-30b expression was significantly reduced in the cerebral cortex of female but not male subjects with schizophrenia. In addition, disease-related changes in miR-30b expression in a subset of female subjects were further modulated by Esr1 genotype. Changes after antipsychotic drug exposure remained insignificant. These preliminary findings point to the possibility that disease-related changes in the expression of small noncoding RNAs such as miR-30b in schizophrenia could be influenced by gender and potentially regulated by estrogen signaling.

Molecular determinants of dysregulated GABAergic gene expression in the prefrontal cortex of subjects with schizophrenia.

BACKGROUND: Prefrontal deficits in gamma-aminobutyric acid (GABA)ergic gene expression, including neuropeptide Y (NPY), somatostatin (SST), and parvalbumin (PV) messenger RNAs (mRNAs), have been reported for multiple schizophrenia cohorts. Preclinical models suggest that a subset of these GABAergic markers (NPY/SST) is regulated by brain-derived neurotrophic factor (BDNF), which in turn is under the inhibitory influence of small noncoding RNAs. However, it remains unclear if these mechanisms are important determinants for dysregulated NPY and SST expression in prefrontal cortex (PFC) of subjects with schizophrenia. METHODS: Using a postmortem case-control design, the association between BDNF protein, NPY/SST/PV mRNAs, and two BDNF-regulating microRNAs (miR-195 and miR-30a-5p) was determined in samples from the PFC of 20 schizophrenia and 20 control subjects. Complementary studies were conducted in cerebral cortex of mice subjected to antipsychotic treatment or a brain-specific ablation of the Bdnf gene. RESULTS: Subjects with schizophrenia showed deficits in NPY and PV mRNAs. Within-pair differences in BDNF protein levels showed strong positive correlations with NPY and SST and a robust inverse association with miR-195 levels, which in turn were not affected by antipsychotic treatment or genetic ablation of Bdnf. CONCLUSIONS: Taken together, these results suggest that prefrontal deficits in a subset of GABAergic mRNAs, including NPY, are dependent on the regional supply of BDNF, which in turn is fine-tuned through a microRNA (miRNA)-mediated mechanism.